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cbfa38a8-4f6e-4ac6-8737-d981ba40cc48
| 4 |
Inhibition was competitive with prephenate (Ki = 0.06 mM) and noncompetitive with nicotinamide adenine dinucleotide. The enzyme was further subject to product inhibition by p-hydroxyphenylpyruvate (Ki = 0.13 mM). Its Km for prephenate was 0.045 mM, and that for nicotinamide adenine dinucleotide was 0.14 mM.
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cbfa38a8-4f6e-4ac6-8737-d981ba40cc48
| 5 |
The pH optimum ranged between 7.0 and 7.6; the temperature optimum was 38 C. It is shown how the sensitive regulation of the entire enzyme system leads to a well-balanced amino acid production.
|
f65002c2-93af-43ac-90cc-e0d1f314663a
| 0 |
Role of deoxyribonucleic acid ligase in a doxyribonucleic acid membrane fraction extracted from pneumococci. Deoxyribonucleic acid (DNA) ligase has been detected in a DNA membrane fraction extracted from Pneumococcus. The specific activity of the enzyme in this fraction is 10-fold greater than in the remaining cell extract. It remains firmly bound (with other enzymes) to the complex after a purification procedure in which a considerable percentage of the macromolecules are dissociated.
|
f65002c2-93af-43ac-90cc-e0d1f314663a
| 1 |
The ligase acts in two ways in the DNA membrane fraction in vitro. One, it catalyzes the linkage of small-molecular-weight pieces of newly synthesized DNA into heavier-molecular-weight DNA strands as shown by others (M Gellert, 1976; R. Okazaki, A. Sugino, S. Hirose, T. Okazaki, Y. Imae, R. Kainuma-Kuroda, T. Ogawa, M. Arisawa, and Y. Kurosowa, 1973; B. Olivera and I. Lehman, 14; and A. Sugino, S. Hirose, and R. Okazaki, 1972) and, two, it protects DNA from degradation by deoxyribonucleases. This latter effect is due to a competition between the ability of the nucleases to degrade DNA and the ability of DNA ligase to seal the nicks produced by these degradative enzymes.
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f65002c2-93af-43ac-90cc-e0d1f314663a
| 2 |
The ligase acts cooperatively with other enzymes in the DNA membrane fraction to synthesize DNA.
|
e4db40e1-0443-4f1d-9621-bc73229fbf7f
| 0 |
Relationship between hemagglutinin and sialidase from Clostridium perfringens CN3870: chromatographic characterization of the biologically active proteins. Biochemical characterization of hemagglutinin and sialidase activities from Clostridium perfringens strain CN3870 revealed that this strain produced three sialidase enzymes that were separable to gel filtration, ion exchange chromatography, and polyacrylamide gel electrophoresis. The molecular weights of sialidase I, II, and III activities were 310,000 +/- 10,000, 105,000 +/- 4,000 and 64,000 +/- 2,000, respectively, the first figure being an approximate value only.
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5037048d-ed23-46dd-9588-a6d754ba65a9
| 0 |
Relationship between hemagglutinin and sialidase from Clostridium perfringens CN3870: gel filtration of mutant and reverant activities. Gel filtration of supernatant fluids, from the wild-type Clostridium perfringens, strain CN3870, and several of the mutants and reverants derived from this strain, showed that these mutants failed to product detectable amounts of still produced sialidase III activity.
|
5037048d-ed23-46dd-9588-a6d754ba65a9
| 1 |
The reverants tested had regained the ability to produce approximately wild-type levels of the I and II forms of both activities. These results showthat there is a direct relationship between the production of the I form and hemagglutinin and sialidase activities and the production of the II form of these biologically active proteins.
|
5037048d-ed23-46dd-9588-a6d754ba65a9
| 2 |
Models that explain the genetic basis for these results are discussed.
|
4b60c9f3-4c97-4cf8-85f5-305dc69aa13c
| 0 |
Development of Microbodies in the yeast Kloeckera growing on methanol. A number of microbodies appear regularly in methanol-grown yeast cells, but rarely in ethanol- or glucose-grown cells. When one of representative methanol-utilizing yeasts, Kloeckera sp.no. 2201 (also known as Candida bodinii), was cultured on glucose and then transferred into a methanol medium, microbodies of small size could be observed in 2-h old cells.
|
4b60c9f3-4c97-4cf8-85f5-305dc69aa13c
| 1 |
The number of microbodies per sectioned cell reached five to six after 4 h of cultivation. Though the number of microbodies did not change during prolonged cultivation, their size became larger with the passage of cultivation time. The activities of catalase and alcohol oxidase were confirmed in the particulate fractions throughout the cultivation period, whereas the activities of formaldehyde dehydrogenase and formate dehydrogenase were not detected in the particles.
|
4b60c9f3-4c97-4cf8-85f5-305dc69aa13c
| 2 |
The activity of isocitrate lyase was detected in the particulate fractions only at the early growth phase.
