Hennara/MedRAG_contents · Datasets at Hugging Face

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0cd8efa9-a8f3-4f27-8894-290053dcfb90	0	Distribution of releasing factors, biogenic amines, and related enzymes in the bovine median eminence. The bovine median eminence was dissected into eight different subdivisions: rostral, anterior internal, anterior external, middle external medial, middle external lateral, middle internal medial, middle internal lateral, and caudal. Thyrotropin-releasing hormone (TRH) was found in the highest concentrations in the middle external medial and lateral subdivisions;
0cd8efa9-a8f3-4f27-8894-290053dcfb90	1	luteinizing hormone-releasing hormone (LHRH) was concentrated in the middle external lateral and anterior internal subdivisions. Among the various neurotransmitters and enzymes assayed, only dopamine and choline acetyltransferase were present in highest concentrations in the same subdivisions of the bovine median eminence found to be rich in TRH and LHRH.
0cd8efa9-a8f3-4f27-8894-290053dcfb90	2	The distributions of norepinephrine, dopamine-beta-hydroxylase, serotonin, tryptophan hydroxylase, phenylethanolamine-N-methyltransferase, glutamic acid decarboxylase, and histamine appeared to correlate poorly with the major distributions of TRH and LHRH. These findings suggest that at the level of the median eminence, central neuroendocrine regulation of TRH and LHRH release may involve an interaction only with dopamine and acetylcholine.
85e0eb7d-c70a-49d1-a0c2-299dd52c3993	0	The effects of neurally active amino acids on prolactin secretion. Several neurally active amino acids were injected into the third ventricle of anesthetized male rats. Two or eight mumole of GABA produced significant increases in the plasma concentrations of prolactin (PRL), indicating increased PRL release from the pituitary. Two mumole of glycine was also effective in elevating PRL levels.
85e0eb7d-c70a-49d1-a0c2-299dd52c3993	1	The intraventricular injection of the lowest dose of GABA (1.0 mumole), glutamate (0.4 or 2.3 mumole), lysine (0.2 or 2.0 mumole), or 0.9% NaCl did not alter PRL levels significantly. Plasma PRL concentrations did not increase following the injection of GABA or glycine directly into the anterior pituitary gland.
85e0eb7d-c70a-49d1-a0c2-299dd52c3993	2	The results suggest that GABA and glycine may play a role in the neural regulation of PRL secretion.
b79e44d6-c488-44cc-b90c-c3ee1f6c92ad	0	Determination of D-3-hydroxybutyrate dehydrogenase in mouse pancreatic islets with a photokinetic technique using bacterial luciferase. A sensitive assay for d-3hydroxybutrate dehydrogenase (EC 1.1.1.30) was developed for use with the minute amounts of material obtained from islets of Langerhans microdissected from freeze-dried pancreatic sections. NADH formed in the enzyme reaction was determined by photokinetic analysis of the luminescence obtained with bacterial luciferase from Achromobacter fishcherii.
b79e44d6-c488-44cc-b90c-c3ee1f6c92ad	1	In this way, accurate determination was obtained with less than 0.1 mug dry weight of islet material. In obese hyperglycemic mice, the islet enzyme had an activity of 4.7 mumoles/min and g dry weight. Optimal enzyme activity was found at pH 8 for the islet enzyme. The enzyme activity was similar in pancreatic islets and acini, while considerably higher activity was found in cardiac muscle, liver and renal cortex.
b79e44d6-c488-44cc-b90c-c3ee1f6c92ad	2	Normal mouse islets showed about equal enzyme activity as the islets from obese hyperglycemic mice.
68e9ce55-7bf2-4f91-866f-a864ba243df8	0	Human platelet glucose-6-phosphate dehydrogenase. Total purification, kinetic studies and relationship with enzyme from other blood cells. Human platelet G-6-PD has been highly purified, to homogeneity, and its kinetic, electrophoretic and immunological characteristics have been studied. Platelet G-6-PD differs from erythrocyte or leukocyte enzymes by an increased Michaelis constant for G-6-P and a slow activity at the acid pHs.
68e9ce55-7bf2-4f91-866f-a864ba243df8	1	By electrofocusing only a main active band (band a) of platelet G-6-PD was found. The incubation at 37 degrees C in the presence of NADP+ and dithiothreitol normalize Km-G-6-P of platelet G-6-PD; the incubation with boiled and ultrafiltered leukemic granulocyte extracts led to an anodisation of G-6-PD active forms, a decrease of the molecular specific activity and a further increase of Km-G-6-P;
68e9ce55-7bf2-4f91-866f-a864ba243df8	2	these last modifications are the same as those undergone by G-6-PD incubated in crude extracts of normal or leukemic granulocytes.
45f35abd-120c-421f-bb10-03a955f47909	0	Clinical evaluation of blood lactate levels in equine colic. Blood lactate levels were evaluated in 36 horses (43 cases) presented with colic. A correlation between increasing blood lactate levels and decreasing percentage survival has been shown. An appreciable anion gap was found in 7 of 10 cases analyzed in detail but in each case the entire gap could not be accounted for by lactate alone.
45f35abd-120c-421f-bb10-03a955f47909	1	Proposals are offered to account for the unmeasured anions. Blood lactate determination is suggested as a prognostic rather than a diagnostic aid for the equine practitioner and should be used to augment other clinical findings in the horse exhibiting colic.
a6135ea2-fb90-4153-b471-9b5041530ee9	0	Equine viral encephalitis. The most important neurotropic viral infections of the horse are the arthropod-borne encephalitides. These include Venezuelan encephalitis (VE), eastern encephalitis (EE) and western encephalitis (WE), which are found in the Americas, and Japanese B encephalitis which occurs in the Far East. All the viruses cause encephalitis in man.
a6135ea2-fb90-4153-b471-9b5041530ee9	1	Between 1969 and 1972 an epidemic of VE occurred in Central America. In 1971 the disease was reported in Texas, where it was brought under control by the vaccination of susceptible horses with an attenuated live virus vaccine and by the reduction of the mosquito population with insecticides sprayed from aircraft.
a6135ea2-fb90-4153-b471-9b5041530ee9	2	A high titre viraemia occurs with VE virus in the horse and epidemics are maintained by a mosquito/horse cycle; infection of man and other species is incidental. EE and WE have been recognised as separate diseases since 1933 and in the U.S.A. horses are protected by routine vaccination.
a6135ea2-fb90-4153-b471-9b5041530ee9	3	Epidemics of these diseases are routine vaccination. Epidemics of these diseases are now uncommon. In contrast with VE, both EE and WE viruses are maintained by a bird/mosquito cycle. The viraemia in the horse is generally considered insufficient to infect mosquito vectors; the horse is a "dead end host".
a6135ea2-fb90-4153-b471-9b5041530ee9	4	Several species of mosquito can act as vectors of VE, WE and EE. The extension of other arthropod-borne diseases to areas originally outside their geographical distribution (e.g. bluetongue in sheep) serves to illustrate the potential of VE, WE and EE to cause disease on other continents.
5a1a3f06-b679-4c86-85ec-c64435112a43	0	The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K-12. Incubation of the enzyme in alkaline conditions: dissociation and disulfide-bridge formation. Aspartokinase I - homoserine dehydrogenase I from Escherichia coli K-12, a homotetrameric enzyme, dissociates into dimers upon alkaline treatment. Both aspartokinase and homoserine dehydrogenase inactivation, as well as desensitazion towards L-threonine, occur in a multi-step process.
5a1a3f06-b679-4c86-85ec-c64435112a43	1	Dithiothreitol stabilizes a dimeric form retaining full activity and sensitivity; L-homoserine stabilizing another dimeric form devoid of aspartokinase activity and retaining a substantial dehydrogenase activity insensitive toward L-threonine. A model is proposed showing that dissociation into dimers occurs in a first step, the resulting dimer losing both aspartokinase and homoserine dehydrogenase sensitivity in two subsequent steps involving the formation of intrachain disulfide bonds.
cdfd2575-3e72-4075-a449-91cf7d318cd4	0	A calorimetric study of the CO Bohr effect of monomeric haemoglobins. A calorimetric study has been made of the heats of CO reaction with the monomeric haemoglobins of Chironomus thummi thummi III and IV as a function of pH. The number of Bohr protons released at pH 7.1 was determined from heats of reaction in different buffers as 0.19
cdfd2575-3e72-4075-a449-91cf7d318cd4	1	and 0.31 mol H+/mol CO for haemoglobin III and IV respectively. The heat of the Bohr ionization process was found to be 6 and 8 kcal/mol H+ (25 and 34 kJ/mol) for the haemoglobins III and IV. These values are consistent with values found for histidine groups.
cdfd2575-3e72-4075-a449-91cf7d318cd4	2	A pH-independent part of the reaction enthalpy was determined as - 19.7 kcal/mol CO (-82.4 kJ/mol). The same reaction with myoglobin is less exothermic. From the combination of deltaG0 and deltaH0 values TdeltaS0 values have been calculated. It was found for both haemoglobins that the entropy of reaction is greater by 2 cal K-1 mol-1 (8.4
cdfd2575-3e72-4075-a449-91cf7d318cd4	3	JK-1 mol-1) at pH 9.5 as compared to pH 6.0.
df89644d-323e-4245-b5e9-e9ae354c90cd	0	Conformational studies of two non-histone chromosomal proteins and their interactions with DNA. The conformational properties of two non-histone chromosomal proteins (high-mobility-group proteins 1 and 2) have been studied by spectroscopic methods. The interaction of high-mobility-group protein 1 with DNA has also been studied.
