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 complete amino acid sequence of winged bean basic agglutinin (WBA I) was obtained by a combination of manual and gas-phase sequencing methods. Peptide fragments for sequence analyses were obtained by enzymatic cleavages using trypsin and Staphylococcus aureus V8 endoproteinase and by chemical cleavages using iodosobenzoic acid, hydroxylamine, and formic acid. COOH-terminal sequence analysis of WBA I and other peptides was performed using carboxypeptidase Y. The primary structure of WBA I was homologous to those of other legume lectins and more so to Erythrina corallodendron. Interestingly, the sequence shows remarkable identities in the regions involved in the association of the two monomers of E. corallodendron lectin. Other conserved regions are the double metal-binding site and residues contributing to the formation of the hydrophobic cavity and the carbohydrate-binding site. Chemical modification studies both in the presence and absence of N-acetylgalactosamine together with sequence analyses of tryptophan-containing tryptic peptides demonstrate that tryptophan 133 is involved in the binding of carbohydrate ligands by the lectin. The location of tryptophan 133 at the active center of WBA I for the first time subserves to explain a role for one of the most conserved residues in legume lectins.
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PMID:Amino acid sequence of the winged bean (Psophocarpus tetragonolobus) basic lectin. Adenine binding and identification of the active-site tryptophan residue. 798 25

The membrane-bound enzyme 3 beta-hydroxysteroid dehydrogenase delta 5-4 isomerase (3 beta-HSD) catalyzes the formation of delta 4-3-ketosteroids from delta 5-3 beta-hydroxysteroids in placental, adrenal, testicular and ovarian tissues. In the present study was investigated the transverse-plane topography of 3 beta-HSD within the human placental microsome membranes employing immune-replica analysis in combination with surface specific proteolysis. The crucial domains of the enzyme for the dehydrogenase and isomerase reactions are inactivated by proteinase treatments under conditions where latency of hexose-6-phosphate dehydrogenase was 95%. The data indicate that these crucial domains face the cytosolic side of the endoplasmic reticulum membrane. Incubation of the intact microsomes with trypsin produces several immune reactive fragments ranging from 29 to 11 kDa in addition to 42 kDa native enzyme, one of them being shielded by the membrane structure and/or by other intrinsic and peripheral membrane proteins. Carboxypeptidase Y degraded the C terminus of the 42 kDa native 3 beta-HSD in intact and detergent-disrupted microsomes, preserving partially a fragment of 31 kDa. The results from the carboxypeptidase Y digestion indicate that the carboxy terminal end of the 3 beta-HSD enzyme is located on the cytoplasmic surface of the endoplasmic reticulum and that only a small fragment of approx. 11 kDa could be removed easily without affecting the enzyme activity. From these data and the predicted hydropathy analysis from the literature, we tried to assign a transmembrane arrangement to the human placental 3 beta-HSD. Our results support a topology model in which practically all the structural 3 beta-HSD enzyme is exposed to the cytoplasmic side of the membrane with one NH2-terminal-anchoring segment and all the 3 beta-HSD enzyme activity facing to the cytoplasmic side within the 31 kDa NH2-terminal peptide.
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PMID:Topography of human placental 3 beta-hydroxysteroid dehydrogenase/delta 5-4 isomerase in microsomal membrane. A study using limited proteolysis and immunoblotting. 804 98

White kidney beans (Phaseolus vulgaris), cv Processor, contain a relatively high content of phaseolin (storage protein), lectins and a special group of glycoproteins as well as a considerable amount of protein-type trypsin inhibitors. Protein digestion of raw 'Processor' beans in monogastrics, for example pigs, is disturbed by poorly digested, phaseolin lectins, which can bind to carbohydrates in brush border membranes of the small intestinal epithelium, and trypsin inhibitors. The effect of the germination of white kidney beans on lectins, phaseolin and trypsin inhibitors was studied in order to achieve a degradation of lectins, phaseolin and trypsin inhibitors and an increase of in vitro enzymatic hydrolysis of the protein of bean flour. Therefore, whole bean extracts were examined throughout a germination period of up to seven days for their lectin and phaseolin pattern, lectin content, binding capacities of functional lectins towards brush border membranes and trypsin inhibitor content. In addition the in vitro enzymatic hydrolysis by pepsin and pancreatin of the protein from flours of (un)germinated white kidney beans was studied. SDS-PAGE demonstrated a degradation of E-lectins and a disappearance of L-lectins and phaseolin during germination. Results indicated a decrease of the lectin content by 85%, a loss of binding capacities of functional lectins towards brush border membranes by 91%, and a decrease of trypsin inhibitors by 76%, in bean flour after germination for seven days. A maximum in in vitro enzymatic hydrolysis of protein from bean flour was already established after germination for half a day.
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PMID:The degradation of lectins, phaseolin and trypsin inhibitors during germination of white kidney beans, Phaseolus vulgaris L. 805 78