|
64650916-1b7c-42ab-bbfa-917d7da02c9c
| 0 |
Phosphatase of Chlamydomonas reinhardi: biochemical and cytochemical approach with specific mutants. The unicellular alga Chlamydomonas reinhardi produces two constitutive acid phosphatases and three depressible phosphatases (a neutral and two alkaline ones) that can utilize napthyl phosphate as a substrate. Specific mutants depressible phosphatase were used to investigate biochemical properties and the cytochemical localization of these enzymes. The two constitutive phosphatases show similar pH optima (about 5.0) and Km values (2 x 10(-3) to 3.3 x 10(-3) M) but differ in their heat sensitivity and affinity for glycerophosphate.
|
d657fa01-57c0-4a15-a5ee-a01a5a42efa1
| 0 |
Autolysis of Neisseria gonorrhoeae. Autolysis of Neisseria gonorrhoeae was studied under different conditions. It was found that low pH and temperature, as well as the presence of divalent cations, spermine, sucrose, and polyvinylpyrrolidone, stabilized nongrowing gonococci. Ethylenediaminetetraacetic acid alone promoted lysis, whereas lysozyme had only a limited additive effect. The autolytic behavior of gonococci appears to be connected with their prolonged cell division process. The relative dependence on the outer membrane and the peptidoglycan layer for the mechanical stability of gonococci is discussed.
|
9c2ac538-d5bd-4964-867b-234f0a0371df
| 0 |
Glucose-6-phosphate dehydrogenase. Purification and partial characterization. Glucose-6-phosphate dehydrogenase has been purified 1000-fold from pig liver. This enzyme exists as an active dimer of molecular weight 133,000 and an inactive monomer of molecular weight 67,500. The pH of maximum activity is 8.5 and the ionic strength maximum is 0.1
|
9c2ac538-d5bd-4964-867b-234f0a0371df
| 1 |
to 0.5 M. Glucose-6-phosphate dehydrogenase is highly specific for NADP+ and glucose 6-phosphate. Apparent Km values of 3.6 muM and 5.4 muM were obtained for glucose 6-phosphate and NADP+. This enzyme is located almost entirely within the soluble portion of the cellular cytoplasm.
|
90169176-ee12-4f8b-8114-63c50c8d4711
| 0 |
A kinetic study of glucose-6-phosphate dehydrogenase. The steady state kinetics of pig liver glucose-6-phosphate dehydrogenase is consistent with an ordered, sequential mechanism in which NADP is bound first and NADPH released last. Kia is 9.0 muM, Ka is 4.8 muM, and Kb is 36 muM.
|
90169176-ee12-4f8b-8114-63c50c8d4711
| 1 |
Glucosamine 6-phosphate, a substrate analogue and competitive inhibitor, is used to help rule out a possible random mechanism. ADP is seen to form a complex with the free form of the enzyme whereas ATP forms a complex with both the free and E-NADP forms of the enzyme.
|
90169176-ee12-4f8b-8114-63c50c8d4711
| 2 |
The KI for the E-ADP complex is 1.9 mM, while the Ki values for the E-ATP and E-NADP-ATP complexes are 7.2 and 4.5 mM, respectively.
|
eb0c118e-f561-48f8-8903-4140f005957b
| 0 |
alpha-Aminomethylglutarate, a beta-amino analog of glutamate that interacts with glutamine synthetase and the enzymes that catalyze glutathione synthesis. The glutamate analog, alpha-aminomethylglutaric acid, was synthetized by Michael addition of ammonia to 2-methylene glutaronitrile followed by hydrolysis of the intermediate alpha-aminomethylglutaryl nitrile; the analog cyclizes readily on heating to 2-piperidone-5-carboxylic acid.
|
eb0c118e-f561-48f8-8903-4140f005957b
| 1 |
Sheep brain glutamine synthetase utilizes one isomer of DL-alpha-aminomethylglutarate at about 10% of the rate with L-glutamate. gamma-Glutamylcysteine synthetase uses both isomers of DL-alpha-aminomethylglutarate, preferentially acting on the same isomer used by glutamine synthetase. gamma-(alpha-Aminomethyl)glutaryl-alpha-aminobutyrate, prepared enzymatically with gamma-glutamylcysteine synthetase, was found to be a substrate and an inhibitor of glutathione synthetase.
|
eb0c118e-f561-48f8-8903-4140f005957b
| 2 |
alpha-Aminomethylglutarate does not inhibit gamma-glutamyl cyclotransferase and gamma-glutamyl transpeptidase appreciably. When alpha-aminomethylglutarate was administered to mice, there were substantial decreases in the levels of glutamine, glutathione, glutamate, and glycine in the kidney, and of glutamine and glutamate in the liver, indicating that this glutamate analog is effective as an inhibitor of glutamine and glutathione synthesis in vivo, and suggesting that it may also inhibit other enzymes.
|
f9063d30-afef-48d6-a5da-127157c5c06f
| 0 |
Studies of human kidney gamma-glutamyl transpeptidase. Purification and structural, kinetic and immunological properties. gamma-Glutamyl transpeptidase, present in various mammalian tissues, transfers the gamma-glutamyl moiety of glutathione to a variety of acceptor amino acids and peptides. This enzyme has been purified from human kidney cortex about 740-fold to a specific activity of 200 units/mg of protein.