df89644d-323e-4245-b5e9-e9ae354c90cd	1	1. Circular dichroism results indicate that in the presence of salt both proteins are 40-50% helical between pH 1 and 9. Above pH 9 denaturation takes place. In the absence of salt the proteins denature below pH 4. 2. Nuclear magnetic resonance spectra show the presence of ring-current shifted peaks and perturbed aromatic resonances, demonstrating that the helix formation is accompanied by specific tertiary folding.
df89644d-323e-4245-b5e9-e9ae354c90cd	2	3. Nuclear magnetic resonance spectra of compelxes between high mobility group protein 1 and DNA demonstrate that a low ionic strength a portion of the molecule rich in lysine and containing all the aromatic residues is bound to DNA, whilst a more acidic region of the chain remains free from the DNA.
12c17b6e-92f4-4f12-84f4-eca70d09b656	0	Succinylation of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus. A reactive threonine residue in the apoenzyme. 1. Glyceraldehyde-3-phosphate dehydrogenase from bacillus stearothermophilus can be extensively succinylated in the presence of substrates and coenzyme without appreciable loss of activity. 2. The apoenzyme in the absence of substrates is rapidly inhibited by small amounts of succinic anhydride.
12c17b6e-92f4-4f12-84f4-eca70d09b656	1	NAD+, glyceraldehyde-3-phosphate and inorganic phosphate all afford protection from inhibition, and inhibition is slowly reversed in the presence of pyrophosphate at pH 8.5. 3. Kinetic and spectral studies have shown that the specific inhibition is associated with the succinylation of the aliphatic hydroxyl group of a serine or threonine residue.
12c17b6e-92f4-4f12-84f4-eca70d09b656	2	4. The residue specifically succinylated has been identified as one of the two threonine residues, most probably Thr-150, adjacent to the activ-site cysteine residue in the primary structure. Its unusual reactivity is discussed in relation to the three-dimensional structure of the enzyme. 5. A second residue, a lysine homologous with Lys-212 in the pig muscle enzyme, can be succinylated in both holoenzyme and apoenzyme with no detectable effect upon the enzymic activity.
a5421409-b983-45e6-8714-84a286bf2eef	0	Membrane-reversible H+-ATPase from Micrococcus lysodeikticus. Treatment of phosphorylating fragments of bacterial membrane from Micrococcus lysodeikticus with trypsin leads to increase ATPase activity. As a result of this treatment, the membrane fragments acquire the ability to transform the ATP energy into transmembrane difference in potential. Dithiothreitol has a similar effect to that of trypsin on the membrane fragments from M.
a5421409-b983-45e6-8714-84a286bf2eef	1	lysodeikticus. Dicyclohexylcarbodimide inhibits ATPase of the membrane fragments of M. lysodeikticus, and also the ATPase-reaction-coupled generation of membrane potential. It has been suggested that the increased ATPase activity of membranes from M. lysodeikticus during treatment with trypsin and dithiothreitol is connected with the effect of these agents on the protein inhibitor of ATPase.
75c44da2-37a4-4c90-a1f9-4ca1d7c8164a	0	Active-site-directed inhibition of the plasma-membrane carrier transporting short-chain, neutral amino acids into Trypanosoma brucei. 1. Glycine chloromethyl inhibited the active-transport of L-serine into bloodstream forms of Trypanosoma brucei. 2. Substrates of the short-chain, neutral amino acid transport system (N1), but not of other amino acid transport systems, protected the carrier protein from inhibition.
75c44da2-37a4-4c90-a1f9-4ca1d7c8164a	1	3. Inhibition was never more than 80% complete. The residual activity might have due to a proportion of N1 carrier active sites which had not reacted with the inhibitor. 4. The inhibition was highly selective for the N1 amino acid transport system. Other amino acid transport systems were not affected and the rate of respiration was only slightly affected.
75c44da2-37a4-4c90-a1f9-4ca1d7c8164a	2	5. The inhibition was first-order with respect to concentration, indicating that one molecule of the inhibitor reacted with each carrier active-site. 6. The high selectivity of this inhibitor should make it a useful labelling agent during the isolation and purification of the N1 amino acid transport carrier protein(s).
bdf1b8ba-b2ae-4fcb-89c9-ffd2f0e08630	0	A quantitative model for partition in aqueous multiphase systems. A model for the partition of charged molecules in aqueous multiphase systems has been developed. The partition coefficient of one component, or the overall partition coefficient of a number of components, between two arbitrary phases is expressed in terms of the difference in electrical potential between the phases (due to electrolytes present in the system), the net charges of the partitioned components and their partition coefficients in a (sometimes hypothetical) uncharged state.
bdf1b8ba-b2ae-4fcb-89c9-ffd2f0e08630	1	The fraction of material in one phase has also been described as a function of the net charges of the partitioned components. The model fits well to experimental data for partition of chromate, pyridine, ribonuclease A, two types of CO-hemoglobin and an enzyme mixture (yeast lysate) in three-phase systems consisting of poly(ethylene glycol), dextran, Ficoll and water.
bdf1b8ba-b2ae-4fcb-89c9-ffd2f0e08630	2	Minor deviations from the model are construed to be a pH-dependent uptake of ions. The data have also been used to detect differences in solvation of similar proteins, as well as the presence of several forms of some glycolytic enzymes present in yeast lysate.
22a6bacd-28a5-42e3-ae90-65e52619ea57	0	Lysis of yeast cell walls. Lytic beta-(1 leads to 6)-glucanase from Bacillus circulans WL-12. When grown in a mineral medium with yeast cell walls or yeast glucan as the sole carbon source, Bacillus circulans WL-12 produces wall-lytic enzymes in addition to non-lytic beta-(1 leads to 3) and beta-(1 leads to 6)-glucananases.
22a6bacd-28a5-42e3-ae90-65e52619ea57	1	The lytic enzymes were isolated from the culture liquid by adsorption on insoluble yeast glucan in batch operation. After digestion of the glucan, the mixture of enzymes was chromatographed on hydroxylapatite on which the lytic activity could be resolved into one lytic beta-(1 leads to 6)glucanase and two lytic beta-(1 leads to 3)-glucanase was further purified by chromatography over diethylamino-ehtyl-agarose and carboxymethyl cellulose.
22a6bacd-28a5-42e3-ae90-65e52619ea57	2	Its specific activity on pustulan was 6.2 units per mg of protein. The enzyme moved as a single protein with a molecular weight of 54000 during sodium dodecylsulphate electrophoresis in slab gels. Hydrolysis of pustulan went thorugh a series of oligosaccharides, leading to a mixture of gentiotriose, gentiobiose and glucose.
22a6bacd-28a5-42e3-ae90-65e52619ea57	3	The enzyme also produced small amounts of gentiobiose from laminarin and pachyman and on this basis its lytic activity on yeast cell walls,was attribut beta-(1 leads to 3)-linked oligosaccharides were not detected. The lytic beta-(1 leads to 6)-glucanase has an optimum pH of 6.0
22a6bacd-28a5-42e3-ae90-65e52619ea57	4	. Pustulan hydrolysis followed Michaelis-Menten kinetics. A Km of 0.29 mg pustulan per ml and a V of 9.1 micro-equivalents of glucose released/min per mg of enzyme were calculated. The enzyme has no metal ion requirement. The lytic beta-(1 leads to 6)-glucanase differs in essence from the non-lytic beta-(1 leads to 6)-glucanase of the same organism by its positive action on yeast cell walls and yeast glucan and its much lower specific activity on soluble pustulan.
a3d8d727-5dfe-4ca2-a032-80a607b9e99b	0	Lysis of yeast cell walls. Lytic beta-(1 leads to 3)-glucanases from Bacillus circulans WL-12. Bacillus circulans WL-12 when grown in a mineral medium with yeast cell walls or yeast glucan as the soli carbon source, produced five beta-glucanases. Two beta-(1 leads to 3)-glucanases (I and II), which are lytic to yeast cell walls, were isolated from the culture liquid by batch adsorption on yeast glucan, and separated by chromatography on hydroxylapatite.
a3d8d727-5dfe-4ca2-a032-80a607b9e99b	1	Lytic beta-(1 leads to 3)-glucanase I was further purified by carboxymethylcellulose chromatography. The specific activity of lytic beta-(1 leads to 3)-glucanase I on laminarin was 4.1 U per mg of protein. The enzyme moved as a single protein with a molecular weight of 40000 during sodium dodecylsulfate electrophoresis in slab gels.
a3d8d727-5dfe-4ca2-a032-80a607b9e99b	2	It was specific for the beta-(1 leads to 3)-glucosidic bond but the enzyme did not hydrolyze laminaribiose. Hydrolysis of laminarin went through a series of oligosaccharides, and laminaribiose and glucose accumulated till the end of the reaction. A small amount of gentibiose was also produced from laminarin.
a3d8d727-5dfe-4ca2-a032-80a607b9e99b	3	Products from yeast cell walls and yeast glucan included laminaripentaose, laminaritriose, laminaribiose, glucose and gentiobiose, but no laminaritetraose was detected. This glucanase has an optimum pH of 5.5.
51d7c3ba-e628-4127-aa60-45f124e46175	0	Algal glyceraldehyde-3-phosphate dehydrogenase. Pyridine-nucleotide requirements of two enzymes purified from Scenedesmus obliquus. Two enzymes with glyceraldehyde-3-phosphate dehydrogenase activity have been purified from heterotrophically grown Scenedesmus obliquus by ion-exchange chromatography and gel filtration. The D-enzyme has a molecular weight of 550000 and a VNADH:
51d7c3ba-e628-4127-aa60-45f124e46175	1	VNADPH ratio of 16 whereas the T-enzyme has a molecular weight of 140000 and a VNADH:VNADPH ratio of 0.15. The two enzymes, however, are very similar with regard to their Michaelis constants for the reduced pyridine nucleotides, pH optimum, subunit size and ultraviolet absorption.
