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d1b8d94f-0e44-489f-bec9-f3f438e4d097
| 0 |
Circulating levels of prolactin in human breast cancer. Serum prolactin concentrations were measured by radioimmunoassays in 98 patients with established carcinoma of breast, 12 patients with cystic mastitis and 10 patients with gynaecomastia and compared with that of age matched normal control women. The serum prolactin levels in the patients with breast cancer, gynaecomastia or cystic mastitis were observed to be similar to that in normal women. It was interesting to note that the levels of prolactin in the luteal phase of the cycle were higher than that in the early follicular phase in normal women.
|
a163f892-609c-4675-bfc7-cacf1e0661ff
| 0 |
Effect of aflatoxin B1 on pyridine nucleotides and NADP linked dehydrogenases. The effect of a single interaperitoneal injection (6 mg/kg body weight) of aflatoxin B1 in propylene glycol on pyridine nucleotides and NDP linked dehydrogenases was studied 24 h after administration of the toxin. The liver showed a decrease in total proteins and pyridine nucleotides though levels of NADP and NADPH remained unchanged.
|
a163f892-609c-4675-bfc7-cacf1e0661ff
| 1 |
Levels of NAD and NADH were decreased. The activities of hepatic of hwpRIX of hepatic malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICDH) were not altered though ICDH showed an increase when expressed on protein basis. However, there was a significance decrease in the activity of combined HMP dehydrogenases.
|
a163f892-609c-4675-bfc7-cacf1e0661ff
| 2 |
Adipose tissue showed increased activities of the HMP dehydrogenasess.
|
3d0f7698-e38d-49e5-8431-01613a820f3f
| 0 |
Esterase activity of zinc neutral proteases. The hydrolysis of a series of depsipeptides demonstrates that the zinc neutral endopeptidases of bacteria are active esterases. Esters such as BzGly-OPhe-Ala, BzGly-OLeu-Ala, and FA-Gly-OLeu-NH2 are hydrolyzed at rates three- to eightfold slower than are their exact peptide analogues, when hydrolyzed by thermolysin, Bacillus subtilis neutral protease and the neutral protease from Aeromonas proteolytica.
|
3d0f7698-e38d-49e5-8431-01613a820f3f
| 1 |
Ester hydrolysis by zinc neutral proteases follows the characteristic preference for hydrophobic amino acids adjacent to the site of cleavage, discerned from the hydrolysis of peptide substrates. Removal of zinc from thermolysin abolishes the esterase activity of the native enzyme. Among the metals examined, only Co2+ and Zn2+ restore esterase activity to any significant extent, Co2+ restoring 50% and Zn2+ 100% of the native thermolysin activity.
|
3d0f7698-e38d-49e5-8431-01613a820f3f
| 2 |
The hydrolysis of esters and peptides by thermolysin does not differ with respect to either the binding or catalytic steps. Substrate specificity, pH-rate profiles, inhibitor, and deuterium isotope effects are identical for both types of substrates.
|
d4d4f845-7d4b-4fd0-9b6f-98cc56c35504
| 0 |
The binding of reduced nicotinamide adenine dinucleotide to citrate synthase of Escherichia coli K12. Citrate synthase from Escherichia coli enhances the fluorescence of its allosteric inhibitor, NADH, and shifts the peak of emission of the coenzyme from 457 to 428 nm. These effects have been used to measure the binding of NADH to this enzyme under various conditions.
|
d4d4f845-7d4b-4fd0-9b6f-98cc56c35504
| 1 |
The dissociation constant for the NADH-citrate synthase complex is about 0.28 muM at pH 6.2, but increases toward alkaline pH as if binding depends on protonation of a group with a pKa of about 7.05. Over the pH range 6.2-8.7, the number of binding sites decreases from about 0.65
|
d4d4f845-7d4b-4fd0-9b6f-98cc56c35504
| 2 |
to about 0.25 per citrate synthase subunit. The midpoint of this transition is at about pH 7.7, and it may be one reflection of the partial depolymerization of the enzyme which is known to occur in this pH range. A gel filtration method has been used to verify that the fluorescence enhancement technique accurately reveals all of the NADH molecules bound to the enzyme in the concentration range of interest.
|
d4d4f845-7d4b-4fd0-9b6f-98cc56c35504
| 3 |
NAD+ and NADP+ were weak competitive inhibitors of NADH binding at pH 7.8 (Ki values greater than 1 mM), but stronger inhibition was shown by 5'-AMP and 3'-AMP, with Ki values of 83 +/- 5 and 65 +/- 4 muM, respectively.
