Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

One of the arginine esteropeptidases in human submaxillary gland was purified from microsomal membranes. The enzyme is inactive in membranes and requires trypsin treatment for its full activation. The trypsin-activated enzyme was purified to homogeneity. Its molecular weight was determined to be 94,000 by SDS-polyacrylamide gel electrophoresis. Among various substrates examined, the obtained enzyme exhibited high specific activities toward Tos-Arg-OMe (esterolysis) and D-Ile-Pro-Arg-pNA (amidolysis). The enzyme was inhibited by some serine proteinase inhibitors, whereas inhibitors of other types of proteinases did not affect or only scarcely affected it. The enzyme appears to be distinct from other arginine esteropeptidases previously described.
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PMID:A new latent arginine esteropeptidase from human submaxillary gland. 187 81

We report identification, biochemical, clinical, and genetic studies of an apparently benign, electrophoretic variant of serum prealbumin (PALB, transthyretin, TTR) in a North American kindred of Swedish ancestry. The variant polypeptide stems from a C to T point mutation in exon 4 which results in methionine instead of threonine at position 119 of the mature molecule. It was discovered incidentally in a girl with classic alpha-1-anti-trypsin (A1AT) deficiency and her father during diagnostic A1AT phenotyping by ISO-DALT high-resolution two-dimensional electrophoresis (2DE). Twelve relatives in the four-generation paternal kindred, including five individuals who were heterozygous for the variant prealbumin, were studied. In each of these five heterozygotes, the variant allele product was equimolar and isoelectric with the normal protein, yet migrated with an apparently lower mass in the SDS-PAGE dimension. The inheritance pattern was consistent with autosomal dominant transmission. Histories and physical examinations showed no evidence of amyloidosis, as has been observed with other variants of prealbumin. Mean values of serum prealbumin and retinol binding protein levels were higher in the carriers as compared to the normal relatives in the family, but the difference was not statistically significant. Thyroid hormone levels and distribution of thyroxine and triiodothyronine among binding proteins in serum were within reference limits. Four members of the lineage had dominant, scalp-restricted keratinaceous cysts, yet only three of these four individuals had the variant. We counseled the family that this is likely a benign variant with regard to amyloidosis-related morbidity or shortened life span, although senile effects cannot be entirely ruled out. The provisional designation assigned to this allele is PALBCHICAGO. The substitution of methionine at position 119, as predicted by the DNA sequence, was confirmed by amino acid sequencing of CNBr and tryptic peptides. This substitution occurs at a CpG dinucleotide that may be a point mutational "hot spot," as has been postulated for the methionine-30 and isoleucine-122 PALB variants. The apparently lower mass of the variant probably results from a more compact conformation in SDS. With the exception of histidine-58, a charge substitution, all other amyloidosis-related prealbumin variant polypeptides had normal mobility in the ISO-DALT 2DE system.
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PMID:Biochemical and clinical characterization of prealbuminCHICAGO: an apparently benign variant of serum prealbumin (transthyretin) discovered with high-resolution two-dimensional electrophoresis. 187 23

To examine the polyamine effects on the fidelity at the aminoacylation level and the physiological significance of the existence of the aminoacyl-tRNA synthetase complex (ARSC) in animal cells, a single-chain Ile-tRNA synthetase (IRSS) was isolated from the complex by treatment with trypsin. Ile-tRNA formation by IRSS was strongly stimulated by spermine, similar to the results with ARSC. Two misacylations (Val-tRNAIle and Ile-tRNAiMet formation) by IRSS were measured. The error frequency was higher in Ile-tRNAiMet formation (tRNA misacylation) than in Val-tRNAIle formation (amino acid misacylation). Spermine did not influence significantly Ile-tRNAiMet formation, but it stimulated Val-tRNAIle formation by IRSS. Accordingly, spermine decreased the error frequency of tRNA misacylation, but not amino acid misacylation. These results suggest that the conformational changes of individual tRNA by spermine differ from each other, meaning that spermine influences the interaction between individual tRNA and aminoacyl-tRNA synthetase variously. When the aminoacylations of tRNAIle from rat liver, yeast, and Escherichia coli were compared with ARSC and IRSS, the relative speed of Ile-tRNA formation with tRNAIle from other species was faster with IRSS than with ARSC. This indicates that ARSC can recognize tRNAIle from the same species more specifically than IRSS. These results show that both spermine and ARSC are involved in the increase of fidelity of rat liver Ile-tRNA formation.
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PMID:Increase in fidelity of rat liver Ile-tRNA formation by both spermine and the aminoacyl-tRNA synthetase complex. 189 44