dc59a6e5-b529-48f8-b2b5-4001b6bed69e	0	Multiple forms of cyclohexanone oxygenase from Nocardia globerula CL1. The cyclohexanone 1,2-monooxygenase of Nocardia globerula CL1 exists as two electrophoretically distinct forms. These are present in crude cell extracts and are not artifacts of enzyme purification or electrophoresis. They have been separated in mg amounts by preparative polyacrylamide gel electrophoresis and shown to have essentially identical kinetic, spectral and physical characteristics.
dc59a6e5-b529-48f8-b2b5-4001b6bed69e	1	They do differ in pH-activity profile and temperature stability. Whether or not they are conformational isoenzymes or arise by gene duplication and divergent evolution has not been established. Cyclohexanone oxygenase constitutes 8% of the soluble protein of induced cells. This high level would correlate well with the presence of duplicate genes.
dc59a6e5-b529-48f8-b2b5-4001b6bed69e	2	It is proposed that the presence of a large amount of cyclohexanone oxygenase may confer an ecological advantage on the organism.
f8a213bb-4c9a-4e3a-b6ef-45785f626e35	0	Purification and properties of cyclopentanone oxygenase of Pseudomonas NCIB 9872. 1. Cyclopentanone oxygenase from Pseudomonas NCIB 9872 has been purified some 40-fold. It gives a single peak in the ultracentrifuge and a single major protein band on polyacrylamide gels contaminated with about 5% of a slower migrating impurity.
f8a213bb-4c9a-4e3a-b6ef-45785f626e35	1	Flavin dissociates from the protein during electrophoresis. 2. The enzyme has a molecular weight of about 200000 and is a homopolymeric assemblage of either three of four subunits of molecular weight 54000-58000. 3. The prosthetic group is FAD and values of about 2.5 are typically obtained for the number of moles bound to each mole of holoenzyme.
f8a213bb-4c9a-4e3a-b6ef-45785f626e35	2	Some FAD probably dissociates during purification and it seems likely that each subunit binds one FAD in the undamaged protein.
481b2e82-5419-4130-b486-2381276c5157	0	Purification and properties of a methanol-oxidizing enzyme in Pseudomonas C. A methanol-oxidizing enzyme has been purified from Pseudomonas C, grown on methanol as a sole source for carbon and energy. The purification procedure involved ammonium sulphate precipitation, ion-exchange chromatography and gel filtration and resulted in a yield of 35.4
481b2e82-5419-4130-b486-2381276c5157	1	%. Enzyme activity can be coupled to phenazine methosulfate and requires the presence of ammonium ions in the assay mixtures. The enzymes possesses a broad specificity for primary alcohols. Formaldehyde is also oxidized by the purified enzyme. The Km value for methanol is 15 muM. The optimum pH for the oxidation of both methanol and formaldehyde is about 10.4
481b2e82-5419-4130-b486-2381276c5157	2	. The enzyme has a molecular weight of about 128000 and consists of two subunits each having a molecular weight of 60000.
6c46ae05-0e2c-49a3-b833-c363607d50b0	0	Temperature-dependent change in rate-limiting step of the magnesium-stimulated ITPase of myosin. The effects of temperature on Mg-ITPase activity of heavy meromyosin and myosin subfragment 1 were measured in 0.1 M KC1. The initial burst of Pi liberation was one mol per mol of heavy meromyosin or two mol of myosin subfragment 1, i.
6c46ae05-0e2c-49a3-b833-c363607d50b0	1	e. one mol per two mol of myosin active sites, at 20 degrees C. However, it was almost zero mol below 8degrees C. Effects of KC1 concentration and pH on ITPase activity of heavy meromyosin at 20 degrees C were different from those below 8 degrees C, suggesting that the rate-limiting step in the Mg-ITP hydrolysis of myosin depends on temperature.
6c46ae05-0e2c-49a3-b833-c363607d50b0	2	The effect of temperature on the actin activation of heavy meromyosin Mg-ITPase was analyzed by measuring the temperature dependence of double-reciprocal plots of ITPase activity against actin concentration. The extent of actin activation was larger at low temperture. The results presented in this paper might be explained by assuming the existence of two kinds of active sites on a myosin molecule.
36f08ac5-3f58-482d-91db-2909cdd58c5e	0	Nuclear-magnetic-resonance study of the active-site structure of yeast phosphoglycerate kinase. The enzyme 3-phosphoglycerate kinase from yeast has been studied by observation of the proton nuclear magnetic resonance spectrum at 270 MHz using Fourier transform techniques. Difference spectroscopy was used to enhance the resolution and to identify specific ligand binding effects and conformational changes. Perturbations involving single protons of amino-acid residues could thus be detected despite the relatively high molecular weight of the protein (47000), particularly in the aromatic (6-9 ppm) and methylene (2-3 ppm) regions of the spectrum.
de7d3a6a-a332-403f-9903-df23564aa306	0	Fluroescence of proteins in 6-M guanidine hydrochloride. A method for the quantitative determination of tryptophan. To determine the tryptophan content in proteins,an analytical ultraviolet fluroescence method is proposed based on making uniform the environment of aromatic chromophores in 6-7 M guanidine hydrochloride. The fluorescence intensity scale is calibrated using standard solutions of free tryptophan.
de7d3a6a-a332-403f-9903-df23564aa306	1	A correlation coefficient between the fluorescence of protein tryptophanyl residues and of free tryptophan was estimated in testing 17 well characterized proteins. This method is particularly suited to proteins carrying groups absorbing in the 290-370 nm region, such as flavin, heme and pyridoxal phosphate and in the presence of substances such as 2-mercaptoethanol which prohibit the use of the spectroscopic or magnetic circular dichroism methods.
de7d3a6a-a332-403f-9903-df23564aa306	2	It is less time-consuming than techniques requiring hydrolysis or chemical reactions.
d2cc608a-864b-43a4-8806-fb9839726e29	0	An extracellular aminopeptidase from Clostridium histolyticum. An aminopeptidase was isolated from the culture filtrate of Clostridium histolyticum and purified to homogeneity. Absence of endopeptidase activity in the purified preparation was demonstrated. Gel filtration on a calibrated column indicates an apparent molecular weight of 340000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of dodecylsulfate in constant acrylamide concentration and in a concentration gradient, resulted in the appearance of a single component for which a molecular weight of 51000 and 59000 respectively, was calculated.
d2cc608a-864b-43a4-8806-fb9839726e29	1	From mobilities of crosslinked and denatured protein species a molecular weight of 56000 was obtained for the monomer. Specificity studies show that the enzyme cleaves all types of N-terminel amino acid residues including proline and hydroxyproline from small peptides and from polypeptides. The peptide bond formed between an N-terminal amino acid residue and proline is not cleaved by the enzyme.
d2cc608a-864b-43a4-8806-fb9839726e29	2	The combined action of aminopeptidase-P and clostridal aminopeptidase leads to complete hydrolysis of the proline-rich nonapeptide bradykinin. Low rates of hydrolysis was observed for charged residues, and amides of amino acids. Kinetic studies with five tripeptides of the general structure X-Gly-Gly, where X stands for Leu, Phe, Val, Ala, or Pro, show a decrease in Km with the increasing size of the hydrophobic side chain of X.
d2cc608a-864b-43a4-8806-fb9839726e29	3	The highest Kcat values are observed with proline and alanine. In the series Pro-Gly, Pro-Gly-Pro, Pro-Gly-Pro-Pro, the last peptide is the best substrate, indicating an active site complementary to at least four amino acid residues. The enzymatic activity is dependent on the presence of divalent cations, maximal activation being reached with Mn2+ and Co2+.
d2cc608a-864b-43a4-8806-fb9839726e29	4	The optimal pH for the Mn2+ and Co2+- activated enzyme is 8.6 and 8.2 respectively. The optimal temperature is 40 degrees C. Inhibition of the aminopeptidase was achieved with Zn2+, Cu2+ and p-mercuribenzoate, but not with diisopropylphosphofluoridate.
da96048a-abab-447a-946b-091316a14d1d	0	Ornithine carbamoyltransferase from Escherichia coli W. Purification, structure and steady-state kinetic analysis. Ornithine carbamoyltransferase from Escherichia coli W was purified to homogeneity. The enzyme has a molecular weight of 105000. It is composed of three apparently identical subunits with molecular weights of 35000. The mechanism of the ornithine carbamoyltransferase enzyme system from E.
da96048a-abab-447a-946b-091316a14d1d	1	coli W was investigated kinetically by using the approach of product inhibition and dead-end inhibition of both forward and reverse reactions. On the basis of the kinetic data and binding studies it appears that the mechanism of the reaction involves a compulsory sequence of substrate binding to the enzyme, in which carbamoylphosphate is the first substrate to bind to the enzyme and phosphate the last product to be released.
da96048a-abab-447a-946b-091316a14d1d	2	The same studies also indicate that the mechanism involves dead-end complexes. The reaction mechanism appears consistent with that proposed by Theorell and Chance. Values have been determined for the Michaelis and dissociation constants involved in the combination of each reactant with the enzyme. Comparison of the values for the kinetic constants which are common to both forward and reverse reaction have shown that they are always of a comparable magnitude.