|
d4d4f845-7d4b-4fd0-9b6f-98cc56c35504
| 4 |
Acetyl-CoA, one of the substrates, and KCl, an activator, also inhibit the binding in a weakly cooperative manner. All of these effects are consistent with kinetic observations on this system. We interpret our results in terms of two types of binding site for nucleotides on citrate synthase:
|
d4d4f845-7d4b-4fd0-9b6f-98cc56c35504
| 5 |
an active site which binds acetyl-CoA, the substrate, or its analogue 3'-AMP; and an allosteric site which binds NADH or its analogue 5'-AMP and has a lesser affinity for other nicotinamide adenine dinucloetides. When the active site is occupied, we propose that NADH cannot bind to the allosteric site, but 5'-AMP can;
|
d4d4f845-7d4b-4fd0-9b6f-98cc56c35504
| 6 |
conversely, when NADH is the in the allosteric site, the active site cannot be occupied. In addition to these two classes of sites, there must be points for interaction with KCl and other salts. Oxaloacetate, the second substrate, and alpha-ketoglutarate, an inhibitor whose mode of action is believed to be allosteric, have no effect on NADH binding to citrate synthase at pH 7.8
|
d4d4f845-7d4b-4fd0-9b6f-98cc56c35504
| 7 |
. When NADH is bound to citrate synthase, it quenches the intrinsic tryptophan fluorescence of the enzyme. The amount of quenching is proportional to the amount of NADH bound, at least up to a binding ratio of 0.50 NADH per enzyme subunit. This amount of binding leads to the quenching of 53 +/- 5% of the enzyme fluorescence, which means that one NADH molecule can quench all the intrinsic fluorescence of the subunit to which it binds.
|
88257cb6-1ddb-4ebd-adc5-dd081629323c
| 0 |
The structure of the covalent adduct formed by the interaction of 3-dimethylamino-1-propyne and the flavine of mitochondrial amine oxidase. 3-Dimethylamino-1-propyne irreversibly inactivates mitochondrial monoamine oxidase from bovine liver. The inactivation results in the loss of absorption in the 450-500-nm region of the flavine spectrum and a concomitant increase in absorbance at 410 nm.
|
88257cb6-1ddb-4ebd-adc5-dd081629323c
| 1 |
For the enzyme-bound adduct epsilon410 = 28000. The spectral properties of the adduct of the liver enzyme with 3-dimethylamino-1-propyne are similar to those observed when the pig kidney enzyme is inactivated with pargyline (Chuang et al. (1974), J. Biol. Chem. 249, 2381).
|
88257cb6-1ddb-4ebd-adc5-dd081629323c
| 2 |
From a proteolytic digest of the enzyme inactivated with labeled inhibitor a flavine peptide has been isolated which contains 1 mol of inactivator/mol of flavine. The chemical and spectral properties of the adduct are those of compounds containing the structure --N--CH==CH--CH==N+ less than.
|
88257cb6-1ddb-4ebd-adc5-dd081629323c
| 3 |
It was concluded that the flavine-inhibtor adduct is a N-5 substituted dihydroflavine and its structure has been determined.
|
b3760baa-2b97-4742-8e0d-94d5033d837a
| 0 |
The absolute configuration of the amino acids in delta-(alpha-aminoadipyl)cysteinylvaline from Penicillium chrysogenum. Radioactive carbon-14 L-alpha-aminoadipic acid, L-cysteine, or L-valine were readily incorporated into the intracellular tripeptide, delta-(alpha-aminoadipyl)cysteinylvaline (ACV), by washed starved cells of Penicillium chrysogenum. The labeled ACV in each case was oxidized with performic acid and isolated as its corresponding sulfonic acid derivative.
|
b3760baa-2b97-4742-8e0d-94d5033d837a
| 1 |
After acid hydrolysis, the configuration of the component acids was determined by L- and D-amino acid oxidases, which showed the tripeptide (ACV) from P. chrysogenum to be delta-(L-aminoadipyl)-L-cysteinyl-D-valine.
|
45fc46dc-7d4b-40e5-a126-780d74d3f73e
| 0 |
Chromatin-bound protease: degradation of chromosomal proteins under chromatin dissociation conditions. A chromatin-bound protease, active in 2 M NaCl-5 M urea or 5 M urea alone, was demonstrated in rat liver, kidney, testes, brain, rabbit bone marrow, chicken reticulocyte, and Ehrlich ascites chromatin.
|
45fc46dc-7d4b-40e5-a126-780d74d3f73e
| 1 |
Chicken erythrocyte chromatin did not possess any detectable proteolytic activity in salt and urea. The proteolytic activity of rat liver chromatin in salt and urea was found to be independent of the methods of chromatin preparation. The protease can be inhibited by the serine specific reagents phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate and the alkylating reagent, carbobenzoxyphenylalanine chloromethyl ketone, in the presence of organic solvents at 1 mM concentration.
|
45fc46dc-7d4b-40e5-a126-780d74d3f73e
| 2 |
The inhibitions of chromatin-bound protease in rat liver by these compounds are irreversible. On the other hand, carbobenzoxyphenylalanine and p-nitrophenyl acetate were shown to be reversible inhibitors of rat liver chromatin-bound protease. The application of these inhibitors during the dissociation of chromatin by salt and urea may be useful to researchers interested in purifying various chromosomal proteins or to those researchers doing reconstitution studies with labile chromatins.
