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)

The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) was previously shown to be an irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase, and MgATP protected against inactivation [Toner-Webb, J., & Taylor, S. S. (1987) Biochemistry 26, 7371]. This inhibition by DCCD indicated that an essential carboxyl group was present at the active site of the enzyme even though identification of that carboxyl group was not possible. This presumably was because a nucleophile on the protein cross-linked to the electrophilic intermediate formed when the carbodiimide reacted with the carboxyl group. To circumvent this problem, the catalytic subunit first was treated with acetic anhydride to block accessible lysine residues, thus preventing intramolecular cross-linking. The DCCD reaction then was carried out in the presence of [14C]glycine ethyl ester in order to trap any electrophilic intermediates that were generated by DCCD. The modified protein was treated with trypsin, and the resulting peptides were separated by HPLC. Two major radioactive peptides were isolated as well as one minor peptide. MgATP protected all three peptides from covalent modification. The two major peaks contained the same modified carboxyl group, which corresponded to Asp-184. The minor peak contained a modified glutamic acid, Glu-91. Both of these acidic residues are conserved in all protein kinases, which is consistent with their playing essential roles. The positions of Asp-184 and Glu-91 have been correlated with the overall domain structure of the molecule. Asp-184 may participate as a general base catalyst at the active site. A third carboxyl group, Glu-230, also was identified.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Identification of aspartate-184 as an essential residue in the catalytic subunit of cAMP-dependent protein kinase. 290 66

The aspartic acid residue at the bottom of the substrate-binding pocket of trypsin was replaced by glutamic acid through site-directed mutagenesis. The wild-type (Asp-189) and mutant (Glu-189) trypsinogens were expressed in E. coli, purified to homogeneity, activated by enterokinase, and tested on a series of fluorogenic tetrapeptide substrates. The substrates were of the general formula succinyl-Ala-Ala-Pro-X-AMC, where AMC is 7-amino-4-methylcoumarin and X is Lys, Arg, or Orn (ornithine). As compared to Asp-189 trypsin, the activity of Glu-189 trypsin on lysyl and arginyl substrates decreased by 3-4 orders of magnitude while its Km values did not significantly change. Lengthening the side-chain of Asp-189 by one methylene group could not be compensated for by shortening the side-chain of the substrate, since Glu-189 trypsin had no measurable activity on the ornithyl substrate. The replacement of Asp-189 with glutamic acid at the base of the substrate-binding pocket of trypsin appears to distort the structure of the critical transition-state complex. This could happen by disrupting interactions normally associated with Asp-189, and by altering the relative position of the scissile peptide bond in the active site of the enzyme.
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PMID:Structural and functional integrity of specificity and catalytic sites of trypsin. 290 52

A cDNA that encodes what appears to be the inhibitory domain of the plasma membrane calcium-pumping ATPase (Ca2+-ATPase) has been isolated by screening a lambda gt11 bovine brain cDNA library with antibodies prepared against the human erythrocyte membrane Ca2+-ATPase. This screening resulted in isolation of a bacteriophage containing a 1.5-kilobase cDNA insert encoding a 71-residue polypeptide, the remainder being a large 3' terminal noncoding region. A portion of this deduced peptide sequence was identical to that of a peptide isolated from a V8 protease digest of the human erythrocyte Ca2+-ATPase except for 1 residue. Antibodies purified by immunoabsorption to the fusion protein containing this cDNA-encoded polypeptide reacted only with those fragments of a limited trypsin digest of the human erythrocyte Ca2+-ATPase that contain the inhibitory domain. Moreover, these antibodies were able to partially stimulate basal enzyme activity and block further activation by calmodulin. The encoded polypeptide bears homology to the glutamic acid-rich regions N-terminal to the Ca2+-binding loops of calmodulin and to a lesser extent with the loops themselves. This encoded polypeptide also represents the C terminus of the Ca2+-ATPase. Portions of the isolated cDNA were homologous to the 3' noncoding region of the sarcoplasmic reticulum Ca2+-ATPase cDNA, indicating a possible mechanism for the evolution of these distinct membrane Ca2+ pumps.
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PMID:A C-terminal, calmodulin-like regulatory domain from the plasma membrane Ca2+-pumping ATPase. 296 97

