Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A protein methylase III responsible for specifically methylating the cytochrome c in Neurospora crassa was partially characterized by using unmethylated horse heart cytochrome c as a substrate. This enzyme utilizes S-adenosyl-L-methionine as the methyl donor. An analysis of the distribution of [14C]methyl groups in the peptides obtained by chymotrypsin digestion of the enzymically methylated cytochrome c showed that all of the radioactivity could be recovered within a single peak after chromatography. This indicates that the enzyme methylates a specific amino acid sequence within cytochrome c. On hydrolysis of the radioactive chymotryptic peptide, Me-14C-labelled epsilon -N-mono-methyl-lysine, epsilon-N-dimethyl-lysine and epsilon-N-trimethyl-lysine were identified. The enzyme can easily be extracted from the N. crassa mycelial pads and was purified approx. 30-fold.
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PMID:Cytochrome c-specific protein methylase III from Neurospora crassa. 19 92

The exposure of apolipoproteins at the surface of human plasma high density lipoproteins (HDL) was assessed by their accessibility to agarose-immobilized forms of trypsin and chymotrypsin. Proteolysis of lipid-free apolipoproteins and the lipoprotein subfractions HDL2 (d = 1.08--1.125 g/ml) and HDL3 (d = 1.125--1.195 g/ml) that differ in lipid-to-protein ratio was compared by polyacrylamide gel electrophoresis and isoelectric focusing of the apolipoproteins and peptide fragments and by quantitation of the various carboxyl-terminal groups formed. Gel filtration of the proteolyzed lipoproteins on Sephadex G-150 column indicated that more than 90% of the apolipoproteins and peptides remain associated with lipoprotein complexes. Proteolysis of lipoproteins occurred more slowly and with less fragmentation of the lipoproteins and apolipoproteins than proteolysis of thelipid-free apolipoproteins or the proteolysis of lipoproteins by soluble proteases reported by other investigators. The difference in lipid content of HDL2 and HDL3 made little difference in their proteolysis. Proteolysis of the lipoproteins by agarose-trypsin was more rapid at 37 degrees C than at 22 degrees C, but the proteolytic products were similar and differed from the products from the lipid free proteins. Peptide fragments from lipoproteins were larger than those from lipid-free proteins, which suggests masking of potentially cleavable groups by lipid. The amounts (mol/g protein) of new carboxyl-terminal tyrosine and phenylalanine released by agarose -chymotrypsin were much greater from the lipid-free proteins, but about 3/4 of the tryptophan residues were inacessible in both lipoproteins and lipid-free proteins. In agarose-trypsin digestion, lysine residues were slightly more masked than arginine in the absence of lipids and much more so in the lipoproteins. However, in the lipoproteins apoA-II, which contains lysine but no arginine, was cleaved more rapidly and extensively by agarose-trypsin than apoA-I.
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PMID:Surface exposure of apolipoproteins in high density lipoproteins. I. Reactivities with agarose-immobilized proteases. 20 44

The catalytic subunit of cyclic AMP-dependent protein kinase (from rabbit skeletal muscle; ATP:protein phosphotransferase, EC 2.7.1.37) was found to be irreversibly inactivated by chloromethyl ketone derivatives of lysine and phenylalanine, chemical reagents originally designed for labeling the active sites of the proteolytic enzymes trypsin and chymotrypsin. This inactivation was shown to occur at pH 7.5 and 22 degrees C, conditions under which chemically related alkylating reagents such as chloroacetamide and chloroacetic acid (which do not possess the amino acid side chain) fail to inactivate the enzyme. In the case of the chloromethyl ketone derivative of N alpha-tosyl-L-lysine, the enzyme could be protected by its nucleotide substrate (MgATP), by one of its protein substrates (histone H2b), and by its regulatory subunit which, upon binding, shields the active site of the catalytic subunit. Differential labeling experiments, together with kinetic studies of the rates of modification of the sulfhydryl groups in the enzyme before and after inactivation with the chloromethyl ketone, suggest that the loss of activity is associated with one (kinetically characterized) sulfhydryl group present either at the active site of the enzyme or at a site intimately associated with it. The general implications of these results regarding the interpretation of affinity labeling experiments carried out in complex mixtures of proteins or under in vivo conditions are discussed.
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PMID:Affinity labeling of the catalytic subunit of cyclic AMP-dependent protein kinase by N alpha-tosyl-L-lysine chloromethyl ketone. 22 53

Elastolytic enzyme was purified and crystallized from culture fluid of Flavobacterium immotum No. 9-35. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight was determined by Sephadex G-100 gel filtration to be 13,000. The isoelectric point was between pH 8.3 and 8.9. The optimum pH of the enzyme was 7.2 for elastolytic activity. The purified enzyme showed not only elastolytic activity, but also non-specific proteolytic activity against various other proteins. Milk-clotting activity was also observed. The enzyme did not act on keratin, collagen, or fourteen amino acid esters, including N-benzoyl-L-alanine methyl ester, N-benzoyl-L-arginine ethyl ester, and N-acetyl-L-tyrosine ethyl ester, which were typical substrates of pancreatic elastase [EC 3.4.21.11], trypsin [EC 3.4.21.4], and chymotrypsin [EC 3.4.21.1], respectively. However, the enzyme selectively hydrolyzed elastin when both elastin and albumin were present in the reaction mixture. The enzyme was inhibited by o-phenanthroline and various heavy metals such as cadmium, lead, zinc, and mercury. Various inhibitors, such as diisopropyl phosphofluoridate, tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalanine chloromethyl ketone, trypsin inhibitor, iodoacetamide, etc., had no effect on the elastolytic activity.
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PMID:Purification and properties of elastolytic enzyme from Flavobacterium immotum. 23 95