|
f1804bae-f289-4748-98c1-ab1ce582fbc0
| 0 |
ATPase of Escherichia coli: purification, dissociation, and reconstitution of the active complex from the isolated subunits. A simple procedure for the purification of Mg2+-stimulated ATPase of Escherichia coli by fractionation with poly(ethylene glycols) and gel filtration is described. The enzyme restores ATPase-linked reactions to membrane preparations lacking these activities.
|
f1804bae-f289-4748-98c1-ab1ce582fbc0
| 1 |
Five different polypeptides (alpha, beta, gamma, delta, epsilon) are observed in sodium dodecyl sulfate electrophoresis. Freezing in salt solutions splits the enzyme complex into subunits which do not possess any catalytic activity. The presence of different subunits is confirmed by electrophoretic and immunological methods. The active enzyme complex can be reconstituted by decreasing the ionic strength in the dissociated sample.
|
f1804bae-f289-4748-98c1-ab1ce582fbc0
| 2 |
Temperature, pH, protein concentration, and the presence of substrate are each important determinants of the rate and extent of reconstitution. The dissociated enzyme has been separated by ion-exchange chromatography into two major fragments. Fragment IA has a molecular weight of about 100000 and contains the alpha, gamma, and epsilon polypeptides.
|
f1804bae-f289-4748-98c1-ab1ce582fbc0
| 3 |
The minor fragment, IB, has about the same molecular weight but contains, besides alpha, gamma, and epsilon, the delta polypeptide. Fragment II, with a molecular weight of about 52000, appears to be identical with the beta polypeptide. ATPase activity can be reconstituted from fragments IA and II, whereas the capacity of the ATPase to drive energy-dependent processes in depleted membrane vesicles is only restored after incubation of these two fractions with fraction IB, which contains the delta subunit.
|
52316842-f0c9-4bf9-9788-808effe4f933
| 0 |
Conformation of gonadotropin releasing hormone. The conformation of the gonadotropin releasing hormone (Gn-RH), whose primary sequence is pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH2, and of several of its structural analogues has been studied by circular dichroism, optical rotatory dispersion, and fluorescence spectroscopy.
|
52316842-f0c9-4bf9-9788-808effe4f933
| 1 |
The effects of pH, guanidine, and temperature on fluorescence emission have also been examined. Titration data demonstrate that the histidine and tyrosine residues are free of any mutual interactions. The similarity of emission spectra in water and in guanidine hydrochloride solutions precludes significant interactions between the fluorescent groups and other residues.
|
52316842-f0c9-4bf9-9788-808effe4f933
| 2 |
Neither the temperature nor the pH profiles of the emission intensities of either tyrosine or tryptophan reveal any fixed secondary structure in Gn-RH. Both the extent of alkaline quenching and the distance of 10-11 A calculated from Förster energy transfer theory are in accord with a randomly coiled structure with only one residue between tyrosine and tryptophan.
|
52316842-f0c9-4bf9-9788-808effe4f933
| 3 |
Furthermore, the circular dichroism spectrum and optical rotatory dispersion do not exhibit any contributions from peptide bonds in an ordered structure, although there is a perturbation of the peptide absorption region due to overlapping bands from side-chain chromophores. Gn-RH, therefore, appears to behave as a random coil polypeptide in water devoid of any intrachain residue interactions.
|
52316842-f0c9-4bf9-9788-808effe4f933
| 4 |
This nonordered structure in Gn-RH and the lack of any significant differences in the physical-chemical properties of the hormone analogues indicate that a predetermined solution conformation is not required for biological activity. In contrast to its behavior in water, Gn-RH in trifluoroethanol exhibits a conformational transition, with the formation of a beta structure.
|
52316842-f0c9-4bf9-9788-808effe4f933
| 5 |
Differences in conformational changes exhibited by several analogues in trifluoroethanol may be relevant to their relative biological activities at the receptor site.
|
a2187156-73c1-4af1-97dd-368253cc7018
| 0 |
Low and high pH form of cadmium carbonic anhydrase determined by nuclear quadrupole interaction. The pH dependence of the nuclear quadrupole interaction between the excited 247-keV state in 111Cd bound to the active site in human carbonic anhydrase B and the nearest protein surroundings has been studied by means of the nuclear spectroscopic technique of perturbed angular correlation of gamma rays.
|
a2187156-73c1-4af1-97dd-368253cc7018
| 1 |
The enzyme has been studied in the pH region 5.6-11.0 at 22 and -196 degrees C. The results show that the Cd enzyme changes from one form at low pH to another form at high pH both at 22 and -196 degrees C. The pK of the transition is 8.9
|
a2187156-73c1-4af1-97dd-368253cc7018
| 2 |
+/- 0.2 at -196 degrees C and close to 9 at 22 degrees C. Parallel to this transformation, the esterase activity of the Cd enzyme for the hydration of p-nitrophenyl acetate exhibits a pH dependency with a pH of 9.1 +/- 0.2. The sulfonamide inhibitor acetazolamide completely inhibits this activity of the Cd enzyme.