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f9063d30-afef-48d6-a5da-127157c5c06f
| 1 |
The purification steps involved incubation of the homogenate at 37 degrees followed by centrifugation and extraction of the sediment with 0.1 M Tris-HCl buffer, pH 8.0, containing 1% sodium deoxycholate; batchwise absorption on DEAE-cellulose; DEAE-cellulose (DE52) column chromatography; Sephadex G-200 gel filtration;
|
f9063d30-afef-48d6-a5da-127157c5c06f
| 2 |
and affinity chromatography using concanavalin A insolubilized on beaded Agarose. Detergents were used throughout the purification of the enzyme. The purified enzyme separated into three protein bands, all of which had enzyme activity, on polyacrylamide disc electrophoresis in the presence of Triton X-100. The enzyme has an apparent molecular weight of about 90,000 as shown by Sephadex G-200 gel filtration, and appears to be a tetramer with subunits of molecular weights of about 21,000.
|
f9063d30-afef-48d6-a5da-127157c5c06f
| 3 |
The Km for gamma-glutamyl transpeptidase using the artificial substrate, gamma-glutamyl-p-nitroanilide, with glycylglycine as the acceptor amino acid was found to be about 0.8 mM. The optimum pH for the enzyme activity is 8.2 and the isoelectric point is 4.5. Both GSH and GSSG competitively inhibited the activity of gamma-glutamyl transpeptidase when gamma-glutamyl-p-nitroanilide was used as the substrate.
|
f9063d30-afef-48d6-a5da-127157c5c06f
| 4 |
Treatment of the purified enzyme with papain has no effect on the enzyme activity or mobility on polyacrylamide disc electrophoresis. The purified gamma-glutamyl transpeptidase had no phosphate-independent glutaminase activity. The ratio of gamma-glutamyl transpeptidase to phosphate-independent glutaminase changed significantly through the initial steps of gamma-glutamyl transpeptidase purification.
|
f9063d30-afef-48d6-a5da-127157c5c06f
| 5 |
These studies indicate that the transpeptidase and phosphate-independent glutaminase activities are not exhibited by the same protein in human kidney.
|
a31a8fe5-f9f0-4029-bd9c-0b9afb0ea355
| 0 |
Kinetics of the hemerythrin-oxygen interaction. The kinetics of the reaction of Golfingia gouldii hemerythrin with O2 have been studied by stopped flow spectrophotometry. For the second order oxygenation process, k1 = 7.4 X 10(6) M-1 s-1, deltaH1++ = 8.2 kcal-mol-1 and deltaS1++ = +1 e.
|
a31a8fe5-f9f0-4029-bd9c-0b9afb0ea355
| 1 |
u. at 25 degrees, pH 8.2, and I = 0.015 M. The rate constant is unchanged when protein concentration is varied from 3 to 25 muM, the ionic strength is increased to 0.07 M, and the pH moved to 6.8. The deoxygenation of oxyhemerythrin is studied with stopped flow by scavenging liberated O2 with S2O4(2-).
|
a31a8fe5-f9f0-4029-bd9c-0b9afb0ea355
| 2 |
For the first order dissociation, k-1 = 51 s-1, deltaH-1++ = 20.6 kcal-mol-1 and deltaS-1++ = +19 e.u. at 25 degrees, pH 8.2, and I = 0.015 M. The value of k-1 is independent of [protein] = 50 to 200 muM, [S2O4(2-)] = 5 to 100 mM I = 0.015
|
a31a8fe5-f9f0-4029-bd9c-0b9afb0ea355
| 3 |
to 0.30 M and pH 6.8 to 9.0. Using myoglobin instead of S2O4(2-) as scavenger gives similar results. Combination of activation parameters for the oxygenation and deoxygenation processes gives K1 = 1.5 X 10(5) M-1, deltaH = -12.4 kcal-mol-1, and deltaS = -18 e.
|
a31a8fe5-f9f0-4029-bd9c-0b9afb0ea355
| 4 |
u., values in good agreement with independent thermodynamic data. Perchlorate ion (0.05 M) enhances k-1 about 3-fold and hardly effects k1. There is no sign of other than a single reaction in either direction, and octameric hemerythrin apparently behaves kinetically as eight single units.
|
cac20a03-b530-473a-981d-83720cc466b6
| 0 |
Guanylate cyclase and cyclic guanosine 3':5'-monophosphate phosphodiesterase activities and cyclic guanosine 3':5'-monophosphate levels in normal and transformed fibroblasts in culture. To investigate the role of guanosine 3':5'-monophosphate (cyclic GMP) in cultured cells we have measured guanylate cyclase and cyclic GMP phosphodiesterase activities and cyclic GMP levels in normal and transformed fibroblastic cells.