It has been shown that the P1 site (the center of the reactive site) of protease inhibitors corresponds to the specificity of the cognate protease, and consequently specificity of Streptomyces subtilisin inhibitor (SSI) can be altered by substitution of a single amino acid at the P1 site. In this paper, to investigate whether similar correlation between inhibitory activity of mutated SSI and substrate preference of protease is observed for subtilisin BPN', which has broad substrate specificity, a complete set of mutants of SSI at the reaction site P1 (position 73) was constructed by cassette and site-directed mutagenesis and their inhibitory activities toward subtilisin BPN' were measured. Mutated SSIs which have a polar (Ser, Thr, Gln, Asn), basic (Lys, Arg), or aromatic amino acid (Tyr, Phe, Trp, His), or Ala or Leu, at the P1 site showed almost the same strong inhibitory activity toward subtilisin as the wild type (Met) SSI. However, the inhibitory activity of SSI variants with an acidic (Glu, Asp), or a beta-branched aliphatic amino acid (Val, Ile), or Gly or Pro, at P1 was decreased. The values of the inhibitor constant (Ki) of mutated SSIs toward subtilisin BPN' were consistent with the substrate preference of subtilisin BPN'. A linear correlation was observed between log(1/Ki) of mutated SSIs and log(1/Km) of synthetic substrates. These results demonstrate that the inhibitory activities of P1 site mutants of SSI are linearly related to the substrate preference of subtilisin BPN', and indicate that the binding mode of the inhibitors with the protease may be similar to that of substrates, as in the case of trypsin and chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibition of subtilisin BPN' by reaction site P1 mutants of Streptomyces subtilisin inhibitor. 190 59

Sarcosine oxidase from Corynebacterium sp. U-96 is inhibited by iodoacetamide (IAM) and the inhibition is prevented by the substrate analog, sodium acetate. To elucidate the mechanism of inhibition of the enzyme by IAM, we determined the amino acid sequences around the IAM-reactive cysteine residues, and the effects of the modification on the enzyme activity and the oxidation-reduction of the FAD moieties of the enzyme. The enzyme was specifically labeled with [14C]IAM, and the labeled subunit B was digested with trypsin and chymotrypsin. The HPLC profiles of the proteolytic digests showed mainly two radioactive peaks. The 14C-labeled peptides were purified, and their N-terminal sequences were determined to be Cys-Gly-Thr-Pro-Gly-Ala-Gly-Tyr (TC-1) and Ala-Gly-Ile-Ala-Cys-Xaa-Asp-Xaa-Val-Ala(-)- (TC-2). Peptide TC-2 contains a covalent FAD-binding sequence [Asx-His-Val-Ala; Shiga et al. (1983) Biochem. Int., 6, 737]. [14C]IAM-incorporation into the TC-1 sequence was strongly inhibited by sodium acetate. The N-terminal amino acid sequence of the CNBr fragment containing the TC-1 sequence (65 residues) was determined. According to the secondary structure predictions, Gly-Thr-Pro-Gly-Ala-Gly of the TC-1 sequence is located between the beta sheet and alpha helix of the sequence, indicating the presence of an AMP-binding site in the TC-1 region. The activity of the enzyme treated with IAM in the presence and absence of sodium acetate was not inhibited by sodium sulfite, which is known to react specifically with covalent FAD.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cysteine residues in the active site of Corynebacterium sarcosine oxidase. 193 12