|
a2187156-73c1-4af1-97dd-368253cc7018
| 3 |
The quadrupole interaction parameters for the Cd enzyme are not significantly different at -196 degrees C from those obtained at 22 degrees C. A measurement at 0 degrees C pH 5.7 shows, however, a form different from those at 22 degrees C pH 5.6 and -196 degrees C pH 5.7
|
a2187156-73c1-4af1-97dd-368253cc7018
| 4 |
. The change in the quadrupole interaction with pH is, in a simple model, consistent with an ionization of a metal-bound water molecule.
|
44af4748-6974-40cf-9c3d-69b639c21789
| 0 |
Oxidation-reduction properties of Chromatium vinosum high potential iron-sulfur protein. The oxidation-reduction properties of the high potential iron-sulfur protein (HIPIP) from Chromatium vinosum have been investigated. Both equilibrium and kinetic measurements demonstrate electron transport by HIPIP is pH independent in the pH range 7-11. The kinetics of reduction (potassium ferrocyanide, SO2, S2O42-, sodium ascorbate, and Rhodospirillum rubrum cytochrome c2) and oxidation (potassium ferricyanide and Rhodospirillium rubrum cytochrome c2) of HIPIP are reported.
|
44af4748-6974-40cf-9c3d-69b639c21789
| 1 |
Based on the data obtained with different reactants and the influence of ionic strength, pH, and temperature on the kinetics of oxidation and reduction, a number of conclusions can be drawn. (1) HIPIP undergoes rapid outer-sphere electron transfer with no evidence of kinetic complexity and no indication of complex formation with various reactants.
|
44af4748-6974-40cf-9c3d-69b639c21789
| 2 |
(2) The site of oxidation of reduced HIPIP has an apparent negative charge while the site of reduction of oxidized HIPIP is uncharged. (3) HIPIP appears to interact with a physiological reactant (R. rubrum cytochrome c2) at the same site as nonphysiological oxidants or reductants suggesting single minimum energy pathways for the oxidation and reduction processes.
|
44af4748-6974-40cf-9c3d-69b639c21789
| 3 |
(4) Based on a comparison of the rates of oxidation and reduction with different reactants, it appears that steric restrictions and differences in oxidation-reduction potential are less important than electrostatic attraction and/or repulsion in determining the absolute rate constants. (5) The thermodynamic activation parameters indicate that both oxidation and reduction by the iron hexacyanides are driven entropically with the enthalpic terms making no contribution to HIPIP oxidation and a small contribution to HIPIP reduction.
|
44af4748-6974-40cf-9c3d-69b639c21789
| 4 |
Based on the data reported here and available structural and physical-chemical information, possible mechanisms of the oxidation and reduction of HIPIP are discussed and their relative merits analyzed. The more likely mechanisms include electron transfer via a tyrosine residue, electron transfer through a nonaqueous media to the iron-sulfur chromophore, and direct interaction between the iron-sulfur chromophore and the different oxidants and reductants.
|
728e2be0-c5a5-4e4a-a83a-7536bbee88ac
| 0 |
Isolation, chemical, and physical properties of alpha-1-antitrypsin. A method of isolation of alpha-1-antitrypsin (alpha-1-AT) in good yield from normal human plasma is described. A key step was affinity chromatography employing an antiserum which had been depleted of alpha-1-AT antibodies.
|
728e2be0-c5a5-4e4a-a83a-7536bbee88ac
| 1 |
The final preparations were homogeneous by immunological and physicochemical criteria. The specific activity of the purified alpha-1-AT was 0.363 mg of active bovine trypsin inhibited per 1.0 mg of inhibitor. Polyacrylamide gel patterns at both alkaline and acid pH of highly pure preparations frequently, but not invariably, showed multiple hands.
|
728e2be0-c5a5-4e4a-a83a-7536bbee88ac
| 2 |
Molecular weight studies by sedimentation equilibrium ultracentrifugation in aqueous buffer and in 6 M guanidine as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis suggest that alpha-1-AT is a single polypeptide chain having a molecular weight of 49,500. Other physical and chemical properties of the inhibitor are described.
|
728e2be0-c5a5-4e4a-a83a-7536bbee88ac
| 3 |
A limited N-terminal sequence (Glu-Asp-Pro-Gln-Gly-Asx-Ala-Ala) was obtained. It was found that alpha-1-AT easily forms polymers and higher aggregates when exposed to denaturing agents such as 8 M urea and 6 M guanidine. The results suggest that aggregation is determined by both covalent and noncovalent forces.
|
cc37744b-9219-4e7a-80a1-21d09d777345
| 0 |
Purification and properties of the thermostable acid protease of Penicillium duponti. An acid protease produced by the thermophilic fungus Penicillium duponti K 1014 has been purified by consecutive ion-exchange and gel permeation chromatography, and crystallized from aqueous acetone solution. The purified endopeptidase gave a symmetrical schlieren peak by sedimentation velocity, and was found to be homogeneous upon disc gel electrophoresis at pH 9.5
|
cc37744b-9219-4e7a-80a1-21d09d777345
| 1 |
. The enzyme was most active at pH 2.5 against milk casein and showed high thermostability. An isoelectric point of 3.81 was found by isoelectric focusing. A minimum molecular weight of 41 590 was calculated from the amino acid composition, adopting an arginine content of one residue per mole of enzyme.