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cac20a03-b530-473a-981d-83720cc466b6
| 1 |
Guanylate cyclase activity is found almost exclusively in the particulate fraction of normal rat kidney (NRK) and BALB 3T3 cells. Enzyme activity is stimulated 3- to 10-fold by treatment with the detergent Lubrol PX. However, enhancement of guanylate cyclase by fibroblast growth factor could not be demonstrated under a variety of assay conditions.
|
cac20a03-b530-473a-981d-83720cc466b6
| 2 |
In both NRK and BALB 3T3 cells guanylate cyclase activity is low during logarithmic growth and increases as the cells crowd together and growth slows. Guanylate cyclase activity is undetectable in homogenates of NRK cells transformed by the Kirsten sarcoma virus (KNRK cells) either in the presence or absence of Lubrol PX.
|
cac20a03-b530-473a-981d-83720cc466b6
| 3 |
Guanylate cyclase activity is also greatly decreased in NRK cells transformed by Moloney, Schmidt-Ruppin, or Harvey viruses. BALB 3T3 cells transformed by RNA viruses (Kirsten, Harvey, or Moloney), by a DNA virus (SV40), by methylcholanthrene, or spontaneously, all have diminished but readily detectable guanylate cyclase activity.
|
cac20a03-b530-473a-981d-83720cc466b6
| 4 |
Cyclic GMP phosphodiesterase activity is found predominately in the soluble fraction of NRK cells. This activity increases slightly as NRK cells enter the stationary growth phase. Cyclic GMP phosphodiesterase activity is undetectable in two clones of KNRK cells under a variety of assay conditions, and is decreased relative to the level present in NRK cells in a third KNRK clone.
|
cac20a03-b530-473a-981d-83720cc466b6
| 5 |
However, both Moloney- and Schmidt-Ruppin-transformed NRK cells have a phosphodiesterase activity similar to that found in NRK cells. Boiled supernatant from both NRK and KNRK cells is observed to appreciably enhance the activity of activator-deficient phosphodiesterase from bovine heart. This result indicates that the absence of cyclic GMP phosphodiesterase activity in KNRK cells is not due to a loss of the phosphodiesterase activator.
|
cac20a03-b530-473a-981d-83720cc466b6
| 6 |
The intracellular concentration of cyclic GMP is found to be very low in transformed NRK cells when compared to levels measured in confluent NRK cells. The low levels of cyclic GMP in transformed NRK cells reflect the greatly decreased guanylate cyclase activity observed in these cells. These results do not appear to support the suggestion that cyclic GMP promotes the growth of fibroblastic cells.
|
14696993-51c3-49d4-b8b8-293a142809f8
| 0 |
Specificity of alpha-chymotrypsin with exposed carboxyl groups blocked. The 15 exposed carboxyl groups of alpha-chymotrypsin were modified with glycine ethyl ester at low pH using barbodiimide reagent. The specificity of the modified enzyme (Chy-15) was studied over the pH range of 4 to 9 with both N-acylated and non-N-acylated amino acid esters.
|
14696993-51c3-49d4-b8b8-293a142809f8
| 1 |
The modified enzyme had lower reactivity toward N-acylated esters than non-N-acylated esters compared to the native enzyme. Typical substances such as acetyl- and benzoyl-L-tyrosine ethyl esters retained 4 and 9% activity, whereas phenylalanine ethyl ester was slightly more reactive with the modified than with the native enzyme.
|
14696993-51c3-49d4-b8b8-293a142809f8
| 2 |
The pH-rate profiles of acetyl-L-phenylalanine ethyl ester and tryptophan ethyl and benzyl esters were investigated in detail. Analysis of these profiles revealed three pKa values of approximately 5, 7, and 9 related to a functional carboxyl, imidazoyl, and an amino group, respectively. Since similar pKa values occur for the native enzyme, modification did not block the carboxyl corresponding to pKa 5.
|
14696993-51c3-49d4-b8b8-293a142809f8
| 3 |
A mechanism is proposed for catalysis which includes both the protonated and unprotonated form of the imidazoyl (His-57) and utilizes water rather than a carboxyl (Asp-102) as the proton sink.
|
80a3e79b-cc0e-4a2a-b5c2-319fc9efaa40
| 0 |
Evidence for the coordinate control of activity of liver glycogen synthase and phosphorylase by a single protein phosphatase. Homogeneous rabbit liver phosphorylase phosphatase (Brandt, H., Capulong, Z. L., and Lee, E. Y. C. (1975) J. Biol. Chem. 250, 8038-8044) also dephosphorylates glycogen synthase b.
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80a3e79b-cc0e-4a2a-b5c2-319fc9efaa40
| 1 |
During purification, phosphorylase phosphatase and glycogen synthase phosphatase co-purified with a constant ratio of activities. The two activities co-migrated on disc gel electrophoresis. Both substrates competed with each other for the phosphatase, and both phosphatase activities were inhibited by lysine ethyl ester. It is concluded that liver phosphorylase phosphatase and glycogen synthase phosphatase have a common identity and that coordinate regulation of the phosphatase-catalyzed activation of glycogen synthase and inactivation of phosphorylase occurs in vivo.
|
80a3e79b-cc0e-4a2a-b5c2-319fc9efaa40
| 2 |
This provides a parallel and opposing mechanism to that mediated by adenosine 3':5'-monophosphate-dependent protein kinase, which coordinately inactivates glycogen synthase and, via phosphorylase kinase, activates phosphorylase. Maximal glycogen synthase phosphatase activity was observed near neutrality. Mg2+ and glucose-6-P activated the glycogen synthase phosphatase reaction and this activation was pH-dependent.
|
80a3e79b-cc0e-4a2a-b5c2-319fc9efaa40
| 3 |
The Km for glycogen synthase b was 0.12 muM.
|
1bde9fe9-e461-4a62-b240-0af64d95f048
| 0 |
Solubilization and characterization of the beta-adrenergic receptor binding sites of frog erythrocytes. Specific beta-adrenergic receptors present in membrane preparations of frog erythrocytes were identified by binding of (-)-[3H]dihydroalprenolol, a potent competitive beta-adrenergic antagonist. The (-)-[3H]dihydroalprenolol binding sites could be solubilized by treatment of a purified erythrocyte membrane fraction with the plant glycoside digitonin but not by treatment with a wide variety of other detergents.