f38ca183-f5d4-4529-a5ee-4a858d2bb31d	0	Reactivity of the sulfhydryl groups of soluble succinate dehydrogenase. Soluble succinate dehydrogenase prepared by butanol extraction reacts with N-ethylmaleimide according to first-order kinetics with respect to both remaining active enzyme and the inhibitor concentration. Binding of the sulfhydryl groups of the enzyme prevents its alkylation by N-ethylmaleimide and inhibition by oxaloacetate.
f38ca183-f5d4-4529-a5ee-4a858d2bb31d	1	A kinetic analysis of the inactivation of alkylating reagent in the presence of succinate or malonate suggests that N-ethylmaleimide acts as a site-directed inhibitor. The apparent first-order rate constant of alkylation increases between pH 5.8 and 7.8 indicating a pKa value for the enzyme sulfhydryl group equal to 7.0
f38ca183-f5d4-4529-a5ee-4a858d2bb31d	2	at 22 degrees C in 50 mM Tris-sufate buffer. Certain anions (phosphate, citrate, maleate and acetate) decrease the reactivity of the enzyme towards the alkylating reagent. Succinate/phenazine methosulfate reductase activity measured in the presence of a saturating concentration of succinate shows the same pH-dependence as the alkylation rate by N-ethylmaleimide.
f38ca183-f5d4-4529-a5ee-4a858d2bb31d	3	The mechanism of the first step of succinate oxidation, including a nucleophilic attack of substrate by the active-site sulfhydryl group, is discussed.
d870a8e1-38bf-4da2-b3b1-a559b1f715ab	0	Identification and properties of 8-hydroxyflavin--adenine dinucleotide in electron-transferring flavoprotein from Peptostreptococcus elsdenii. 1. A new flavin prosthetic group has been isolated in pure form from the electron-transferring flavoprotein of Peptostreptococcus elsdenni. Its structure has been established as the FAD derivative of 7-methyl-8-hydroxyisoalloxazine:
d870a8e1-38bf-4da2-b3b1-a559b1f715ab	1	(see article). Proof of this structure has been obtained by chemical syntehsis of 7-methyl-8-hydroxyisoalloxazine models, and by stepwise degradation of the native compound to 7-methy-8-hydroxyalloxazine. The orange chromophore is characterized by a strong absorption band with a maximum at 472 nm (xi = 41 000 M-1 CM-1) and a pK at 4.8
d870a8e1-38bf-4da2-b3b1-a559b1f715ab	2	due to the ionisation of the C(8)-OH group. 2. The properties of a series of functionally substituted derivatives of 8-hydroxy flavins and lumichromes have been investigated to provide a basis for interpreting the effects of pH on the spectroscopic properties of the 8-hydroxy derivatives of FAD and FMN.
d870a8e1-38bf-4da2-b3b1-a559b1f715ab	3	3. The 8-hydroxy derivative of FAD is bound by apo-D-amino acid oxidase; the complex shows no catalytic activity. The 8-hydroxy derivative of FMN is bound by apoflavodoxin to give a complex which has catalytic activity similar to that of native flavodoxin. The complex is reversibly reduced by dithionite, first to a relatively stable semiquinone and further to the dihydroflavin form.
f6da3cc8-8270-4103-a7ea-259fd4fa7202	0	Kinetics of reassociation and reactivation of pig-muscle lactic dehydrogenase after acid dissociation. Lactic dehydrogenase from pig skeletal muscle (M4) can be reversibly dissociated to the monomer at pH 4-5 depending on the anion applied. Using identical experimental conditions the pH-depending profiles of dissociation, denaturation, and deactivation coincide with each other.
f6da3cc8-8270-4103-a7ea-259fd4fa7202	1	Deviations in the pH-dependence of protein fluorescence reflect changes in the microenvironment of specific chromophores rather than significant differences in the structure-function relationship as pH is changed. The dissociation state is characterized by the homogeneous inactive monomer of 35000. The reassociated material consists of up to 85% fully active tetramers, indistinguishable from the initial native enzymes, as shown by hydrodynamic, spectroscopic and enzymic properties.
f6da3cc8-8270-4103-a7ea-259fd4fa7202	2	The rest represents a mixture of irreversibly denatured high aggregates. Under optimum conditions of reactivation both recovery of enzymic activity and native fluorescence obey strict second order kinetics with an activation energy of 44 kcal/mol (184 kJ/mol). NAD+ and NADH do not show any significant influence on the yield and kinetics of the refolding process and the recovery of enzymic activity.
f6da3cc8-8270-4103-a7ea-259fd4fa7202	3	The kinetic results suggest the reassociation of inactive monomers to be rate-limiting in the refolding and reactivation processes being considered.
309f68cf-c1e9-46c6-af62-8cdeecec7c1d	0	Alkylation of estradiol 17beta-dehydrogenase from human placenta with 3-chloroacetylpyridine--adenine dinucleotide. 3-Chloroacetylpyridine--adenine dinucleotide, which is active as a hydride acceptor (Km = 0.6 mM), inactivates and alkylates estradiol 17beta-dehydrogenase. The kinetics of inactivation by 3-chloroacetylpyridine--adenine dinucleotide and the absence of inactivation by 3-chloroacetylpyridine ribose phosphate show that the alkylation follows the formation of a binary complex (Kd = 4.5
309f68cf-c1e9-46c6-af62-8cdeecec7c1d	1	X 10(-4) M). Studies of the labelling by 3-chloro[2-14C]acetylpyridine--adenine dinucleotide and the rate of alkylation as a function of pH, give evidence to the alkylation of a cysteine, the stoichiometry being one mole per subunit. The 14C label is distributed between three chymotryptic peptides, one of which accounts for about 50% of the radioactive label.
6758e54e-664f-4437-97bb-a6daf7a721e2	0	Regulation of respiration and nitrogen fixation in different types of Azotobacter vinelandii. The levels of the adenine nucleotides, pyridine nucleotides and the kinetical parameters of the enzymes of the Entner-Doudoroff pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) were determined in Azotobacter vinelandii cells, grown under O2- or N2-limiting conditions.
6758e54e-664f-4437-97bb-a6daf7a721e2	1	It was concluced that the levels of both the adenine nucleotides and pyridine nucleotides do not limit the rate of sucrose oxidation. Experiments with radioactive pyruvate and sucrose show that the rate of sucrose oxidation of Azotobacter cells is associated with an increase in the rate of sucrose uptake.
6758e54e-664f-4437-97bb-a6daf7a721e2	2	The sites of oxidative phosphorylation and the composition of the respiratory membranes with respect to cytochromes c4 + c5, b and d differ in cells growth either O2- or N2-limited. It was possible to show that the respiration protection of the nitrogen-fixing system in Azotobacter is mainly independent of the oxidation capacity of the cells.
6758e54e-664f-4437-97bb-a6daf7a721e2	3	The oxidation capacity intrinsically depends on the type of substrate and can be partly adapted. The maximum activity of the nitrogenase in Azotobacter depends on the type of substrate oxidized. Although the level of energy charge is somewhat dependent on the type of substrate used, no obvious relation can be derived between changes in energy charge and nitrogenase activity.
6758e54e-664f-4437-97bb-a6daf7a721e2	4	An alternative proposal is given.
53cab9fd-f782-4e38-9f3f-c77cd8f08406	0	The proton electrochemical gradient in Escherichia coli cells. The internal pH of Escherichia coli cells was estimated from the distribution of either 5,5-[14C]dimethyl-2,4-oxazolidinedione or [14C]methylamine. EDTA/valinomycin treatment of cells was employed to estimate delta psi from 86Rb+ distribution concomitant with the delta pH for calculation of delta muH.
53cab9fd-f782-4e38-9f3f-c77cd8f08406	1	Respiring intact cells maintained an internal pH more alkaline by 0.63-0.75 unit than that of the milieu at extracellular pH 7, both in growth medium and KCl solutions. The delta pH decreased when respiration was inhibited by anaerobiosis or in the presence of KCN. The delta muH, established by EDTA/valinomycin-treated cells, was constant (122-129 mV) over extracellular potassium concentration of 0.01
53cab9fd-f782-4e38-9f3f-c77cd8f08406	2	mM-1 mM. At the lower potassium concentration delta psi (110-120 mV) was the predominant component, and at the higher concentration delta pH increased to 0.7 units (42 mV). At 150 mM potassium delta muH was reduced to 70 mV mostly due to a delta pH component of 0.89
53cab9fd-f782-4e38-9f3f-c77cd8f08406	3	(53 mV). The interchangeability of the delta muH components is consistent with an electronic proton pump and with potassium serving as a counter ion in the presence of valinomycin. Indeed both parameters of delta muH decreased in the presence of carbonylcyanide p-trifluoromethoxyphenylhydrazone. The highest delta pH of 2 units was observed in the intact cells at pH 6;
53cab9fd-f782-4e38-9f3f-c77cd8f08406	4	increasing the extracellular pH decreased the delta pH to 0 at pH 7.65 and to -0.51 at pH 9. A similar pattern of dependence of delta pH on extracellular pH was observed in EDTA/valinomycin-treated cells but the delta psi was almost constant over the whole range of extracellular pH values (6-8) implying electroneutral proton movement.
53cab9fd-f782-4e38-9f3f-c77cd8f08406	5	Potassium is specifically required for respiration of EDTA-treated E. coli K12 cells since other monovalent or divalent cations could not replace potassium and valinomycin was not required.
ddc50302-0508-423b-86a5-bffa0f5e0cf2	0	[Spectral properties of porcine plasminogen: study of the acidic transition (author's transl)]. The acidic transition of porcine plasminogen, prepared by affinity chromatography, was studied by non-destructive methods. These methods are based on the analysis of the behaviour of the tryptophyls under various conditions. The perturbation of the absorption and emission spectra by pH or temperature and the dynamic quenching of the intrinsic fluorescence are used to obtain information on structural changes which affect the environment of these residues.