|
cc37744b-9219-4e7a-80a1-21d09d777345
| 2 |
This minimum molecular weight is in good agreement with the value of 41 000 previously found by gel permeation (Hashimoto, H., Iwaasa, T., and Yokotsuka, T. (1973), Appl. Microbiol. 25, 578). Besides the thermostability, the purified P. duponti protease differs from other well-characterized acid proteases in that it contains carbohydrate, 4.33
|
cc37744b-9219-4e7a-80a1-21d09d777345
| 3 |
% expressed as glucose. The enzyme was not affected by p-bromophenacyl bromide, but was completely inactivated by alpha-diazo-p-bromoacetophenone, diazoacetyl-DL-norleucine methyl ester, and diazoacetylglycine ethyl ester, in the presence of Cu2+. The complete inactivation of the protease by diazoacetyl-DL-norleucine methyl ester resulted in the specific incorporation of 1 mol of norleucine/mol of enzyme.
|
cc37744b-9219-4e7a-80a1-21d09d777345
| 4 |
On the basis of similar behavior of other acid proteases toward this inactivator, the results suggest the presence at the active site of an unusually reactive carboxyl group, involved in the catalytic function. The naturally occurring pepsin inhibitor of Streptomyces naniwaensis [Murao, S., and Satoi, S.
|
cc37744b-9219-4e7a-80a1-21d09d777345
| 5 |
(1970), Agric. Biol. Chem. 34, 1265] inhibited also the protease, at a threefold molar excess with respect to the enzyme.
|
5e81cdb6-480a-459e-a059-827e849ef5ac
| 0 |
Biosynthesis of bacterial glycogen. Purification and properties of the Escherichia coli B ADPglucose:1,4-alpha-D-glucan 4-alpha-glucosyltransferase. The Escherichia coli B glycogen synthase has been purified to apparent homogeneity with the use of a 4-aminobutyl-Sepharose column. Two fractions of the enzyme were obtained:
|
5e81cdb6-480a-459e-a059-827e849ef5ac
| 1 |
glycogen synthase I with a specific activity of 380 mumol mg-1 and devoid of branching enzyme activity and glycogen synthase II having a specific activity of 505 mumol mg-1 and containing branching enzyme activity which was 0.1% of the activity observed for the glycogen synthase. Only one protein band was found in disc gel electrophoresis for each glycogen synthase fraction and they were coincident with glycogen synthase activity.
|
5e81cdb6-480a-459e-a059-827e849ef5ac
| 2 |
One major protein band and one very faint protein band which hardly moved into the gel were observed in sodium dodecyl sulfate gel electrophoresis of the glycogen synthase fractions. The subunit molecular weight of the major protein band in sodium dodecyl sulfate gel electrophoresis of both glycogen synthase fractions was determined to be 49 000 +/- 2 000.
|
5e81cdb6-480a-459e-a059-827e849ef5ac
| 3 |
The molecular weights of the native enzymes were determined by sucrose density gradient ultracentrifugation. Glycogen synthase I had a molecular weight of 93 000 while glycogen synthase II had a molecular weight of 200 000. On standing at 4 degrees C or at -85 degrees C both enzymes transform into species having molecular weights of 98 000, 135 000, and 185 000.
|
5e81cdb6-480a-459e-a059-827e849ef5ac
| 4 |
Thus active forms of the E. coli B glycogen synthase can exist as dimers, trimers, and tetramers of the subunit. The enzyme was shown to catalyze transfer of glucose from ADPglucose to maltose and to higher oligosaccharides of the maltodextrin series but not to glucose. 1,5-Gluconolactone was shown to be a potent inhibitor of the glycogen synthase reaction.
|
5e81cdb6-480a-459e-a059-827e849ef5ac
| 5 |
The glycogen synthase reaction was shown to be reversible. Formation of labeled ADPglucose occurred from either [14C]ADP or [14C]glycogen. The ratio of ADP to ADPglucose at equilibrium at 37 degrees C was determined and was found to vary threefold in the pH range of 5.27-6.82
|
5e81cdb6-480a-459e-a059-827e849ef5ac
| 6 |
. From these data the ratio of ADP2- to ADPglucose at equilibrium was determined to be 45.8 +/- 4.5. Assuming that deltaF degrees of the hydrolysis of the alpha-1,4-glucosidic linkage is -4.0 kcal the deltaF degrees of hydrolysis of the glucosidic linkage in ADPglucose is -6.3
|
5e81cdb6-480a-459e-a059-827e849ef5ac
| 7 |
kcal.