Singly modified soybean trypsin inhibitors (STIs), Tia* [Tia cleaved at Arg(63)-Ile(64)] and Tib* [Tib cleaved at Arg(63)-Ile(64)], were prepared by limited proteolysis with trypsin at pH 3.0. These singly modified inhibitors were further modified to yield doubly modified inhibitors, Tia** and Tib**, by limited proteolysis with subtilisin BPN', which cleaved the Met(84)-Leu(85) bonds of Tia* and Tib*, respectively. The doubly modified inhibitors could be separated into two parts: protein moiety A and peptide moiety a (IRFIAEGHPLSLKFDS-FAVIM) for Tia**, and protein moiety B and peptide moiety b (IRFIAEGNPLRLKFDS-FAVIM) for Tib**. These protein and peptide moieties showed no trypsin inhibitory activities alone. However, the inhibitors can be reconstituted through the mutual exchange of the protein and peptide moieties isolated from STIs. The reconstituted inhibitor which has tyrosine at position 62 and histidine at position 71 shows the highest inhibitory activity. Its Ki value for bovine trypsin is around 10(-10) M, which is almost the same as that of Tia for bovine trypsin. The inhibitor possessing either tyrosine at position 62 or histidine at position 71 exhibits a Ki value of around 10(-9) M, which is between those of Tia and Tib. The inhibitor having phenylalanine and asparagine at positions 62 and 71, respectively, shows the weakest inhibitory activity of around 10(-8) M similar to that of Tib for bovine trypsin.
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PMID:Reconstitution of new inhibitors through mutual exchange of Ile(64)-Met(84) peptides of soybean trypsin inhibitors, Tia and Tib. 193 14

Site-specific mutagenesis techniques have been used to construct active site variants of the Kunitz-type protease inhibitor domain present in the Alzheimer's beta-amyloid precursor protein (APP-KD). Striking alteration of its protease inhibitory properties were obtained when the putative P1 residue, arginine, was replaced with the small hydrophobic residue valine. The altered protein was no longer inhibitory toward bovine pancreatic trypsin, human Factor XIa, mouse epidermal growth factor-binding protein, or bovine chymotrypsin, all of which are strongly inhibited by the unaltered APP-KD (Sinha, S., Dovey, H. F., Seubert, P., Ward, P. J., Blacher, R. W., Blaber, M., Bradshaw, R. A., Arici, M., Mobley, W. C., and Lieberburg, I. (1990) J. Biol. Chem. 265, 8983-8985). Instead, the P1-Val-APP-KD was a potent inhibitor of human neutrophil elastase, with a Ki = 0.8 nM, as estimated by the inhibition of the activity of human neutrophil elastase measured using a chromogenic substrate. It also inhibited the degradation of insoluble elastin by the enzyme virtually stoichiometrically. Replacement of the P1' (Ala) and P2' (Met) residues of P1-Val-MKD with the corresponding residues (Ser, Ile) from alpha 1-proteinase inhibitor resulted in an inactive protein, underscoring the mechanistic differences between the serpins from the Kunitz-type protease inhibitor family. These results confirm the importance of the P1 arginine residue of APP-KD in determining inhibitory specificity, and are also the first time that a single amino acid replacement has been shown to generate a specific potent human neutrophil elastase inhibitor from a human KD sequence.
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PMID:Conversion of the Alzheimer's beta-amyloid precursor protein (APP) Kunitz domain into a potent human neutrophil elastase inhibitor. 193 50

The reactive sites and the C-terminal sequences of three trypsin chymotrypsin inhibitors from fenugreek seeds (TFI-B2, TFI-N2, and TFI-A8) were determined by chemical modification and carboxypeptidase degradation of native und enzymatically modified inhibitors. TFI-B2 contained lysine and leucine in the trypsin- and chymotrypsin-reactive sites, respectively, and -(Lys)-Phe-Leu-Ile was the C-terminal sequence. TFI-N2 possessed arginine and leucine in the trypsin- and chymotrypsin-reactive sites, respectively, and -(Tyr)-Lys-Ile-Leu at the C-terminus. TFI-A8 contained two arginines, one in each of the two reactive sites. At least one of these sites, although mainly directed against trypsin, could also bind some chymotrypsin. -(Leu)-Phe-Ile-Arg was found to be the C-terminus in TFI-A8. These results confirmed that all three fenugreek inhibitors belong to the Bowman-Birk proteinase inhibitor family.
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PMID:Inhibitors of human and bovine trypsin and chymotrypsin in fenugreek (Trigonella foenum-graecum L.) seeds. Reactive sites and C-terminal sequences. 196 6