Kunitz bovine trypsin inhibitor gave with alpha-chymotrypsin a stoichiometric complex stable at neutral pH. The complex has been characteristized by amino acid composition, molecular sieving and zone electrophoresis. Complete dissociation occurred at pH 4.0 as shown by gel filtration, alpha-Chymotrypsin was displaced from the complex by trypsin either in solution or by affinity chromatography on trypsin-Sepharos: alpha-chymotrypsin was recovered in the filtrate (yield about 100%) and the inhibitor was eluted from trypsin-Sepharose with 0.1 M HCl (yield: 83%). Lysine-15 of the inhibitor was shown to be involved in the interaction between alpha-chymotrypsin and the inhibitor. When the complex was maleylated, the maleylated chymotrypsin-bound inhibitor was displaced by affinity chromatography on trypsin-Sepharose. Teh recovered derivative was oxidized, subjected to tryptic hydrolysis and the products separated by peptide mapping and analyzed. The peptides were compared with those obtained with non-maleylated inhibitor and fully maleylated free inhibitor. In the fully maleylated inhibitor, the four lysyl residues of the molecule were blocked but in the maleylated chymotrypsin-bound inhibitor, Lys-15 was unmodified in contrast to Lys-26, Lys-41 and Lys-46; therefore Lys-15 is shielded by chymotrypsin in the complex. On the other hand, when inhibitor with a selectively reduced carboxamidomethylated Cys-14-Cys-38 dislufide bridge was allowed to react with chymotrypsin, cleavage occurred not only at Tyr-21, Tyr-35 and Phe-45 but also at Lys-15, cleavage not observed in the case of the fully oxidized inhibitor. This result shows that under particular conditions the bond Lys-15-Ala-16 can be the substrate for chymotrypsin and the side chain of Lys-15 can be inserted in the chymotrypsin specificity pocket. Apparently the contact area of inhibitor with chymotrypsin seems to be similar to that with trypsin [J. Chauvet and R. Acher (1967) J. Biol. Chem. 242, 4274-4275].
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PMID:The reactive sites of Kunitz bovine-trypsin inhibitor. Role of lysine-15 in the interaction with chymotrypsin. 23 47

Chemical modifications of human plasma alpha1-antitrypsin with reagents which modify lysyl residues (citraconic anhydride, acetic anhydride, formaldehyde and 2,4,6-trinitrobenzenesulfonic acid) and arginyl residued (1,2-cyclohexanedione) were examined with regard to their effect upon the elastase inhibitory capacity of the glycoprotein. 2,4,6-Trinitrobenzenesulfonic acid was employed to quantitate the remaining free amino groups (epsilon-NH2 groups of lysine) and the extent of modifications. Amino acid analysis was utilized in the same capacity for the guanidino groups of arginyl residues. The elastase inhibitory capacity of alpha1-antitrypsin was destroyed following trinitrophenylation, citraconylation and acetylation. Circular dichroism of the native and modified derivatives revealed major changes in conformation following trinitrophenylation and citraconylation while CD profiles of acetylated and reductively methylated derivatives differed from that of the native profile considerably less. Reductively methylated alpha1-antitrypsin retained its elastatse inhibitory capacity. The reaction of 1,2-cyclohexanedione with alpha1-antitrypsin did not effect in a loss in inhibitory capacity. Gel filtration studies of native and modified alpha1-antitrypsin on Sephadex G-100 demonstrated an increased molecular weight presumably through molecular aggregation, in the citraconylated and trinitrophenylated derivatives, but not in the cases of the other derivatives. Based upon these studies and previous investigations of our laboratory, it was concluded that (1) alpha1-antitrypsin is a lysyl inhibitor type (i.e., the reactive site is a Lys-X bond), (2) its interaction with elastase follows a pattern similar to trypsin and chymotrypsin, and (3) the positively charged epsilon-NH2 group of lysine is essential for the maintenance of elastase inhibitory capacity.
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PMID:Circular dichroism of chemically modified human plasma alpha1-antitrypsin. Interaction with porcine elastase. 31 Mar 16