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1bde9fe9-e461-4a62-b240-0af64d95f048
| 1 |
The binding sites appeared to be soluble by several independent experimental criteria including (a) failure to sediment of 105,000 X g for 2 hours; (b) passage through 0.22-mu Millipore filters; (c) chromatography on Sepharose 6B gels; and (d) electron microscopy. The soluble receptor sites retained all of the essential characteristics of the membrane-bound sites, namely rapid and reversible binding of beta-adrenergic agonists and antagonists;
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1bde9fe9-e461-4a62-b240-0af64d95f048
| 2 |
strict stereospecificity toward both beta-adrenergic agonists and antagonists; appropriate structure-activity relationships; saturability of the sites at low concentrations of ligand; no affinity for alpha-adrenergic drugs, nonphysiologically active catechol compounds, and catecholamine metabolites. Based on gel chromatography in the presence of detergent, the molecular weight of the soluble receptor is estimated to be no greater than 130,000 to 150,000.
|
1bde9fe9-e461-4a62-b240-0af64d95f048
| 3 |
Equilibrium binding studies indicated a KD for the soluble receptor of 2 nM. Hill coefficients (nH) of 0.77 and curved Scatchard plots suggested the presence of negatively cooperative interactions among the solubilized receptors in agreement with previous findings with the membrane-bound sites. Kinetic studies indicated an association rate constant K1 = 3.8
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1bde9fe9-e461-4a62-b240-0af64d95f048
| 4 |
X 10(6) M-1 min-1 and a reverse rate constant k2 = 2.3 X 10(-3) min-1 at 4 degrees. The kinetically derived KD (k2/k1) of 0.6 nM is in reasonable agreement with that determined by equilibrium studies. The soluble receptors were labile at temperature greater than 4 degrees but could be stabilized with high concentrations of EDTA.
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1bde9fe9-e461-4a62-b240-0af64d95f048
| 5 |
Guanidine hydrochloride and urea produced concentration-dependent losses of binding activity which were partially reversible upon dialysis. Trypsin and phospholipase A both degraded the soluble receptors but a variety of other proteases and phospholipases as well as DNase and RNase were without effect. Experiments with group-specific reagents indicated that free lysine, tryptophan, serine, and sulfhydryl groups may be important for receptor binding.
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1bde9fe9-e461-4a62-b240-0af64d95f048
| 6 |
These studies suggest that the receptor is probably a protein which requires lipids for functional integrity. Data obtained with the solubilized binding sites are consistent with the contention that these sites represent the physiologically relevant beta-adrenergic receptors which have been extracted from the membranes with full retention of their properties.
|
9c177e68-1734-43ca-8e54-a1bc4161f10a
| 0 |
Reconstitution and characterization of the adenine nucleotide transporter derived from bovine heart mitochondria. 1. Adenine nucleotide exchange-transport was reconstituted in vesicles prepared from phospholipids and protein fractions derived from bovine heart submitochondrial particles. The transport, which was specific for ATP and ADP was measured either as ADP/ADP, ATP/ATP, or ADP/ATP exchange.
|
9c177e68-1734-43ca-8e54-a1bc4161f10a
| 1 |
The highest specific activity (370 nanomoles of ADP/ADP exchange/min/mg of protein at room temperature) was obtained with a protein fraction prepared by cholate extraction of partly resolved submitochondrial particles followed by ammonium sulfate fractionation. 2. At 200 muM external nucleotide, the exchange reactions were inhibited by low concentrations of bongkrekate, atractyloside, and palmitoyl-CoA, with Ki values of 1.8
|
9c177e68-1734-43ca-8e54-a1bc4161f10a
| 2 |
, 3.0, and 7.5 muM, respectively. The ADP/ADP nucleotide exchange was stimulated about 5-fold by 500 muM MgCl2 or MnCl2(km of 40 muM) and about 3-fold by 500 muM CaCl2(Km of 90 muM). It was optimal between pH 6.0 and 7.0 and decreased rapidly above pH 7.5
|
9c177e68-1734-43ca-8e54-a1bc4161f10a
| 3 |
. Arrhenius plots between 0 degrees and 40 degrees showed a break point at 15 degrees with soybean phospholipids and an activation energy of 29.5 kcal/mole from 0 degrees-15 degrees and 9.0 kcal/mole from 15 degrees-40 degrees. With mitochondrial phospholipids the break point was at 9 degrees and activation energies were 42.4
|
9c177e68-1734-43ca-8e54-a1bc4161f10a
| 4 |
kcal/mole from 0 degrees-9 degrees and 7.6 kcal/mole from 9 degrees-40 degrees. 3. The phospholipid requirements for adenine nucleotide exchange were similar to those of oxidative phosphorylation. Optimal rates were observed with a phosphatidylethanolamine to phosphatidylcholine ratio of 4:1. Cardiolipin had a slight stimulatory effect.