The sesquiterpene antibiotic koningic acid (heptelidic acid) has been previously demonstrated to modify glyceraldehyde-3-phosphate dehydrogenase in specific manner, probably by binding to the sulfhydryl residue at the active site of the enzyme (Sakai, K., Hasumi, K. and Endo, A. (1988) Biochim. Biophys. Acta 952, 297-303). Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase labeled with [3H]koningic acid was digested with trypsin. Reverse-phase HPLC revealed that the label is associated exclusively with a tryptic peptide having 17 amino acid residues. Microsequencing and fast atom bombardment mass spectrometry demonstrated that the peptide has the sequence Ile-Var-Ser-Asn-Ala-Ser-Cys-Thr-Thr-Asn-Cys-Leu-Ala-Pro-Leu-Ala-Lys. In comparison to the amino acid sequence of glyceraldehyde-3-phosphate dehydrogenase from other species, this peptide is in a highly conserved region and is part of the active site of the enzyme. The cysteine residue corresponding to the Cys-149 in the pig muscle enzyme, which has been shown to be an essential residue for the enzyme activity, was shown to be the site modified by koningic acid. Structural analyses of the reaction product of koningic acid and L-cysteine suggested that the epoxide of koningic acid reacts with the sulfhydryl group of cysteine residue, resulting in a thioether.
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PMID:Identification of koningic acid (heptelidic acid)-modified site in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. 201 92

Tryptase was purified 13,000-fold to apparent homogeneity from rat skin. The two-step procedure involved ammonium sulfate fractionation of the initial extract followed by combined sequential affinity chromatography on agarose-glycyl-glycyl-p-aminobenzamidine and concanavalin A-agarose. The purified enzyme had a specific activity toward N-benzoylarginine ethyl ester (BzArgOEt) of 170 mumol/min mg-1 and was obtained in a yield of 28% as determined by the specific substrate, H-D-Ile-Pro-Arg-p-nitroanilide. Rat skin tryptase was thermal labile, losing 50% of its activity when preincubated for 30 min at 30 degrees C. The presence of NaCl (1 M) improved thermal stability and was necessary for long-term storage. Heparin did not stabilize the enzyme against thermal denaturation, and heparin-agarose failed to bind the enzyme. Rat skin tryptase was inhibited by diisopropylphosphofluoridate, antipain, leupeptin, and aprotinin but not by alpha 1-antitrypsin, ovomucoid, or soybean or lima bean trypsin inhibitors. Substrate specificity studies using a series of tri- and tetrapeptidyl-p-nitroanilide and peptidyl-7-amino-4-methylcoumarin substrates demonstrated the existence of an extended substrate binding site. Rat skin tryptase hydrolyzed [Arg8]vasopressin, neurotensin, and the oxidized B-chain of insulin at the -Arg8-Gly9-NH2, -Arg8-Arg9-, and -Arg22-Gly23-bonds, respectively. No general proteinase activity was observed toward casein, hemoglobin, or azocoll. Rat skin tryptase had a Mr of 145,000 by gel filtration. The subunit Mr was either 34,000 or 30,000 depending on the electrophoretic technique used. Treatment of the enzyme with peptide N-glycosidase F (N-glycanase) decreased the subunit Mr by 4000. The enzyme exhibited multiple isoelectric forms (pI's of 4.5-4.9). Rat skin tryptase was found to be related statistically to other tryptases on the basis of amino acid composition. The N-terminal amino acid sequence was Ile1-Val2-Gly3-Gly4-Gln5-Glu6-Ala7-+ ++Ser8-Gly9-Asn10-Lys11-Trp12-Pro13- Trp14- Gln15-Val16-Ser17-Leu18-Arg19-Val20- --21-Asp-22Thr23-Tyr24-Typ25-, with a putative glycosylation site at residue 21. This sequence was 72-80% homologous with the N-terminus of other tryptases but only 40% homologous with that of bovine trypsin.
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PMID:Tryptase from rat skin: purification and properties. 203 67


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