|
3d1f4ee0-54c5-4f09-ab64-9d3f9dd2f380
| 0 |
Bovine procarboxypeptidase A: kinetics of peptide and ester hydrolysis. Bovine procarboxypeptidase A exhibits intrinsic hydrolytic activity toward haloacyl amino acids (Behnke and Vallee, 1972), as well as toward conventional peptide and ester substrates for carboxypeptidase A (Bezzone, 1974; Uren and Neurath, 1974). The kinetics of hydrolysis of a series of such substrates by native procarboxypeptidase has now been examined in detail in order to ascertain the extent to which the binding and catalytic sites of carboxypeptidase preexist inthe zymogen.
|
3d1f4ee0-54c5-4f09-ab64-9d3f9dd2f380
| 1 |
Distinct differences in the substrate binding sites of the zymogen compared with the enzyme are apparent from their respective kinetic profiles as well as from the effects of modifiers on their activities. Substrate activation with the dipeptides BzGly-L-Phe and CbzGly-L-Phe, well known for carboxypeptidase, is exhibited also by the zymogen, but the corresponding substrate inhibition by CbzGly-L-Phe and BzGly-Ophe is absent.
|
3d1f4ee0-54c5-4f09-ab64-9d3f9dd2f380
| 2 |
Moreover, the substrate inhibition of carboxypeptidase by CbzGlyGly-L-Phe and BzGly-Ophe is replaced by substrate activation in the zymogen...
|
ffe5737a-c965-4e66-bada-567b61b9a899
| 0 |
Ionic influences on the phase transition of dipalmitoylphosphatidylserine. The ionization and phase behavior of 1,2-dipalmitoyl-sn-glycero-3-phosphoserine have been investigated under a variety of condtions by several different methods. As measured by turbidity changes, the temperature of the crystal-liquid crystal phase transition of this lipid is influenced by pH and mono- and divalent cation concentrations.
|
ffe5737a-c965-4e66-bada-567b61b9a899
| 1 |
The pH-transition temperature curve is congruent with the curve relating temperature to the degree of ionization of the carboxyl group of the crystalline form. The transition temperature falls from an upper plateau of 72 degrees C at low pH values, where the carboxyl group is fully protonated, to a lower plateau of 55 degrees C at high pH values, where this group is fully ionized.
|
ffe5737a-c965-4e66-bada-567b61b9a899
| 2 |
The apparent pK (pH at 50% ionization) of the crystalline form shifts from 6.0 to 4.6 to 3.7 with an increase of NaCl concentration from 10(-3) to 0.1 to l.0 M, respectively. These observations are in accord with a simple theoretical analysis that utilizes diffuse double layer theory and the influence of surface potential on surface concentration of protons.
|
ffe5737a-c965-4e66-bada-567b61b9a899
| 3 |
In qualitative terms, an increase in electrolyte concentration reduces the surface potential, the result of which is a diminution of the surface-bulk pH difference and a lowering of the apparent pK. Assuming an area of 50 A2/molecule, the intrinsic pKa (apparent pK corrected for surface pH) of the carboxyl group is 2.7
|
ffe5737a-c965-4e66-bada-567b61b9a899
| 4 |
. A 1000-fold change of NaCl concentration produces a very large change in surface potential without influencing the transition temperature of the ionized form of the lipid.
|
23c6e6db-72ae-491a-8752-3c4397b7d8a5
| 0 |
Light scattering from suspensions of membrane fragments derived from sonication of beef heart mitochondria. The intensity of light scattering from suspensions of membrane fragments prepared by sonication of beef heart mitochondria in the presence of EDTA at alkaline pH (ESMP) was determined at 45, 90, and 135 degrees with light of wavelength 546 nm.
|
23c6e6db-72ae-491a-8752-3c4397b7d8a5
| 1 |
The dissymmetry ratio Z = I45 degrees c/I135 degrees c, where I45 degrees c and I135 degrees c are the scattering intensities at 45 and 135 degrees extrapolated to zero particle concentration and corrected for reflectance effects, was used to calculate particle size from the Rayleigh-Gans-Debye theory.
|
23c6e6db-72ae-491a-8752-3c4397b7d8a5
| 2 |
An average particle diameter D of 184-190 nm was obtained, within the range of particle diameter 50-300 nm determined previously by electron microscopy. This average diameter determined by light scattering is a useful parameter for characterization of ESMP particle size. We propose the term: light scattering average particle diameter, DLS, for this parameter.
|
23c6e6db-72ae-491a-8752-3c4397b7d8a5
| 3 |
The refractive index of ESMP was determined to be 1.443 by measurement of scattering intensity in buffer solutions of varying sucrose concentration. The value of Z was independent of sucrose concentration in this determination, showing that the particles are osmotically inactive toward sucrose. The values of average particle diameter DLS and of refractive index fall within the range of validity of the Rayleigh-Gans-Debye theory, for which light scattering changes are attributable solely to dimension change, rather than to change in particle refractive index.
|
23c6e6db-72ae-491a-8752-3c4397b7d8a5
| 4 |
Uptake of water accompanying energy-linked salt uptake in ESMP was calculated from light scattering changes to be 0.18 mul of H2O/mg of protein, compared with 0.49 mul of H2O/mg of protein measured by dextran inaccessibility. Measurement of light scattering changes provides a rapid and sensitive method for determining volume changes of ESMP.
