scholarly journals Structure and enzymic activity of ribonuclease-A esterified at glutamic acid-49 and aspartic acid-53

1978 ◽  
Vol 173 (3) ◽  
pp. 821-830 ◽  
Author(s):  
A. Seetharama Acharya ◽  
Belur N. Manjula ◽  
Paul J. Vithayathil
Keyword(s):  
Methyl Ester ◽  
Glutamic Acid ◽  
Aspartic Acid ◽  
Spectral Characteristics ◽  
Dimethyl Ester ◽  
Limited Proteolysis ◽  
Ribonuclease A ◽  
Enzymic Activity ◽  
S Protein ◽  
Hydrolysis Of

The dimethyl ester of bovine pancreatic ribonuclease-A (dimethyl RNAase-A), the initial product of esterification of RNAase-A in anhydrous methanolic HCl, was isolated in a homogeneous form. The two carboxy functions esterified in this derivative are those of glutamic acid-49 and aspartic acid-53. There were no changes in the u.v.-absorption spectral characteristics, the accessibility of the methionine residues, the resistance of the protein to proteolysis by trypsin and the antigenic behaviour of RNAase-A as a result of the esterification of these two carboxy groups. Dimethyl RNAase-A exhibited only 65% of the specific activity of RNAase-A, but still had the same Km value for both RNA and 2′:3′-cyclic CMP. However, the Vmax. was decreased by about 35%. On careful hydrolysis of the methyl ester groups at pH9.5, dimethyl RNAase-A was converted back into RNAase-A. Limited proteolysis of dimethyl RNAase-A by subtilisin resulted in the formation of an active RNAase-S-type derivative, namely dimethyl RNAase-S, which was chromatographically distinct from dimethyl RNAase-A and had very nearly the same enzymic activity as dimethyl RNAase-A. Fractionation of dimethyl RNAase-S by trichloroacetic acid yielded dimethyl RNAase-S-protein and dimethyl RNAase-S-peptide, both of which were inactive by themselves but regenerated dimethyl RNAase-S when mixed together. Dimethyl RNAase-A-peptide was identical with RNAase-S-peptide. RNAase-S-protein could be generated from dimethyl RNAase-S-protein by careful hydrolysis of the methyl ester groups at pH9.5. The interaction of dimethyl RNAase-S-protein with RNAase-S-peptide appears to be about 4-fold weaker than that between the RNAase-S-protein and RNAase-S-peptide. Conceivably, the binding of the S-peptide ‘tail’ of dimethyl RNAase-A with the remainder of the molecule is similarly weaker than that in RNAase-A, and this brings about subtle changes in the geometrical orientation of the active-site amino acid residues of these modified methyl ester derivatives. It is suggested that these changes could be responsible for the generation of the catalytically less-efficient RNAase-A and RNAase-S molecules (dimethyl RNAase-A and dimethyl RNAase-S respectively).

scholarly journals Aspartic Acid, Asparagine, Glutamic Acid, and Glutamine Contents of Wool and two Derived Protein Fractions

1971 ◽  
Vol 24 (3) ◽  
pp. 509 ◽  
Author(s):  
LA Holt ◽  
B Milligan ◽  
CM Roxburgh
Keyword(s):  
Glutamic Acid ◽  
Aspartic Acid ◽  
Leucine Aminopeptidase ◽  
Ribonuclease A ◽  
Protein Fractions ◽  
Hydrolysis Of ◽  
Successive Reduction

A method is described for the hydrolysis of wool which entails successive reduction, carboxymethylation, and digestion with the three enzymes, Pronase, prolidase, and leucine aminopeptidase. The reliability of the method has been checked using two proteins of known composition, viz. ribonuclease A and insulin.

scholarly journals Subtilisin modification of monodeamidated ribonuclease-A

1977 ◽  
Vol 165 (2) ◽  
pp. 337-345 ◽  
Author(s):  
B. N. Manjula ◽  
A. Seetharama Acharya ◽  
Paul J. Vithayathil
Keyword(s):  
Acid Treatment ◽  
Structural Changes ◽  
Limited Proteolysis ◽  
Ribonuclease A ◽  
Enzymic Activity ◽  
Initial Reaction ◽  
S Protein ◽  
Acidic Solutions ◽  
Peptide Region ◽  
Protein Part