0cd8efa9-a8f3-4f27-8894-290053dcfb90

0

Distribution of releasing factors, biogenic amines, and related enzymes in the bovine median eminence. The bovine median eminence was dissected into eight different subdivisions: rostral, anterior internal, anterior external, middle external medial, middle external lateral, middle internal medial, middle internal lateral, and caudal. Thyrotropin-releasing hormone (TRH) was found in the highest concentrations in the middle external medial and lateral subdivisions;

0cd8efa9-a8f3-4f27-8894-290053dcfb90

1

luteinizing hormone-releasing hormone (LHRH) was concentrated in the middle external lateral and anterior internal subdivisions. Among the various neurotransmitters and enzymes assayed, only dopamine and choline acetyltransferase were present in highest concentrations in the same subdivisions of the bovine median eminence found to be rich in TRH and LHRH.

0cd8efa9-a8f3-4f27-8894-290053dcfb90

2

The distributions of norepinephrine, dopamine-beta-hydroxylase, serotonin, tryptophan hydroxylase, phenylethanolamine-N-methyltransferase, glutamic acid decarboxylase, and histamine appeared to correlate poorly with the major distributions of TRH and LHRH. These findings suggest that at the level of the median eminence, central neuroendocrine regulation of TRH and LHRH release may involve an interaction only with dopamine and acetylcholine.

85e0eb7d-c70a-49d1-a0c2-299dd52c3993

0

The effects of neurally active amino acids on prolactin secretion. Several neurally active amino acids were injected into the third ventricle of anesthetized male rats. Two or eight mumole of GABA produced significant increases in the plasma concentrations of prolactin (PRL), indicating increased PRL release from the pituitary. Two mumole of glycine was also effective in elevating PRL levels.

85e0eb7d-c70a-49d1-a0c2-299dd52c3993

1

The intraventricular injection of the lowest dose of GABA (1.0 mumole), glutamate (0.4 or 2.3 mumole), lysine (0.2 or 2.0 mumole), or 0.9% NaCl did not alter PRL levels significantly. Plasma PRL concentrations did not increase following the injection of GABA or glycine directly into the anterior pituitary gland.

85e0eb7d-c70a-49d1-a0c2-299dd52c3993

2

The results suggest that GABA and glycine may play a role in the neural regulation of PRL secretion.

b79e44d6-c488-44cc-b90c-c3ee1f6c92ad

0

Determination of D-3-hydroxybutyrate dehydrogenase in mouse pancreatic islets with a photokinetic technique using bacterial luciferase. A sensitive assay for d-3hydroxybutrate dehydrogenase (EC 1.1.1.30) was developed for use with the minute amounts of material obtained from islets of Langerhans microdissected from freeze-dried pancreatic sections. NADH formed in the enzyme reaction was determined by photokinetic analysis of the luminescence obtained with bacterial luciferase from Achromobacter fishcherii.

b79e44d6-c488-44cc-b90c-c3ee1f6c92ad

1

In this way, accurate determination was obtained with less than 0.1 mug dry weight of islet material. In obese hyperglycemic mice, the islet enzyme had an activity of 4.7 mumoles/min and g dry weight. Optimal enzyme activity was found at pH 8 for the islet enzyme. The enzyme activity was similar in pancreatic islets and acini, while considerably higher activity was found in cardiac muscle, liver and renal cortex.

b79e44d6-c488-44cc-b90c-c3ee1f6c92ad

2

Normal mouse islets showed about equal enzyme activity as the islets from obese hyperglycemic mice.

68e9ce55-7bf2-4f91-866f-a864ba243df8

0

Human platelet glucose-6-phosphate dehydrogenase. Total purification, kinetic studies and relationship with enzyme from other blood cells. Human platelet G-6-PD has been highly purified, to homogeneity, and its kinetic, electrophoretic and immunological characteristics have been studied. Platelet G-6-PD differs from erythrocyte or leukocyte enzymes by an increased Michaelis constant for G-6-P and a slow activity at the acid pHs.

68e9ce55-7bf2-4f91-866f-a864ba243df8

1

By electrofocusing only a main active band (band a) of platelet G-6-PD was found. The incubation at 37 degrees C in the presence of NADP+ and dithiothreitol normalize Km-G-6-P of platelet G-6-PD; the incubation with boiled and ultrafiltered leukemic granulocyte extracts led to an anodisation of G-6-PD active forms, a decrease of the molecular specific activity and a further increase of Km-G-6-P;

68e9ce55-7bf2-4f91-866f-a864ba243df8

2

these last modifications are the same as those undergone by G-6-PD incubated in crude extracts of normal or leukemic granulocytes.

45f35abd-120c-421f-bb10-03a955f47909

0

Clinical evaluation of blood lactate levels in equine colic. Blood lactate levels were evaluated in 36 horses (43 cases) presented with colic. A correlation between increasing blood lactate levels and decreasing percentage survival has been shown. An appreciable anion gap was found in 7 of 10 cases analyzed in detail but in each case the entire gap could not be accounted for by lactate alone.

45f35abd-120c-421f-bb10-03a955f47909

1

Proposals are offered to account for the unmeasured anions. Blood lactate determination is suggested as a prognostic rather than a diagnostic aid for the equine practitioner and should be used to augment other clinical findings in the horse exhibiting colic.

a6135ea2-fb90-4153-b471-9b5041530ee9

0

Equine viral encephalitis. The most important neurotropic viral infections of the horse are the arthropod-borne encephalitides. These include Venezuelan encephalitis (VE), eastern encephalitis (EE) and western encephalitis (WE), which are found in the Americas, and Japanese B encephalitis which occurs in the Far East. All the viruses cause encephalitis in man.

a6135ea2-fb90-4153-b471-9b5041530ee9

1

Between 1969 and 1972 an epidemic of VE occurred in Central America. In 1971 the disease was reported in Texas, where it was brought under control by the vaccination of susceptible horses with an attenuated live virus vaccine and by the reduction of the mosquito population with insecticides sprayed from aircraft.

a6135ea2-fb90-4153-b471-9b5041530ee9

2

A high titre viraemia occurs with VE virus in the horse and epidemics are maintained by a mosquito/horse cycle; infection of man and other species is incidental. EE and WE have been recognised as separate diseases since 1933 and in the U.S.A. horses are protected by routine vaccination.

a6135ea2-fb90-4153-b471-9b5041530ee9

3

Epidemics of these diseases are routine vaccination. Epidemics of these diseases are now uncommon. In contrast with VE, both EE and WE viruses are maintained by a bird/mosquito cycle. The viraemia in the horse is generally considered insufficient to infect mosquito vectors; the horse is a "dead end host".

a6135ea2-fb90-4153-b471-9b5041530ee9

4

Several species of mosquito can act as vectors of VE, WE and EE. The extension of other arthropod-borne diseases to areas originally outside their geographical distribution (e.g. bluetongue in sheep) serves to illustrate the potential of VE, WE and EE to cause disease on other continents.

5a1a3f06-b679-4c86-85ec-c64435112a43

0

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K-12. Incubation of the enzyme in alkaline conditions: dissociation and disulfide-bridge formation. Aspartokinase I - homoserine dehydrogenase I from Escherichia coli K-12, a homotetrameric enzyme, dissociates into dimers upon alkaline treatment. Both aspartokinase and homoserine dehydrogenase inactivation, as well as desensitazion towards L-threonine, occur in a multi-step process.