Enzymatic formation of acid-stable trypsin-plasmin inhibitors (ASTPIs) in human plasma with several proteinases, particularly SH-proteinases, was demonstrated. The maximal activity obtained with bromelain was 40 U/ml plasma, which corresponded to about a 10-fold increase as compared to the untreated control plasma (4.2 U/ml). Gel filtration revealed at least two ASTPI activity peaks of molecular weight 16,000 (main peak) and 8000 (minor peak). The main ASTPI was further purified by trypsin-Sepharose affinity chromatography, isoelectric focusing and gel filtration on Sephadex G-75 superfine. The purified inhibitor was found to be identical to the active fragment of plasma ASTPI or urinary trypsin inhibitor (UTI) formed by bromelain treatment. It had an isoelectric point (pI) of 3.7, a molecular weight of 16,000 by SDS-polyacrylamide gel electrophoresis and was a glycine- and glutamic acid-rich protein lacking histidine. The NH2-terminal amino acid sequence was H2N-(Lys)-Glu-Asp-Ser-X-Gln-Leu-Gly-Tyr-Ser-Ala-Gly-Pro-X-Met-Gly-Met-Th r-X-Arg - Tyr-Phe-Tyr-... COOH, which was homologous to the Lys22-Met36 part (or Glu23-Met36 part; 30% of the total) of the plasma ASTPI or UTI molecule (molecular weight 70,000-80,000 by gel filtration). The purified ASTPI displayed the same antigenicity as UTI and exerted strong inhibitory effects on trypsin, chymotrypsin and plasmin amidolysis, but had a much lesser effect on plasmin fibrinolysis. It also strongly inhibited non-plasmic fibrinolysis with human leukocyte proteinase and earthworm proteinase.
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PMID:Acid-stable trypsin-plasmin inhibitors formed enzymatically from plasma precursor protein. 296 15

A glutamic acid residue at the active-site of bovine lung angiotensin I-converting enzyme was esterified with p-[N,N-bis-(chloroethyl)amino]phenylbutyryl-L-[U-14]-Proline (chlorambucyl-L-[U-14C]-L-Proline), an affinity label for this enzyme. The radiolabeled enzyme was digested with BrCN and only 1 of the 30 cleavage peptides resolved by reverse-phase HPLC contained the bound radiolabel. This active-site peptide (Mr approximately 16,000) was digested with trypsin, and the labeled peptide (T-2) was further degraded with thermolysin. The enzyme digest peptides were also resolved by reverse-phase HPLC. Only 1 of the 5 peptides obtained after thermolysin digestion (Th-1, Mr 1290) contained the bound radiolabel. Th-1 (12 residues) was subjected to manual Edman degradation and the following partial sequence was determined: H2N-Phe-Thr-Glu-Leu-Ala-Asp-Ser-Glu. The radiolabel was released at cycle 3 and the amount recovered was equivalent to the amount of PTH-Glu detected on HPLC. Thus, glutamic acid is esterified with chlorambucyl-L-[U-14C]-Proline which confirms our earlier findings. The sequence that we determined is homologous in five residues with the corresponding sequences of carboxypeptidase A and B, two other mammalian zinc-proteases. There is little sequence homology with thermolysin, a bacterial zinc-protease that also contains an essential active-site glutamic acid residue.
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PMID:Isolation and sequencing of an active-site peptide from angiotensin I-converting enzyme. 302 71

The UP1 single-stranded nucleic acid binding protein from calf thymus (Herrick, G. & Alberts, B.M. (1976) J. Biol. Chem. 251, 2124-2132) has recently been shown to be a proteolytic fragment derived from the A1 heterogeneous nuclear ribonucleoprotein (hnRNP) (Pandolfo et al. (1985) Nucleic Acids Res. 13, 6577-6590). The NH2-terminus of the 22,162 dalton UP1 protein appears to be blocked, which suggests that UP1 represents the NH2-terminal two thirds of this 32,000 dalton hnRNP protein. The complete amino acid sequence for UP1 was derived from automated sequencing of peptides that were purified by HPLC from digests with trypsin, chymotrypsin, Staphylococcus aureus protease, endoproteinase Lys-C, and cyanogen bromide. Trichloroacetic acid precipitation followed by enzymatic digestion in 2 M urea proved to be the best approach for generating UP1 peptides. By carboxymethylating after, rather than before, digestion it was possible to avoid problems associated with the insolubility of the carboxymethylated UP1. All of the resulting peptides in amounts varying from 2 to 15 nmol were coupled to aminopolystyrene prior to solid-phase sequencing. Using these methods, no difficulties were encountered in assigning glutamic acid residues or in completely sequencing peptides that contained up to 25-30 residues. The relative ease with which the UP1 protein was sequenced, requiring only about a year to complete, and the comparatively modest amount of protein required, less than 5 mg, attests to the usefulness of water soluble carbodiimide coupling and solid-phase sequencing for determining the primary structures of proteins. In addition to serving as a basis for determining structural relationships among various mammalian single-stranded nucleic acid binding proteins, the amino acid sequence of UP1 reveals that the A1 hnRNP protein contains a region of internal sequence homology that apparently corresponds to two independent nucleic acid binding sites.
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PMID:Amino acid sequence of UP1, an hnRNP-derived single-stranded nucleic acid binding protein from calf thymus. 303 34