Limited proteolysis of RNAase-Aa1 (monodeamidated ribonuclease-A) by subtilisin results in the formation of an active RNAase-S type of derivative, namely RNAase-Aa1S. RNAase-Aa1S was chromatographically distinct from RNAase-S, but exhibited very nearly the same enzymic activity, antigenic conformation and susceptibility to trypsin as did RNAase-S. Fractionation of RNAase-Aa1S by trichloroacetic acid yielded RNAase-Aa1S-protein and RNAase-Aa1S-peptide, both of which are inactive by themselves, but regenerate active RNAase-Aa1S′ when mixed together. RNAase-Aa1S-peptide was identical with RNAase-S-peptide, whereas the protein part was distinct from that of RNAase-S-protein. Titration of RNAase-Aa1S-protein with S-peptide exhibited slight but noticeably weaker binding of the peptide to the deamidated S-protein as compared with that of native protein. Unlike the subtilisin digestion of RNAase-A, which gives nearly 100% conversion into RNAase-S, the digestion of RNAase-Aa1 gives only a 50% conversion. The resistance of RNAase-Aa1 to further subtilisin modification after 50% conversion is apparently due to the interaction of RNAase-Aa1 with its subtilisin-modified product. RNAase-S was also found to undergo activity and structural changes in acidic solutions, similar to those of RNAase-A. The initial reaction product (RNAase-Sa1) isolated by chromatography was not homogeneous. Unlike the acid treatment of RNAase-A, which affected only the S-protein part, the acid treatment of RNAase-S affected both the S-protein and the S-peptide region of the molecule.

scholarly journals A spectrophotometric method for the microdetermination of periodate

1968 ◽  
Vol 108 (5) ◽  
pp. 883-887 ◽  
Author(s):  
Robert Fields ◽  
H. B. F. Dixon
Keyword(s):  
Methyl Ester ◽  
Spectrophotometric Method ◽  
Ribonuclease A ◽  
Sample Solution ◽  
S Protein

1. A method is described for measuring the concentration of periodate over the range 0·2–20μm by adding 1,2-di-(p-dimethylaminophenyl)ethane-1,2-diol to a sample solution. Periodate cleaves this compound to from two molecules of p-dimethylaminobenzaldehyde, the extinction of which is then read at 352mμ. 2. The method has been used to follow the course of periodate oxidations of serine methyl ester, ribonuclease A and ribonuclease S-protein. Addition of the reagent stops further periodate reaction by reducing the remaining periodate to iodate. 3. The presence of protein does not interfere with the assay.

Conversion of phenylalanine to tyrosine by microorganisms

1968 ◽  
Vol 14 (5) ◽  
pp. 573-578 ◽  
Author(s):  
P. Chandra ◽  
L. C. Vining
Keyword(s):  
Glutamic Acid ◽  
Aspartic Acid ◽  
Marine Bacterium ◽  
Specific Activities ◽  
Hydrolysis Of ◽  
Preferential Incorporation

Fourteen microorganisms of different genera were examined for their ability to convert L-phenylalanine directly to tyrosine. Comparison of the specific activities of phenylalanine, tyrosine, aspartic acid, and glutamic acid isolated after hydrolysis of cells grown in the presence of L-phenylalanine-U-14C indicated that p-hydroxylation of phenylalanine had occurred in all seven species of microfungi tested, and in the marine bacterium, Pseudomonas atlantica. In two basidiomycetes, two yeasts, an actinomycete, and a bacillus, there was no preferential incorporation of radioactivity into tyrosine.

Novel high-yield synthesis of .gamma. esters of glutamic acid and .beta. esters of aspartic acid by the copper-catalyzed hydrolysis of their diesters

1975 ◽  
Vol 40 (22) ◽  
pp. 3287-3288 ◽  
Author(s):  
R. L. Prestidge ◽  
D. R. K. Harding ◽  
J. E. Battersby ◽  
W. S. Hancock
Keyword(s):  
Glutamic Acid ◽  
Aspartic Acid ◽  
High Yield ◽  
Hydrolysis Of

Uptake of Dipeptides Containing Basic and Acidic Amino Acids by Rat Small Intestine in Vitro

1972 ◽  
Vol 43 (6) ◽  
pp. 823-837 ◽  
Author(s):  
D. Burston ◽  
Jill M. Addison ◽  
D. M. Matthews
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glutamic Acid ◽  
Aspartic Acid ◽  
Free Amino Acids ◽  
Free Amino ◽  
Tissue Uptake ◽  
Acidic Amino Acids ◽  
Hydrolysis Of