5a1a3f06-b679-4c86-85ec-c64435112a43

1

Dithiothreitol stabilizes a dimeric form retaining full activity and sensitivity; L-homoserine stabilizing another dimeric form devoid of aspartokinase activity and retaining a substantial dehydrogenase activity insensitive toward L-threonine. A model is proposed showing that dissociation into dimers occurs in a first step, the resulting dimer losing both aspartokinase and homoserine dehydrogenase sensitivity in two subsequent steps involving the formation of intrachain disulfide bonds.

cdfd2575-3e72-4075-a449-91cf7d318cd4

0

A calorimetric study of the CO Bohr effect of monomeric haemoglobins. A calorimetric study has been made of the heats of CO reaction with the monomeric haemoglobins of Chironomus thummi thummi III and IV as a function of pH. The number of Bohr protons released at pH 7.1 was determined from heats of reaction in different buffers as 0.19

cdfd2575-3e72-4075-a449-91cf7d318cd4

1

and 0.31 mol H+/mol CO for haemoglobin III and IV respectively. The heat of the Bohr ionization process was found to be 6 and 8 kcal/mol H+ (25 and 34 kJ/mol) for the haemoglobins III and IV. These values are consistent with values found for histidine groups.

cdfd2575-3e72-4075-a449-91cf7d318cd4

2

A pH-independent part of the reaction enthalpy was determined as - 19.7 kcal/mol CO (-82.4 kJ/mol). The same reaction with myoglobin is less exothermic. From the combination of deltaG0 and deltaH0 values TdeltaS0 values have been calculated. It was found for both haemoglobins that the entropy of reaction is greater by 2 cal K-1 mol-1 (8.4

cdfd2575-3e72-4075-a449-91cf7d318cd4

3

JK-1 mol-1) at pH 9.5 as compared to pH 6.0.

df89644d-323e-4245-b5e9-e9ae354c90cd

0

Conformational studies of two non-histone chromosomal proteins and their interactions with DNA. The conformational properties of two non-histone chromosomal proteins (high-mobility-group proteins 1 and 2) have been studied by spectroscopic methods. The interaction of high-mobility-group protein 1 with DNA has also been studied.

df89644d-323e-4245-b5e9-e9ae354c90cd

1

1. Circular dichroism results indicate that in the presence of salt both proteins are 40-50% helical between pH 1 and 9. Above pH 9 denaturation takes place. In the absence of salt the proteins denature below pH 4. 2. Nuclear magnetic resonance spectra show the presence of ring-current shifted peaks and perturbed aromatic resonances, demonstrating that the helix formation is accompanied by specific tertiary folding.

df89644d-323e-4245-b5e9-e9ae354c90cd

2

3. Nuclear magnetic resonance spectra of compelxes between high mobility group protein 1 and DNA demonstrate that a low ionic strength a portion of the molecule rich in lysine and containing all the aromatic residues is bound to DNA, whilst a more acidic region of the chain remains free from the DNA.

12c17b6e-92f4-4f12-84f4-eca70d09b656

0

Succinylation of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus. A reactive threonine residue in the apoenzyme. 1. Glyceraldehyde-3-phosphate dehydrogenase from bacillus stearothermophilus can be extensively succinylated in the presence of substrates and coenzyme without appreciable loss of activity. 2. The apoenzyme in the absence of substrates is rapidly inhibited by small amounts of succinic anhydride.

12c17b6e-92f4-4f12-84f4-eca70d09b656

1

NAD+, glyceraldehyde-3-phosphate and inorganic phosphate all afford protection from inhibition, and inhibition is slowly reversed in the presence of pyrophosphate at pH 8.5. 3. Kinetic and spectral studies have shown that the specific inhibition is associated with the succinylation of the aliphatic hydroxyl group of a serine or threonine residue.

12c17b6e-92f4-4f12-84f4-eca70d09b656

2

4. The residue specifically succinylated has been identified as one of the two threonine residues, most probably Thr-150, adjacent to the activ-site cysteine residue in the primary structure. Its unusual reactivity is discussed in relation to the three-dimensional structure of the enzyme. 5. A second residue, a lysine homologous with Lys-212 in the pig muscle enzyme, can be succinylated in both holoenzyme and apoenzyme with no detectable effect upon the enzymic activity.

a5421409-b983-45e6-8714-84a286bf2eef

0

Membrane-reversible H+-ATPase from Micrococcus lysodeikticus. Treatment of phosphorylating fragments of bacterial membrane from Micrococcus lysodeikticus with trypsin leads to increase ATPase activity. As a result of this treatment, the membrane fragments acquire the ability to transform the ATP energy into transmembrane difference in potential. Dithiothreitol has a similar effect to that of trypsin on the membrane fragments from M.

a5421409-b983-45e6-8714-84a286bf2eef

1

lysodeikticus. Dicyclohexylcarbodimide inhibits ATPase of the membrane fragments of M. lysodeikticus, and also the ATPase-reaction-coupled generation of membrane potential. It has been suggested that the increased ATPase activity of membranes from M. lysodeikticus during treatment with trypsin and dithiothreitol is connected with the effect of these agents on the protein inhibitor of ATPase.

75c44da2-37a4-4c90-a1f9-4ca1d7c8164a

0

Active-site-directed inhibition of the plasma-membrane carrier transporting short-chain, neutral amino acids into Trypanosoma brucei. 1. Glycine chloromethyl inhibited the active-transport of L-serine into bloodstream forms of Trypanosoma brucei. 2. Substrates of the short-chain, neutral amino acid transport system (N1), but not of other amino acid transport systems, protected the carrier protein from inhibition.

75c44da2-37a4-4c90-a1f9-4ca1d7c8164a

1

3. Inhibition was never more than 80% complete. The residual activity might have due to a proportion of N1 carrier active sites which had not reacted with the inhibitor. 4. The inhibition was highly selective for the N1 amino acid transport system. Other amino acid transport systems were not affected and the rate of respiration was only slightly affected.

75c44da2-37a4-4c90-a1f9-4ca1d7c8164a

2

5. The inhibition was first-order with respect to concentration, indicating that one molecule of the inhibitor reacted with each carrier active-site. 6. The high selectivity of this inhibitor should make it a useful labelling agent during the isolation and purification of the N1 amino acid transport carrier protein(s).

bdf1b8ba-b2ae-4fcb-89c9-ffd2f0e08630

0

A quantitative model for partition in aqueous multiphase systems. A model for the partition of charged molecules in aqueous multiphase systems has been developed. The partition coefficient of one component, or the overall partition coefficient of a number of components, between two arbitrary phases is expressed in terms of the difference in electrical potential between the phases (due to electrolytes present in the system), the net charges of the partitioned components and their partition coefficients in a (sometimes hypothetical) uncharged state.

bdf1b8ba-b2ae-4fcb-89c9-ffd2f0e08630

1

The fraction of material in one phase has also been described as a function of the net charges of the partitioned components. The model fits well to experimental data for partition of chromate, pyridine, ribonuclease A, two types of CO-hemoglobin and an enzyme mixture (yeast lysate) in three-phase systems consisting of poly(ethylene glycol), dextran, Ficoll and water.

bdf1b8ba-b2ae-4fcb-89c9-ffd2f0e08630

2

Minor deviations from the model are construed to be a pH-dependent uptake of ions. The data have also been used to detect differences in solvation of similar proteins, as well as the presence of several forms of some glycolytic enzymes present in yeast lysate.

22a6bacd-28a5-42e3-ae90-65e52619ea57

0

Lysis of yeast cell walls. Lytic beta-(1 leads to 6)-glucanase from Bacillus circulans WL-12. When grown in a mineral medium with yeast cell walls or yeast glucan as the sole carbon source, Bacillus circulans WL-12 produces wall-lytic enzymes in addition to non-lytic beta-(1 leads to 3) and beta-(1 leads to 6)-glucananases.

22a6bacd-28a5-42e3-ae90-65e52619ea57

1

The lytic enzymes were isolated from the culture liquid by adsorption on insoluble yeast glucan in batch operation. After digestion of the glucan, the mixture of enzymes was chromatographed on hydroxylapatite on which the lytic activity could be resolved into one lytic beta-(1 leads to 6)glucanase and two lytic beta-(1 leads to 3)-glucanase was further purified by chromatography over diethylamino-ehtyl-agarose and carboxymethyl cellulose.

22a6bacd-28a5-42e3-ae90-65e52619ea57

2

Its specific activity on pustulan was 6.2 units per mg of protein. The enzyme moved as a single protein with a molecular weight of 54000 during sodium dodecylsulphate electrophoresis in slab gels. Hydrolysis of pustulan went thorugh a series of oligosaccharides, leading to a mixture of gentiotriose, gentiobiose and glucose.