|
9c177e68-1734-43ca-8e54-a1bc4161f10a
| 5 |
4. The uptake of ADP into vesicles containing ATP was stimulated by KCl or by KPi as well as by hexafluoracetonylacetone, and uncoupler of oxidative phosphorylation. The uptake of ATP into vesicles containing ADP was inhibited by KCl or by KPi, but was also stimulated by hexafluoracetonylacetone. In both cases valinomycin reversed the effects of KCl, while mersalyl or N-ethylmaleimide prevented the effects of KPi.
|
9c177e68-1734-43ca-8e54-a1bc4161f10a
| 6 |
In contrast, none of these salts nor hexafluoracetonylactone affected the ADP/ADP or ATP/ATP exchange. These findings suggest that in the reconstituted system the ADP/ATP exchange is electrogenic.
|
63768a38-173e-4eb0-82d1-1109f602649f
| 0 |
Solubilization of thyroid peroxidase by nonionic detergents. We have examined the ability of nonionic detergents to solubilize thyroid peroxidase from a porcine thyroid particulate fraction, as measured by the release of peroxidase activity into the supernatant fraction after centrifugation at 105,000 X g for 1 hour and the retardation of the supernatant peroxidase of Sepharose 6B.
|
63768a38-173e-4eb0-82d1-1109f602649f
| 1 |
The parameters of peroxidase solubilization by Triton X-100 have been investigated in detail. Under optimum conditions, 60 to 95% of the thryoid peroxidase and about 50% of the total protein is released into the 105,000 X g, 1-hour supernatant. Under the optimum conditions established with Triton X-100, a series of Brij detergents of different chemical structure were equally effective in releasing peroxidase and protein.
|
63768a38-173e-4eb0-82d1-1109f602649f
| 2 |
The protein patterns of the supernatants obtained with these detergents were similar on sodium dodecyl sulfate-polyacrylamide electrophoresis gels, suggesting that the detergents studied release similar membrane proteins. The Triton X-100 and Brij 58 supernatants were chromatographed separately on Sepharose 6B equilibrated with 0.1% Triton X-100 or Brij 58, respectively.
|
63768a38-173e-4eb0-82d1-1109f602649f
| 3 |
In both cases, 75 to 80% of the peroxidase activity was retarded, thereby indicating that the nonionic detergents effect solubilization of the peroxidase rather than dispersal of nonsedimentable membrane fragments. These studies report the first successful solubilization of thyroid peroxidase by nonionic detergents. Together with previous evidence from our laboratory, these experiments indicate that thyroid peroxidase is an integral membrane protein.
|
591be421-b2ab-4b2d-82ef-88a5c8acbd61
| 0 |
Carbon 13 magnetic resonance studies of DL-2-(alpha-hydroxyethyl) thiamin and related compounds. Relation of kinetic acidity to electronic factors in thiamin catalysis. Carbon 13 NMR spectra have been obtained for aqueous solutions of DL-2-(alpha-hydroxyethyl)thiamin, DL-2-(alpha-hydroxybenzyl)thiamin, DL-2-(alpha-hydroxybenzyl)oxythiamin, and related N-3 methyl and N-3 benzyl analogs.
|
591be421-b2ab-4b2d-82ef-88a5c8acbd61
| 1 |
The unusually large downfield shift of the 13C resonance of C-2 of hydroxyethylthiamin suggests that this carbon bears a partial positive charge. This result stands in contrast to results of x-ray crystallographic studies of hydroxyethylthiamin, which place a partial negative charge on C-2 (Pletcher, J.
|
591be421-b2ab-4b2d-82ef-88a5c8acbd61
| 2 |
, and Sax, M. (1974) J. Am. Chem. Soc. 96, 155-165). A partial positive charge on C-2 helps to explain the facility of carbanion formation at the alpha carbon both enzymatically and in model systems. The rates of proton-deuteron exchange of (C-alpha)-H with solvent deuterium, and of release of aldehyde to regenerate thiamin have been measured for hydroxyethylthiamin and analogs.
|
591be421-b2ab-4b2d-82ef-88a5c8acbd61
| 3 |
The differences in kinetic acidity of (C-alpha)-H and of rates of aldehyde release are rationalized in terms of differing electron-withdrawing abilities of the substituents attached to N-3, and appear not to be related to intramolecular basic catalysis of these processes by the C-4' amino group.
|
e78ce6e4-3283-4378-9c94-c2a1079dc9e1
| 0 |
Oxygen and carbon monoxide kinetics of Glycera dibranchiata monomeric hemoglobin. Oxygen and carbon monoxide kinetics of Glycera dibranchiata monomeric hemoglobin have been studied using laser photolysis, air flash, and stopped flow techniques. The reactions of this hemoglobin with both ligands were found to be more rapid than the corresponding reactions involving myoglobin and were also biphasic in nature, the rate constants being approximately an order of magnitude different for the fast and slow phases in each case.
|
e78ce6e4-3283-4378-9c94-c2a1079dc9e1
| 1 |
No pH or hemoglobin concentration dependence of the pseudo-first order rate constants was apparent between pH 6 and 9 and in the concentration range of 1.25 to 40 muM heme. Both fast and slow pseudo-first order oxygen combination rate constants varied linearly with oxygen concentration between 16 and 1300 muM.