1. The characteristics of transport and hydrolysis of twenty-two dipeptides containing basic and acidic amino acids by rat ileal rings were investigated in vitro. The peptides included combinations of basic and neutral, basic and basic, basic and acidic, acidic and acidic, and acidic and neutral amino acids. 2. All peptides studied were removed intact from the bulk phase of the incubation medium, though, in general, only free amino acids appeared in the tissue. Uptake of one or both constituent amino acids was greater from the peptide than from the equivalent amino acid or amino acid mixture in the case of at least one peptide from each group and in eighteen of the twenty-two peptides studied. In general, there was no relationship between the extent of uptake of amino acids from peptides and the extent of their hydrolysis by the system. The results support the hypothesis that there is more than one mode of uptake of amino acids from peptides. 3. Hydrolysis of γ-glutamyl-l-glutamic acid by intact intestine or intestinal homogenate was slight, and intact peptide was taken up by the tissue. Uptake of free glutamic acid from this peptide was poor. Comparison of γ-glutamyl-l-glutamic acid with three other slowly hydrolysed dipeptides, glycyl-d-valine, sarcosylglycine and glycylsarcosine, suggested that all four were transported into the mucosal cells and hydrolysed intracellularly. The results indicate that the presence of a γ-linkage or a d-amino acid, or methylation of the free amino group as in sarcosylglycine, impair both transport and hydrolysis of peptide, but that attachment of a methyl group to the N of the peptide bond, as in glycylsarcosine, impairs hydrolysis but has no effect on peptide transport. 4. l-Aspartic acid and l-glutamic acid were extensively transaminated by the intestine, whether presented as free amino acids or in peptides. Evidence was obtained suggesting that production of alanine from aspartic acid resulted from direct transamination of aspartic acid with pyruvic acid, rather than from a sequence of two reactions involving aspartate and alanine aminotransferases. 5. The results show that more rapid uptake of amino acids from peptides than from free amino acids is not confined to peptides made up of neutral amino acids, and probably occurs with many small peptides. Uptake of lysine and the dicarboxylic amino acids, which are particularly slowly absorbed from free solution, was much greater from several dipeptides than from the free amino acids. The results suggest the importance of mucosal peptide uptake in protein absorption.

scholarly journals Bovine pepsinogens and pepsins. The sequence around a reactive aspartyl residue

1971 ◽  
Vol 124 (4) ◽  
pp. 673-676 ◽  
Author(s):  
Patricia A. Meitner
Keyword(s):  
Methyl Ester ◽  
Aspartic Acid ◽  
Specific Inhibitor ◽  
Enzymic Activity ◽  
Active Centre ◽  
Aspartic Acid Residue ◽  
Porcine Pepsin ◽  
Simultaneous Loss ◽  
Aspartyl Residue

The specific inhibitor, N-diazoacetylnorleucine methyl ester reacts stoicheiometrically with bovine pepsin resulting in a simultaneous loss of all enzymic activity. A peptide containing a modified aspartyl group was isolated from bovine pepsin labelled with 14C-labelled inhibitor. The aspartic acid residue is presumed to be part of the active centre and is in the same heptapeptide sequence as in porcine pepsin: Ile-Val-Asp-Thr-Gly-Thr-Ser.

An extracellular proteolytic enzyme from Scopalariopsis brevicaulis. II. Hydrolysis of poly amino acids

1972 ◽  
Vol 18 (7) ◽  
pp. 1165-1167 ◽  
Author(s):  
Kartar Singh ◽  
Claude Vézina
Keyword(s):  
Amino Acids ◽  
Glutamic Acid ◽  
Aspartic Acid ◽  
Proteolytic Enzyme ◽  
Ph Values ◽  
Optimum Ph ◽  
Extracellular Proteolytic Enzyme ◽  
Scopulariopsis Brevicaulis ◽  
Hydrolysis Of

Scopulariopsis brevicaulis protease hydrolyzed poly-L-lysine and poly-L-glutamic acid; optimum pH values for hydrolysis were 10.6 and 4.7 respectively. Final products of poly-L-lysine digestion by the protease were intermediate peptides from tetramer upwards. Pentalysine was not hydrolyzed by the enzyme. The protease had no action on poly-L-aspartic acid, poly-L-alanine, poly-L-glycine, poly-L-valine, or poly-L-leucine.

The Hydrolysis of Some N-Acylaspartic and N-Acylglutamic Monoamides in Dilute Mineral Acid

1975 ◽  
Vol 53 (11) ◽  
pp. 1137-1144 ◽  
Author(s):  
M. Ali ◽  
J. B. Capindale
Keyword(s):  
Hydrochloric Acid ◽  
Glutamic Acid ◽  
Aspartic Acid ◽  
Rate Constants ◽  
Mineral Acid ◽  
First Order ◽  
Order Rate ◽  
Succinamic Acid ◽  
Aqueous Hydrochloric Acid ◽  
Hydrolysis Of

The release of ammonia by hydrolysis of N-benzoyl-L-asparagine, glycyl-DL-asparagine, L-asparagine, and succinamic acid, and of aniline from N-benzoyl-L-glutamic-α-anilide, N-benzoyl-L-aspartic-α-anilide, L-aspartic-α-anilide, and the monoanilides of succinic and glutaric acids is first-order with respect to substrate in dilute (0.4–0.03 M) aqueous hydrochloric acid at 100 °C. The first-order rate constants (kobs) for these reactions can be expressed as kobs = kintra + k2[H+]. The above hydrolyses are used as models for developing a tentative mechanism to account for the selective release of aspartic acid from proteins under these conditions. The data are also used to suggest reasons why glutamic acid is not released with equal facility.

Sign in / Sign up

Export Citation Format

Share Document