1. The cytosolic and mitochondrial fumarases (EC 4.2.1.2) from baker's yeast (Saccharomyces cerevisiae) have been purified to homogeneity. 2. Subunit molecular weights for the cytosolic and mitochondrial isoenzymes were 53,000 and 48,000 respectively. 3. Peptide maps obtained after digestion of the two isoenzymes with trypsin were almost identical but showed significant small differences. The same was true of peptide maps obtained after digestion with the glutamic acid-specific proteinase from S. aureus.
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PMID:Purification and structural comparisons of the cytosolic and mitochondrial fumarases from baker's yeast. 307 79

We have previously reported that pS2 mRNA expressed in cultured epithelial cells derived from a hormone-dependent breast carcinoma (MCF-7 cells) is also expressed in mucosa cells of normal human stomach. This mRNA encodes a putative 84 amino-acid-long protein, which is secreted by both cell types after elimination of a signal peptide. We report here the purification of the pS2 protein, its trypsin digestion and amino-acid sequencing. The MCF-7 cell-secreted protein is 60 amino-acid-long and its sequence is in complete agreement with that deduced from the mRNA sequence. The presence of an N-terminal glutamic acid indicates that the signal peptidase releases a 24 amino-acid-long signal peptide. Analysis of tryptic peptides derived from the secreted gastric pS2 protein indicates that the signal peptide and the sequence of the first 48 amino-acids are identical to those of secreted MCF-7 pS2 protein, although the N-terminal amino-acid of the gastric protein may be cyclized as a pyroglumatic acid.
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PMID:[Primary structure of human protein pS2]. 314 13

Carotenoid-carrying lipoprotein (CCL) was rapidly isolated from the high density lipoprotein (HDL) fraction of the upstream migrating male chum salmon (Oncorhynchus keta) by a single-step density gradient ultracentrifugation. The two apolipoproteins (Mr = 24,000 and 12,000; designated apo-I and apo-II, respectively) were readily dissociated and separated in 0.1% SDS by gel filtration chromatography. Prominent features of the amino acid composition in the CCL included the relative high levels of glutamic acid, alanine, leucine, and lysine, and the low cysteine content. Apo-I, as well as the CCL, was rich in glutamic acid, alanine, leucine, and lysine. Compared to the amino acid composition of apo-I, apo-II included relatively high levels of glycine and tyrosine, and low threonine, serine, and arginine contents. When the intact CCL particle was treated with trypsin, apo-I was rapidly proteolyzed, while apo-II was resistant. However, both apo-I and apo-II isolated from the CCL particle were readily digested with trypsin. This suggested that a different structural arrangement rather than the amino acid compositions of the apolipoproteins was associated with the limited trypsin digestion of the CCL particle. Apo-II may be sheltered from the aqueous environment and lie partly within the CCL particle. The properties of both the HDL fraction and apolipoproteins from pink salmon (Oncorhynchus gorbuscha) were similar to those of the CCL from chum salmon.
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PMID:Isolation of apolipoproteins from carotenoid-carrying lipoprotein in the serum of chum salmon, Oncorhynchus keta. 323 13

Human lung lavage proteins were fractionated by centrifugation and molecular sieving. An antiserum to the post-albumin fraction of the soluble proteins reacted with a 10 KD protein and this protein was isolated by conventional chromatography. The protein, which has a pI of 4.8, consists of two 5 KD polypeptides and is rich in glutamic acid, leucine, serine, and aspartic acid amino acids. The protein does not bind to concanavalin A, pancreatic elastase, leukocyte elastase, or trypsin, and lacks anti-protease activity. It constitutes about 0.15% of the soluble proteins in lung lavage. Antibodies to the 10 KD protein specifically and exclusively stain Clara cells in human, dog, and rat. Staining of granules of Clara cells was prominent in the distal bronchioles; however, the non-ciliated cells of respiratory bronchioles did not stain for the 10 KD protein. This 10 KD protein appears in fetal lungs at 21 weeks of gestation, and was present in about 10% of the primary pulmonary adenocarcinomas. As a specific marker for Clara cells, this protein could be useful in the study of development, regulation of secretion, and pathobiology of these cells.
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PMID:Identification, cellular localization, isolation, and characterization of human Clara cell-specific 10 KD protein. 327 12


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