|
23c6e6db-72ae-491a-8752-3c4397b7d8a5
| 5 |
The magnitude of the volume change observed during energy-linked water and salt uptake and the initial degree of hydration suggests that ESMP are analogous to polyelectrolyte gels with regard to sorption of strong electrolytes and that the Donnan formulation for ion exchange equilibria may be usefully applied to these processes in ESMP.
|
43ff4ada-da98-49c3-89ac-b5c475c3c4cb
| 0 |
Isolation and characterization of two alkaline ribonucleases from calf serum. Treatment of calf serum at 60 degrees C and pH 3.5 followed by chromatography on carboxymethyl (CM) cellulose resulted in the separation of two major peaks of alkaline RNAse activity. One was eluted from CM-cellulose at 0.075 M KCl with an overall purification of 5400-fold and the other was eluted at 0.25
|
43ff4ada-da98-49c3-89ac-b5c475c3c4cb
| 1 |
M KCl with a 6700-fold purification. The RNAse eluted from CM-cellulose at 0.075 M KCl was almost completely inhibited by anti-RNAse A serum and by the endogenous RNAse inhibitor and a 33% inhibition was observed in the presence of 5 mM MgCl2. This enzyme seems to be similar or identical to RNAse A.
|
43ff4ada-da98-49c3-89ac-b5c475c3c4cb
| 2 |
The other RNAse, eluted from CM-cellulose at 0.25 M KCl was not inhibited by anti-RNAse A or 5 mM MgCl2 and was much less sensitive to the endogenous inhibitor. Both enzymes degraded RNA endonucleolytically and the nucleoside monophosphates obtained after partial hydrolysis of RNA by the two serum RNAases were primarily 2'- or 3' -CMP and 2'- or 3' -UMP.
|
43ff4ada-da98-49c3-89ac-b5c475c3c4cb
| 3 |
Poly(A), native DNA and denatured DNA were degraded slowly or not at all. The RNAase A-like enzyme degraded poly(C) at a significantly faster rate, and poly(U) at a slower rate, than RNA. However, the other serum RNAase was more active with poly(U) than with RNA and almost inactive with poly(C) as the substrate.
|
e218db34-840c-4822-8fa2-d9d4ed711bdb
| 0 |
Isolation and characterization of the brush border fraction from newborn rat renal proximal tubule cells. A renal brush border fraction was isolated from newborn Sprague-Dawley rats, and its morphological and enzymatic characteristics were studied in comparison to that from the adult. Definite microvillar structures are seen by electron microscopy, and border preparations from the newborn are enriched in known marker enzymes.
|
e218db34-840c-4822-8fa2-d9d4ed711bdb
| 1 |
Though morphological development is more advanced and enzyme specific activities are greater in the adult, polyacrylamide gel electrophoresis of membrane proteins reveals no significant change in pattern with increasing age. These studies suggest that the brush border of the proximal tubule cell is present at birth as a significantly developed structure.
|
50e238bf-cc45-4edf-b13a-4757c211f874
| 0 |
Glial fibrillary acidic protein from bovine and rat brain. Degradation in tissues and homogenates. Compared with human material glial fibrillary acidic protein isolated from bovine, rat and mouse brain was remarkably homogeneous and migrated as a single band at 54 000 mol. wt. on sodium dodecyl sulfate gel electrophoresis.
|
50e238bf-cc45-4edf-b13a-4757c211f874
| 1 |
The protein was extremely susceptible to proteolysis and lower molecular weight components were invariably isolated together with the major species when the brain was not rapidly frozen. Further degradation of the 54 000 mol wt. polypeptide in bovine tissues incubated at 24 degrees C resulted in preparations essentially identical to those previously isolated from human autopsy material and separating into a series of immunologically active polypeptides ranging in molecular weight from 54 000 to approximately 40 500.
|
50e238bf-cc45-4edf-b13a-4757c211f874
| 2 |
The gel band pattern obtained after progressively longer periods of autolysis suggested that small fragments were cleaved from the original polypeptide in successive steps of degradation. As in human brain, the lower molecular weight products in the 45 000-40 500 range were more resistant to proteolysis and still present after prolonged periods of tissue autolysis.
|
50e238bf-cc45-4edf-b13a-4757c211f874
| 3 |
The effect of the pH and of proteinase inhibitors on degradation was studied in homogenates of bovine brain stem incubated at 37 degrees C. At pH 8.0 PROTEOLYSIS OF The glial fibrillary acidic protein followed essentially the same pattern as in tissue. Cleavage of the major species was not prevented by the addition of proteinase inhibitors.
|
50e238bf-cc45-4edf-b13a-4757c211f874
| 4 |
At pH 6.0 and 6.5 a different type of degradation was observed, with rapid breakdown of the protein and loss of immunological activity. Increased solubility in buffer solutions was another effect of autolysis. Compared with cerebral cortex and brain stem, where most of the protein was water soluble, only a small fraction was extracted with buffer from bovine white matter.