|
e78ce6e4-3283-4378-9c94-c2a1079dc9e1
| 2 |
A first order slow relaxation was also noted which was linearly dependent on heme concentration and inversely dependent on oxygen concentration. This reaction has been shown to be due to a replacement of oxygen by carbon monoxide. The presence of this reaction is a result of the high affinity of Glycera monomer for carbon monoxide as shown by the partition coefficient Mr = approximately 20,000 ana an equilibrium dissociation constant of the order L = 1.1
|
e78ce6e4-3283-4378-9c94-c2a1079dc9e1
| 3 |
X 10(-9) M.
|
712a0fd6-38c5-4108-be96-7290cf2c0509
| 0 |
Analysis of phosphate metabolites, the intracellular pH, and the state of adenosine triphosphate in intact muscle by phosphorus nuclear magnetic resonance. 31P nuclear magnetic resonance spectra recorded from intact muophosphate, and the sugar phosphates. Quantitation of these metabolites by 31P nuclear magnetic resonance was in good agreement with values obtained by chemical analyses.
|
712a0fd6-38c5-4108-be96-7290cf2c0509
| 1 |
The spectra obtained from various muscles showed considerable variation in their phosphorus profile. Thus, differences could be detected between (a) normal and diseased muscle; (b) vertebrates and invertebrates; (c) different species of the same animal. The time course of change in phosphate metabolites in frog muscle showed that ATP level remains unchanged until phosphocreatine is nearly depleted.
|
712a0fd6-38c5-4108-be96-7290cf2c0509
| 2 |
Comparative studies revealed that under anaerobic conditions the Northern frog maintains its ATP content for 7 hours, while other types of amphibian, bird, and mammalian muscles begin to show an appreciable decay in ATP after 2 hours. Several lines of evidence indicated that ATP forms a complex with magnesium in the muscle water:
|
712a0fd6-38c5-4108-be96-7290cf2c0509
| 3 |
(a) the phosphate resonances of ATP in the muscle were shifted downfield as compared to those in the alkaline earth metal-free perchloric acid extract of the muscle; (b) the coupling constants of ATP measured in various live muscles closely corresponded to those for MgATP in a solution resembling the composition of the muscle water;
|
712a0fd6-38c5-4108-be96-7290cf2c0509
| 4 |
(c) in the muscle the gamma-phosphate group of ATP exhibited no shift change over a period of 10 hours under conditions where resonances of other phosphate compounds could be titrated. This behavior is similar to that of MgATP in model solutions in the physiological pH range, and it is different from that of CaATP.
|
712a0fd6-38c5-4108-be96-7290cf2c0509
| 5 |
The chemical shifts of the phosphate metabolites were determined in several relevant solutions as a function of pH. Under all conditions only inorganic orthophosphate showed an invariant titration curve. From the chemical shift of inorganic phosphate observed during aging of intact muscle the intracellular pH of frog muscle was estimated to be 7.2
|
712a0fd6-38c5-4108-be96-7290cf2c0509
| 6 |
.
|
f5852a8f-d026-4d54-9fea-200b51d4c9cb
| 0 |
5' leads to 3'-Exonucleases of bacteriophage T4. Two enzyme activities which release nucleotides preferentially from the 5' termini of DNA were found in T4-infected Escherichia coli. Since no corresponding activities were found in uninfected cells, the activities appeared to be induced by T4. Both activities are capable of excising pyrimidine dimers from ultraviolet-irradiated DNA which has been treated with T4 endonuclease V.
|
f5852a8f-d026-4d54-9fea-200b51d4c9cb
| 1 |
One of the activities , referred to as T4 exonuclease B, was purified 400-fold from an extract of T4v 1- infected cells. The enzyme initiates hydrolysis of DNA specifically at the 5' termini to yield products which are mainly oligonucleotides of varying length. The hydrolysis reaction proceeds in a limited manner.
|
f5852a8f-d026-4d54-9fea-200b51d4c9cb
| 2 |
The enzyme shows optimal activity at pH 7.0 and absolutely requires Mg2+. The molecular weight of the enzyme , as estimated by gel filtration, is approximately 35,000. Another activity, referred to as T4 exonuclease C, was purified 240-fold from the extract. This activity also excises pyrimidine dimers from ultraviolet-irradiated, incised DNA and releases nucleotides at 5' termini.
|
f5852a8f-d026-4d54-9fea-200b51d4c9cb
| 3 |
It has a pH optimum at 7.5 and requires Mg2+. The molecular weight of the enzyme is approximately 20,000.
|
59623347-1f21-4d25-b477-f8805e84f2b7
| 0 |
Nuclear magnetic resonance titration curves of histidine ring protons. A direct assignment of the resonances of the active site histidine residues of ribonuclease. One of the four titrating histidine ring C-2 proton resonances of bovine pancreatic ribonuclease has been assigned to histidine residue 12.
|
59623347-1f21-4d25-b477-f8805e84f2b7
| 1 |
This was accomplished by a direct comparison of the rate of tritium incorporation into position C-2 of histidine 12 of S-peptide (residues 1 to 20) derived from ribonuclease S, with the rates of deuterium exchange of the four histidine C-2 proton resonances of ribonuclease S under the same experimental conditions.