22a6bacd-28a5-42e3-ae90-65e52619ea57

3

The enzyme also produced small amounts of gentiobiose from laminarin and pachyman and on this basis its lytic activity on yeast cell walls,was attribut beta-(1 leads to 3)-linked oligosaccharides were not detected. The lytic beta-(1 leads to 6)-glucanase has an optimum pH of 6.0

22a6bacd-28a5-42e3-ae90-65e52619ea57

4

. Pustulan hydrolysis followed Michaelis-Menten kinetics. A Km of 0.29 mg pustulan per ml and a V of 9.1 micro-equivalents of glucose released/min per mg of enzyme were calculated. The enzyme has no metal ion requirement. The lytic beta-(1 leads to 6)-glucanase differs in essence from the non-lytic beta-(1 leads to 6)-glucanase of the same organism by its positive action on yeast cell walls and yeast glucan and its much lower specific activity on soluble pustulan.

a3d8d727-5dfe-4ca2-a032-80a607b9e99b

0

Lysis of yeast cell walls. Lytic beta-(1 leads to 3)-glucanases from Bacillus circulans WL-12. Bacillus circulans WL-12 when grown in a mineral medium with yeast cell walls or yeast glucan as the soli carbon source, produced five beta-glucanases. Two beta-(1 leads to 3)-glucanases (I and II), which are lytic to yeast cell walls, were isolated from the culture liquid by batch adsorption on yeast glucan, and separated by chromatography on hydroxylapatite.

a3d8d727-5dfe-4ca2-a032-80a607b9e99b

1

Lytic beta-(1 leads to 3)-glucanase I was further purified by carboxymethylcellulose chromatography. The specific activity of lytic beta-(1 leads to 3)-glucanase I on laminarin was 4.1 U per mg of protein. The enzyme moved as a single protein with a molecular weight of 40000 during sodium dodecylsulfate electrophoresis in slab gels.

a3d8d727-5dfe-4ca2-a032-80a607b9e99b

2

It was specific for the beta-(1 leads to 3)-glucosidic bond but the enzyme did not hydrolyze laminaribiose. Hydrolysis of laminarin went through a series of oligosaccharides, and laminaribiose and glucose accumulated till the end of the reaction. A small amount of gentibiose was also produced from laminarin.

a3d8d727-5dfe-4ca2-a032-80a607b9e99b

3

Products from yeast cell walls and yeast glucan included laminaripentaose, laminaritriose, laminaribiose, glucose and gentiobiose, but no laminaritetraose was detected. This glucanase has an optimum pH of 5.5.

51d7c3ba-e628-4127-aa60-45f124e46175

0

Algal glyceraldehyde-3-phosphate dehydrogenase. Pyridine-nucleotide requirements of two enzymes purified from Scenedesmus obliquus. Two enzymes with glyceraldehyde-3-phosphate dehydrogenase activity have been purified from heterotrophically grown Scenedesmus obliquus by ion-exchange chromatography and gel filtration. The D-enzyme has a molecular weight of 550000 and a VNADH:

51d7c3ba-e628-4127-aa60-45f124e46175

1

VNADPH ratio of 16 whereas the T-enzyme has a molecular weight of 140000 and a VNADH:VNADPH ratio of 0.15. The two enzymes, however, are very similar with regard to their Michaelis constants for the reduced pyridine nucleotides, pH optimum, subunit size and ultraviolet absorption.

dc59a6e5-b529-48f8-b2b5-4001b6bed69e

0

Multiple forms of cyclohexanone oxygenase from Nocardia globerula CL1. The cyclohexanone 1,2-monooxygenase of Nocardia globerula CL1 exists as two electrophoretically distinct forms. These are present in crude cell extracts and are not artifacts of enzyme purification or electrophoresis. They have been separated in mg amounts by preparative polyacrylamide gel electrophoresis and shown to have essentially identical kinetic, spectral and physical characteristics.

dc59a6e5-b529-48f8-b2b5-4001b6bed69e

1

They do differ in pH-activity profile and temperature stability. Whether or not they are conformational isoenzymes or arise by gene duplication and divergent evolution has not been established. Cyclohexanone oxygenase constitutes 8% of the soluble protein of induced cells. This high level would correlate well with the presence of duplicate genes.

dc59a6e5-b529-48f8-b2b5-4001b6bed69e

2

It is proposed that the presence of a large amount of cyclohexanone oxygenase may confer an ecological advantage on the organism.

f8a213bb-4c9a-4e3a-b6ef-45785f626e35

0

Purification and properties of cyclopentanone oxygenase of Pseudomonas NCIB 9872. 1. Cyclopentanone oxygenase from Pseudomonas NCIB 9872 has been purified some 40-fold. It gives a single peak in the ultracentrifuge and a single major protein band on polyacrylamide gels contaminated with about 5% of a slower migrating impurity.

f8a213bb-4c9a-4e3a-b6ef-45785f626e35

1

Flavin dissociates from the protein during electrophoresis. 2. The enzyme has a molecular weight of about 200000 and is a homopolymeric assemblage of either three of four subunits of molecular weight 54000-58000. 3. The prosthetic group is FAD and values of about 2.5 are typically obtained for the number of moles bound to each mole of holoenzyme.

f8a213bb-4c9a-4e3a-b6ef-45785f626e35

2

Some FAD probably dissociates during purification and it seems likely that each subunit binds one FAD in the undamaged protein.

481b2e82-5419-4130-b486-2381276c5157

0

Purification and properties of a methanol-oxidizing enzyme in Pseudomonas C. A methanol-oxidizing enzyme has been purified from Pseudomonas C, grown on methanol as a sole source for carbon and energy. The purification procedure involved ammonium sulphate precipitation, ion-exchange chromatography and gel filtration and resulted in a yield of 35.4

481b2e82-5419-4130-b486-2381276c5157

1

%. Enzyme activity can be coupled to phenazine methosulfate and requires the presence of ammonium ions in the assay mixtures. The enzymes possesses a broad specificity for primary alcohols. Formaldehyde is also oxidized by the purified enzyme. The Km value for methanol is 15 muM. The optimum pH for the oxidation of both methanol and formaldehyde is about 10.4

481b2e82-5419-4130-b486-2381276c5157

2

. The enzyme has a molecular weight of about 128000 and consists of two subunits each having a molecular weight of 60000.

6c46ae05-0e2c-49a3-b833-c363607d50b0

0

Temperature-dependent change in rate-limiting step of the magnesium-stimulated ITPase of myosin. The effects of temperature on Mg-ITPase activity of heavy meromyosin and myosin subfragment 1 were measured in 0.1 M KC1. The initial burst of Pi liberation was one mol per mol of heavy meromyosin or two mol of myosin subfragment 1, i.

6c46ae05-0e2c-49a3-b833-c363607d50b0

1

e. one mol per two mol of myosin active sites, at 20 degrees C. However, it was almost zero mol below 8degrees C. Effects of KC1 concentration and pH on ITPase activity of heavy meromyosin at 20 degrees C were different from those below 8 degrees C, suggesting that the rate-limiting step in the Mg-ITP hydrolysis of myosin depends on temperature.

6c46ae05-0e2c-49a3-b833-c363607d50b0

2

The effect of temperature on the actin activation of heavy meromyosin Mg-ITPase was analyzed by measuring the temperature dependence of double-reciprocal plots of ITPase activity against actin concentration. The extent of actin activation was larger at low temperture. The results presented in this paper might be explained by assuming the existence of two kinds of active sites on a myosin molecule.

36f08ac5-3f58-482d-91db-2909cdd58c5e

0

Nuclear-magnetic-resonance study of the active-site structure of yeast phosphoglycerate kinase. The enzyme 3-phosphoglycerate kinase from yeast has been studied by observation of the proton nuclear magnetic resonance spectrum at 270 MHz using Fourier transform techniques. Difference spectroscopy was used to enhance the resolution and to identify specific ligand binding effects and conformational changes. Perturbations involving single protons of amino-acid residues could thus be detected despite the relatively high molecular weight of the protein (47000), particularly in the aromatic (6-9 ppm) and methylene (2-3 ppm) regions of the spectrum.

de7d3a6a-a332-403f-9903-df23564aa306

0

Fluroescence of proteins in 6-M guanidine hydrochloride. A method for the quantitative determination of tryptophan. To determine the tryptophan content in proteins,an analytical ultraviolet fluroescence method is proposed based on making uniform the environment of aromatic chromophores in 6-7 M guanidine hydrochloride. The fluorescence intensity scale is calibrated using standard solutions of free tryptophan.

de7d3a6a-a332-403f-9903-df23564aa306

1

A correlation coefficient between the fluorescence of protein tryptophanyl residues and of free tryptophan was estimated in testing 17 well characterized proteins. This method is particularly suited to proteins carrying groups absorbing in the 290-370 nm region, such as flavin, heme and pyridoxal phosphate and in the presence of substances such as 2-mercaptoethanol which prohibit the use of the spectroscopic or magnetic circular dichroism methods.

de7d3a6a-a332-403f-9903-df23564aa306

2

It is less time-consuming than techniques requiring hydrolysis or chemical reactions.

d2cc608a-864b-43a4-8806-fb9839726e29

0

An extracellular aminopeptidase from Clostridium histolyticum. An aminopeptidase was isolated from the culture filtrate of Clostridium histolyticum and purified to homogeneity. Absence of endopeptidase activity in the purified preparation was demonstrated. Gel filtration on a calibrated column indicates an apparent molecular weight of 340000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of dodecylsulfate in constant acrylamide concentration and in a concentration gradient, resulted in the appearance of a single component for which a molecular weight of 51000 and 59000 respectively, was calculated.

d2cc608a-864b-43a4-8806-fb9839726e29

1

From mobilities of crosslinked and denatured protein species a molecular weight of 56000 was obtained for the monomer. Specificity studies show that the enzyme cleaves all types of N-terminel amino acid residues including proline and hydroxyproline from small peptides and from polypeptides. The peptide bond formed between an N-terminal amino acid residue and proline is not cleaved by the enzyme.

d2cc608a-864b-43a4-8806-fb9839726e29

2

The combined action of aminopeptidase-P and clostridal aminopeptidase leads to complete hydrolysis of the proline-rich nonapeptide bradykinin. Low rates of hydrolysis was observed for charged residues, and amides of amino acids. Kinetic studies with five tripeptides of the general structure X-Gly-Gly, where X stands for Leu, Phe, Val, Ala, or Pro, show a decrease in Km with the increasing size of the hydrophobic side chain of X.