The release of a peptide (molecular weight: about 3,600) was observed during complex formation between human alpha 1-antitrypsin (alpha 1-AT) and bovine alpha-chymotrypsin, when monitored by gel-electrophoresis in the presence of sodium lauryl sulfate. Release of the peptide was proportional to the extent of complex formation. Peptides of the same molecular weight were also released during the complex formation of alpha 1-AT with bovine trypsin or porcine elastase. The peptide released from the complex with bovine alpha-chymotrypsin was composed of 32 amino acid residues, which did not correspond to the composition of any 32 amino acid segment in the bovine alpha-chymotrypsin sequence. The N- and C-terminal sequences of the peptide were determined to be H-(Ser)-Ile-Pro-Pro-Glu- and -Gln-Lys-OH, respectively. Though there was some uncertainty as to the N-terminal sequence, it is quite different from that of the original alpha-AT molecule, and showed a similarity to the sequences of the leaving group sides of the reactive sites in some legume proteinase inhibitors. The C-terminal 2 residues were identical with those of native alpha 1-AT. These results suggest that the peptide was released from the C-terminal region of alpha 1-AT uon interaction with alpha-chymotrypsin. It is tempting to suggest that alpha 1-AT inhibits a serine proteinase by the acyl enzyme mechanism at a residue adjacent to the amino group of the N-terminus of this peptide and that this peptide is liberated as a leaving group in the enzymic process.
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PMID:Characterization of a peptide released during the reaction of human alpha 1-antitrypsin and bovine alpha-chymotrypsin. 31 7

1. The reactivities of phenylglyoxal (PGO), glyoxal (GO), and/or methylglyoxal (MGO) with several proteins, including ribonuclease A [EC 3.1.4.22] and its derivatives, alpha-chymotrypsin [EC 3.4.21.1], trypsin [EC 3.4.21.4], lysozyme [EC 3.2.1.17], pepsin [EC 3.4.23.1], rennin [EC 3.4.23.4], thermolysin, and insulin and its B chain, have been examined. From analyses of the reaction products, PGO was shown to be the most specific for arginine residues. GO and MGO also reacted rapidly with arginine residues, but they also reacted with lysine residues to a significant extent. A side reaction with N-terminal alpha-amino groups was observed with each of these reagents. 2. Two arginine residues out of four in ribonuclease A, two out of three in alpha-chymotrypsin, one out of two in trypsin, one out of two in pepsin, and one out of five in rennin appeared to react with PGO fairly rapidly, indicating a difference in the relative accessibility of these residues by the reagent. Extensive modification of the arginine residues by PGO occurred with RCM-derivatives of ribonuclease A and insulin B chain. The N-terminal isoleucine residues of alpha-chymotrypsin and trypsin appeared to be unreactive with PGO because of salt bridge formation with an aspartyl residue. The activity of alpha-chymotrypsin toward N-benzoyl-L-tyrosine ethyl ester and the lytic activity of lysozyme were lost rapidly on treatment with PGO, as in the case of ribonuclease A. Pepsin and rennin were only partially inactivated by reaction with PGO.
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PMID:Further studies on the reactions of phenylglyoxal and related reagents with proteins. 32 41

This paper presents the experimental details which led to the elucidation of the complete primary structure of S16, a protein which belongs to the small subunit of E. coli ribosomes. Protein S16 was digested with trypsin, alpha-chymotrypsin, and the staphylococcal protease. The resulting peptides were purified on paper and their amino acid composition and sequence were determined. Automatic Edman degradation with a modified sequenator on the complete protein yielded information from the 56N-terminal residues. The combination of all these results led to the following complete amino acid sequence: Met-Val-Thr-Ile-Arg-Leu-Ala-Arg-His-Gly-Ala-Lys-Lys-Arg-Pro-Phe-Tyr-Gln-Val-Val-Val-Ala-Asp-Ser-Arg--Asn-Ala-Arg-Asn-Gly-Arg-Phe-Ile-Glu-Arg-Val-Gly-Phe-Phe-Asn-Pro-Ile-Ala-Ser-Glu-Lys-Glu-Glu-Gly-Thr-Arg-Leu-Asp-Leu-Asp-Arg-Ile-Ala-His-Trp-Val-Gly-Gln-Gly-Ala-Thr-Ile-Ser-Asp-Arg-Val-Ala-Ala-Leu-Ile-Lys-Glu-Val-Asn-Lys-Ala-Ala. The molecular weight derived from the sequence amounts to 9 162.
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PMID:The complete amino acid sequence of protein S16 from Escherichia coli. 33 10

The amino acid sequence of the heavy-chain variable region of the human immunoglobulin. New has been determined. Since the amino terminus of the heavy chain was blocked, the sequence of residues 1-69 was established by digesting the appropriate CNBr fragment separately with trypsin, chymotrypsin, and thermolysin and sequencing the resulting peptides. The region from residues 70 to 120 was present in another CNBr fragment which was submitted directly to automatic Edman degradation. The result of this experiment extended the sequence to residue 100. The primary structure of the remaining portion of the VH region was determined by automatic Edman degradation of a lysine-blocked tryptic peptide derived from this region which included residues 98-214. The sequence of the VH region of New corresponds most closely to VH sequences of proteins in the VH II subgroup. This primary structure makes it possible to construct a model from the high-resolution electron-density map of protein New.
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PMID:Amino acid sequence of the VH region of a human myeloma immunoglobulin (IgG New). 40 27


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