|
50e238bf-cc45-4edf-b13a-4757c211f874
| 5 |
However, the solubility markedly increased following incubation and comparable amounts were extracted in buffer and in 6 M urea.
|
b598998a-c9a3-48a4-827d-2abaa5a23d07
| 0 |
Studies on the binding of haemoglobin by haptoglobin using electrofocusing and gradient electrophoresis. 1. Gel electrofocusing followed by gel gradient electrophoresis separated the haptoglobins and their complexes with haemoglobin into characteristic two-dimensional patterns of protein bands. 2. Molecular weights of 107 000, 139 000 and 168 000 were obtained for the three bands seen after a purified preparation of haptoglobin type 1 was partially saturated with haemoglobin.
|
b598998a-c9a3-48a4-827d-2abaa5a23d07
| 1 |
This indicated that free haptoglobin, the intermediate haptoglobin-haemoglobin complex containing one half-haemoglobin and the saturated complex with two half-haemoglobins were present. 3. The three proteins showed considerable microheterogeneity and gave a number of isoelectric points in the pH ranges 4.58-4.77, 5.20-5.40 and 5.74
|
b598998a-c9a3-48a4-827d-2abaa5a23d07
| 2 |
-5.93, free haptoglobin type 1 being the lowest group. These ranges were all 0.15-0.30pH units lower if other values were taken for the isoelectric points of markers used to calibrate the pH gradient. 4. All three proteins were present over a wide range of haemoglobin concentrations, from 0.5
|
b598998a-c9a3-48a4-827d-2abaa5a23d07
| 3 |
% to 92% of that required for saturation. This would be expected if both binding sites have similar affinities for haemoglobin.
|
bb9308df-c5a3-46d7-9d07-6a0b6099148b
| 0 |
Cytochrome P450cam and its complexes. Mössbauer parameters of the heme iron. Mössbauer spectroscopy has been used to study the heme iron in various states of cytochrome P450cam from the camphor-hydroxylating system of the bacterium Pseudomonas putida. Native, camphor-free P450cam contains low-spin ferric iron, part of which (approx.
|
bb9308df-c5a3-46d7-9d07-6a0b6099148b
| 1 |
50-70%) is converted to the high-spin ferric state upon addition of camphor. The Mössbauer spectra of the camphor-free enzyme (S equals 1/2) and of the high-spin component (S equals 5/2) of the camphor complex have been successfully simulated using a model based on crystal-field theory and simple convalency considerations.
|
bb9308df-c5a3-46d7-9d07-6a0b6099148b
| 2 |
The native low-spin ferric state of P450cam forms a complex with 2-phenylimidazole, with small changes in the g values and Mössbauer spectra. These changes can be accounted for consistently in the crystal-field model referred to above. The addition of putidaredoxin to the camphor-complexed, oxidized P450cam decreases the intensity of the high-spin component and changes its quadrupole splitting.
|
bb9308df-c5a3-46d7-9d07-6a0b6099148b
| 3 |
The reduced form of P450cam contrins high-spin ferrous iron, both in the presence and absence of camphor. The complex of reduced P450cam with molecular oxygen is diamagnetic and has a combination of quadrupole splitting and isomer shift that is unusual for a ferrous complex, but strongly resembles that of oxyhemoglobin.
|
bb9308df-c5a3-46d7-9d07-6a0b6099148b
| 4 |
These results are compatible with the bound superoxide, Fe3+-O-2, model proposed for oxyhemoglobin (Weiss, J. J. (1964) Nature 202, 83-84). Reduced P450cam and its complexes, oxyP450cam-CO, are all found to be analogous in some respects to the corresponding hemoglobin complexes.
|
cb1e1f08-a858-4d2c-84c2-8c41d040bc02
| 0 |
Cross partition and determination of net charge of the isoenzymes of enolase. Enolase from bakers' yeast was separated into three isoenzymes by countercurrent distribution. The isoenzymes were partitioned in aqueous polymer two-phase systems containing positively charged trimethylamino poly(ethylene glycol) or negatively charged poly(ethylene glycol) sulphonate.
|
cb1e1f08-a858-4d2c-84c2-8c41d040bc02
| 1 |
The plots of the partition coefficient of each isoenzyme versus pH in the two biphasic systems intersect at pH equal to the isoelectric point. From slopes of the plots, the net charge of the isoenzymes at pH 6.57 was determined to be +2, -3, and -8 respectively.
|
0145255f-035e-4060-94a2-944d744fcfef
| 0 |
Avoidance of strongly chaotropic eluents for immunoaffinity chromatography by chemical modification of immobilized ligand. The need for chaotropic eluents in immunoaffinity chromatography is a consequence of the high affinities of antibodies towards their antigens. This affinity is decreased and elution of antiglucagon antibodies from a column of immobilized glucagon can be achieved under mild conditions when the steric complementarity to the antibody binding site is perturbed by selective chemical modification of the hormone.
|
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