d2cc608a-864b-43a4-8806-fb9839726e29

3

The highest Kcat values are observed with proline and alanine. In the series Pro-Gly, Pro-Gly-Pro, Pro-Gly-Pro-Pro, the last peptide is the best substrate, indicating an active site complementary to at least four amino acid residues. The enzymatic activity is dependent on the presence of divalent cations, maximal activation being reached with Mn2+ and Co2+.

d2cc608a-864b-43a4-8806-fb9839726e29

4

The optimal pH for the Mn2+ and Co2+- activated enzyme is 8.6 and 8.2 respectively. The optimal temperature is 40 degrees C. Inhibition of the aminopeptidase was achieved with Zn2+, Cu2+ and p-mercuribenzoate, but not with diisopropylphosphofluoridate.

da96048a-abab-447a-946b-091316a14d1d

0

Ornithine carbamoyltransferase from Escherichia coli W. Purification, structure and steady-state kinetic analysis. Ornithine carbamoyltransferase from Escherichia coli W was purified to homogeneity. The enzyme has a molecular weight of 105000. It is composed of three apparently identical subunits with molecular weights of 35000. The mechanism of the ornithine carbamoyltransferase enzyme system from E.

da96048a-abab-447a-946b-091316a14d1d

1

coli W was investigated kinetically by using the approach of product inhibition and dead-end inhibition of both forward and reverse reactions. On the basis of the kinetic data and binding studies it appears that the mechanism of the reaction involves a compulsory sequence of substrate binding to the enzyme, in which carbamoylphosphate is the first substrate to bind to the enzyme and phosphate the last product to be released.

da96048a-abab-447a-946b-091316a14d1d

2

The same studies also indicate that the mechanism involves dead-end complexes. The reaction mechanism appears consistent with that proposed by Theorell and Chance. Values have been determined for the Michaelis and dissociation constants involved in the combination of each reactant with the enzyme. Comparison of the values for the kinetic constants which are common to both forward and reverse reaction have shown that they are always of a comparable magnitude.

f38ca183-f5d4-4529-a5ee-4a858d2bb31d

0

Reactivity of the sulfhydryl groups of soluble succinate dehydrogenase. Soluble succinate dehydrogenase prepared by butanol extraction reacts with N-ethylmaleimide according to first-order kinetics with respect to both remaining active enzyme and the inhibitor concentration. Binding of the sulfhydryl groups of the enzyme prevents its alkylation by N-ethylmaleimide and inhibition by oxaloacetate.

f38ca183-f5d4-4529-a5ee-4a858d2bb31d

1

A kinetic analysis of the inactivation of alkylating reagent in the presence of succinate or malonate suggests that N-ethylmaleimide acts as a site-directed inhibitor. The apparent first-order rate constant of alkylation increases between pH 5.8 and 7.8 indicating a pKa value for the enzyme sulfhydryl group equal to 7.0

f38ca183-f5d4-4529-a5ee-4a858d2bb31d

2

at 22 degrees C in 50 mM Tris-sufate buffer. Certain anions (phosphate, citrate, maleate and acetate) decrease the reactivity of the enzyme towards the alkylating reagent. Succinate/phenazine methosulfate reductase activity measured in the presence of a saturating concentration of succinate shows the same pH-dependence as the alkylation rate by N-ethylmaleimide.

f38ca183-f5d4-4529-a5ee-4a858d2bb31d

3

The mechanism of the first step of succinate oxidation, including a nucleophilic attack of substrate by the active-site sulfhydryl group, is discussed.

d870a8e1-38bf-4da2-b3b1-a559b1f715ab

0

Identification and properties of 8-hydroxyflavin--adenine dinucleotide in electron-transferring flavoprotein from Peptostreptococcus elsdenii. 1. A new flavin prosthetic group has been isolated in pure form from the electron-transferring flavoprotein of Peptostreptococcus elsdenni. Its structure has been established as the FAD derivative of 7-methyl-8-hydroxyisoalloxazine:

d870a8e1-38bf-4da2-b3b1-a559b1f715ab

1

(see article). Proof of this structure has been obtained by chemical syntehsis of 7-methyl-8-hydroxyisoalloxazine models, and by stepwise degradation of the native compound to 7-methy-8-hydroxyalloxazine. The orange chromophore is characterized by a strong absorption band with a maximum at 472 nm (xi = 41 000 M-1 CM-1) and a pK at 4.8

d870a8e1-38bf-4da2-b3b1-a559b1f715ab

2

due to the ionisation of the C(8)-OH group. 2. The properties of a series of functionally substituted derivatives of 8-hydroxy flavins and lumichromes have been investigated to provide a basis for interpreting the effects of pH on the spectroscopic properties of the 8-hydroxy derivatives of FAD and FMN.

d870a8e1-38bf-4da2-b3b1-a559b1f715ab

3

3. The 8-hydroxy derivative of FAD is bound by apo-D-amino acid oxidase; the complex shows no catalytic activity. The 8-hydroxy derivative of FMN is bound by apoflavodoxin to give a complex which has catalytic activity similar to that of native flavodoxin. The complex is reversibly reduced by dithionite, first to a relatively stable semiquinone and further to the dihydroflavin form.

f6da3cc8-8270-4103-a7ea-259fd4fa7202

0

Kinetics of reassociation and reactivation of pig-muscle lactic dehydrogenase after acid dissociation. Lactic dehydrogenase from pig skeletal muscle (M4) can be reversibly dissociated to the monomer at pH 4-5 depending on the anion applied. Using identical experimental conditions the pH-depending profiles of dissociation, denaturation, and deactivation coincide with each other.

f6da3cc8-8270-4103-a7ea-259fd4fa7202

1

Deviations in the pH-dependence of protein fluorescence reflect changes in the microenvironment of specific chromophores rather than significant differences in the structure-function relationship as pH is changed. The dissociation state is characterized by the homogeneous inactive monomer of 35000. The reassociated material consists of up to 85% fully active tetramers, indistinguishable from the initial native enzymes, as shown by hydrodynamic, spectroscopic and enzymic properties.

f6da3cc8-8270-4103-a7ea-259fd4fa7202

2

The rest represents a mixture of irreversibly denatured high aggregates. Under optimum conditions of reactivation both recovery of enzymic activity and native fluorescence obey strict second order kinetics with an activation energy of 44 kcal/mol (184 kJ/mol). NAD+ and NADH do not show any significant influence on the yield and kinetics of the refolding process and the recovery of enzymic activity.

f6da3cc8-8270-4103-a7ea-259fd4fa7202

3

The kinetic results suggest the reassociation of inactive monomers to be rate-limiting in the refolding and reactivation processes being considered.

309f68cf-c1e9-46c6-af62-8cdeecec7c1d

0

Alkylation of estradiol 17beta-dehydrogenase from human placenta with 3-chloroacetylpyridine--adenine dinucleotide. 3-Chloroacetylpyridine--adenine dinucleotide, which is active as a hydride acceptor (Km = 0.6 mM), inactivates and alkylates estradiol 17beta-dehydrogenase. The kinetics of inactivation by 3-chloroacetylpyridine--adenine dinucleotide and the absence of inactivation by 3-chloroacetylpyridine ribose phosphate show that the alkylation follows the formation of a binary complex (Kd = 4.5

309f68cf-c1e9-46c6-af62-8cdeecec7c1d

1

X 10(-4) M). Studies of the labelling by 3-chloro[2-14C]acetylpyridine--adenine dinucleotide and the rate of alkylation as a function of pH, give evidence to the alkylation of a cysteine, the stoichiometry being one mole per subunit. The 14C label is distributed between three chymotryptic peptides, one of which accounts for about 50% of the radioactive label.

6758e54e-664f-4437-97bb-a6daf7a721e2

0

Regulation of respiration and nitrogen fixation in different types of Azotobacter vinelandii. The levels of the adenine nucleotides, pyridine nucleotides and the kinetical parameters of the enzymes of the Entner-Doudoroff pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) were determined in Azotobacter vinelandii cells, grown under O2- or N2-limiting conditions.

6758e54e-664f-4437-97bb-a6daf7a721e2

1

It was concluced that the levels of both the adenine nucleotides and pyridine nucleotides do not limit the rate of sucrose oxidation. Experiments with radioactive pyruvate and sucrose show that the rate of sucrose oxidation of Azotobacter cells is associated with an increase in the rate of sucrose uptake.

6758e54e-664f-4437-97bb-a6daf7a721e2

2

The sites of oxidative phosphorylation and the composition of the respiratory membranes with respect to cytochromes c4 + c5, b and d differ in cells growth either O2- or N2-limited. It was possible to show that the respiration protection of the nitrogen-fixing system in Azotobacter is mainly independent of the oxidation capacity of the cells.

6758e54e-664f-4437-97bb-a6daf7a721e2

3

The oxidation capacity intrinsically depends on the type of substrate and can be partly adapted. The maximum activity of the nitrogenase in Azotobacter depends on the type of substrate oxidized. Although the level of energy charge is somewhat dependent on the type of substrate used, no obvious relation can be derived between changes in energy charge and nitrogenase activity.

6758e54e-664f-4437-97bb-a6daf7a721e2

4

An alternative proposal is given.