|
59623347-1f21-4d25-b477-f8805e84f2b7
| 2 |
The same assignment was obtained by a comparison of the NMR titration curves of ribonuclease S, the noncovalent complex of S-peptide and S-protein (residues 21 to 124) with the results for the recombined complex in which position C-2 of histidine 12 was fully deuterated. The second active site histidine resonance was assigned to histidine residue 119 by consideration of the NMR titration results fro carboxymethylated histidines and 1-carboxymethylhistidine 119 ribonuclease.
|
59623347-1f21-4d25-b477-f8805e84f2b7
| 3 |
This assignment is a reversal of that originally reported, and has important implications for the interpretation of NMR titration data of ribonuclease.
|
13231c69-e1d4-40ba-a4c8-832243945f7d
| 0 |
Nuclear magnetic resonance titration curves of histidine ring protons. Ribonuclease S-peptide and S-proteins. The histidine C-2 proton NMR titration curves of ribonuclease S-peptide (residues 1 to 20) and S-protein (residues 21 to 124) are reported. Although S-protein contains 3 histidine residues, four discrete resonances are observed to titrate.
|
13231c69-e1d4-40ba-a4c8-832243945f7d
| 1 |
One of these arises from the equivalent histidine residues of unfolded S-protein. The variation in area of the four resonances indicate that there is a reversible pH-dependent equilibrium between the folded and unfolded forms of S-protein, with some unfolded material being present at most pH values.
|
13231c69-e1d4-40ba-a4c8-832243945f7d
| 2 |
Two of the resonances of the folded S-protein can be assigned to 2 of the histidine residues, 48 and 105, from the close similarity of their titration curves to those in ribonuclease. These similarities indicate a homology of portions of the folded conformation of S-protein to that of ribonuclease in solution.
|
13231c69-e1d4-40ba-a4c8-832243945f7d
| 3 |
These results indicate that the complete amino acid sequence is not required to produce a folded conformation similar to the native globular protein, and they appear to eliminate the possibility that proteins fold from their NH2 terminus during protein synthesis. The low pH inflection present in the titration curve assigned to histidine residue 48 in ribonuclease is absent from this curve in S-protein.
|
13231c69-e1d4-40ba-a4c8-832243945f7d
| 4 |
This is consistent with our previous conclusion that this inflection arises from the interaction of histidine 48 with aspartic acid residue 14, which is also absent in S-protein. The third titrating resonance of native S-protein is assigned to the remaining histidine residue at position 119. The properties of this resonance are not identical with either of the titration curves of the active site histidine residues 12 and 119 of ribonuclease.
|
13231c69-e1d4-40ba-a4c8-832243945f7d
| 5 |
The resonance assigned to histidine 119 is the only one significantly affected on the addition of sodium phosphate to S-protein, indicating that some degree of phosphate binding occurs. In both the absence and presence of phosphate this curve also lacks the low pH inflection observed in the histidine 119 NMR titration curve in ribonuclease.
|
13231c69-e1d4-40ba-a4c8-832243945f7d
| 6 |
This difference presumably arise from a conformational between ribonuclease and the folded S-protein involving a carboxyl group.
|
7242ec22-7fe9-494b-a7c9-1222815ae523
| 0 |
Effects of formylation of vinyl side chains of heme on optical and ligand binding properties of horse heart ferric myoglobin. Effects of substitution of vinyl groups of hemin with formyl groups on the optical and ligand binding properties of horse heart ferric myoglobin were investigated. The peak positions as well as the line shapes of the absorption spectra of the ferric derivatives of three kinds of formylmyoglobin, 2-vinyl-4-formyl-, 2-formyl-4-vinyl-, and 2,4-diformylmyoglobins depend on the number and the position of the formyl groups.
|
7242ec22-7fe9-494b-a7c9-1222815ae523
| 1 |
Absorption maxima in the Soret region of the acid forms of these ferric formylmyoglobins in 0.1 M potassium phosphate buffer, pH 6.0, at 20 degrees were 415.2, 422, and 429 nm, respectively. The acid forms of these formylmyoglobins exhibit absorption spectra of the mixture of high- and low spin states at ambient temperature.
|
7242ec22-7fe9-494b-a7c9-1222815ae523
| 2 |
Since proto-, deutero- and mesomyoglobins have a high spin state under the same condition, the increase of the low spin iron in these formylmyoglobins may be due to the strong electron withdrawal by the formyl groups toward the periphery of the porphyrin ring. The affinities of these ferric formylmyoglobins and protomyoglobin for N3-, F-, OCN-, and SCN- increased in the order of proto-, monoformyl-monovinyl-, 2,4-diformyl-myoglobin, which corresponds to the increasing order of electron-withdrawing power of the porphyrin side chains.
|
7242ec22-7fe9-494b-a7c9-1222815ae523
| 3 |
The pKa values of the acid-alkaline transition decreased in the same order. Although the ferric forms of the two isomeric monoformyl-monovinylmyoglobins exhibited different optical spectra, the dissociation constants of the complexes of these isomers for various ligands were similar to each other. The pKa values of the acid-alkaline transition were also similar.
|
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