Titration of Escherichia coli DNA topoisomerase I with PMPS and 65Zn(II) binding showed independent release and binding of the three Zn(II) in each enzyme molecule. Removal of Zn(II) from topoisomerase I or top85 (truncated topoisomerase I with the Zn(II) binding domain at the carboxyl terminal) affected their sensitivity to Glu-C and Asp-N endoproteases but there was no significant effect on their rate of proteolysis by trypsin or Lys-C endoprotease. This suggested that Zn(II) removal did not result in complete unfolding of topoisomerase enzyme structure but only affected folding of small local regions. Digestion with carboxypeptidase Y further demonstrated that the folding of the zinc binding region itself was altered upon Zn(II) removal.
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PMID:Binding of Zn(II) to Escherichia coli DNA topoisomerase I. 808 Dec 8

A purification procedure for guanylate kinase from pig brain has been developed consisting of ammonium sulfate precipitation and heptane extraction of the crude extract, hydrophobic-interaction chromatography, affinity chromatography and chromatofocussing. From 1.75 kg pig brain, 1.2 mg enzyme was isolated with a yield of 18% and a purity of about 90%. For sequence determination, the protein was cleaved with trypsin, cyanogen bromide and endoproteinase Glu-C. Some of the isolated peptides were subcleaved with chymotrypsin, thermolysin or trifluoroacetic acid. The blocked N-terminus was analyzed by mass spectrometry and by amino acid analysis of a tryptic peptide, while the C-terminus was found in a tryptic and a chymotryptic peptide and confirmed by a carboxypeptidase Y digestion. The sequence contains 197 amino acids with a M(r) of 21,831, one tryptophan and one cysteine residue. It has been compared to those of the homologous enzymes of yeast and Escherichia coli, as well as to proteins from sequence data banks that show similarities. The sequence is discussed in the light of the known spatial structure of yeast guanylate kinase.
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PMID:Purification and sequence determination of guanylate kinase from pig brain. 809 61

Digestion of renal Na/K-ATPase with trypsin, in the presence of rubidium and absence of calcium ions, produces so-called "19-kDa membranes," containing a C-terminal 19-kDa and smaller fragments (8-12 kDa) of the alpha chain, and a beta chain either intact or split into two fragments (Karlish, S. J. D., Goldshleger, R., and Stein, W.D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4566-4570). Cation occlusion is intact. The cation sites are thought to be located within trans-membrane segments, but the identity and number of segments involved is unknown. Analysis of Ca(2+)-induced sensitization of 19-kDa membranes to proteolysis, and characterization of the limit membrane-embedded fragments, has provided some insight into this question. Calcium ions have been shown to compete with two rubidium ions for occlusion sites on 19-kDa membranes, with a high affinity (KD approximately 2.8 microM, pH 7.5, 20 degrees C). The kinetics of displacement of rubidium by calcium ions indicate that competition is direct and is not an allosteric antagonism. At 37 degrees C, reversible displacement of rubidium ions by calcium ions is followed by an irreversible thermal inactivation of rubidium occlusion. Calcium ions partially protect rubidium occlusion sites against modification by the carboxyl reagent, N,N'-dicyclohexylcarbodiimide. We propose that calcium ions, like rubidium ions, recognize carboxyl groups at the entrance to the cation sites, but the calcium ions do not become occluded and thus fail to protect 19-kDa membranes against further proteolysis or thermal inactivation. Upon displacement of occluded rubidium, trypsin digests the Ca(2+)-bound and thermally inactivated 19-kDa membranes, and all of the membrane-embedded fragments are truncated or are split in these conditions. A related finding is that the C-terminal sequence of the 19-kDa fragment (and alpha chain), E-T-Y-Y, is digested by carboxypeptidase Y only when the rubidium occlusion is inactivated. Identification of the limit tryptic fragments indicates that polypeptide loops and the C-terminal tail of the 19-kDa fragment, N and C termini of the smaller fragments of the alpha chain, and both N and C termini of a 16-kDa fragment of the beta chain are split by proteolytic enzymes upon displacement of occluded rubidium.4+ We conclude that all fragments of 19-kDa membranes form a complex, which is stabilized and protected against proteolytic enzymes upon occlusion of rubidium ions, and which relaxes upon displacement of occluded rubidium. The cation occlusion "cage" presumably consists of litigating groups from several trans-membrane segments.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Evidence that the cation occlusion domain of Na/K-ATPase consists of a complex of membrane-spanning segments. Analysis of limit membrane-embedded tryptic fragments. 814 67

The carboxyl function of pepstatin has been coupled, through an amide bond, to methoxypoly(ethylene glycol) (5 kDa), to which an amino function had been previously grafted. The mPEG-pepstatin conjugate inhibits hog pepsin (aspartic proteinase) in vitro as pepstatin itself, however, with a 400 times higher apparent Ki. The conjugate apparently does not inhibit proteinases belonging to other proteinase families such as serine (trypsin, carboxypeptidase Y), cysteine (Papaya proteinase III), or metallo (collagenase) proteinases.
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PMID:Preparation and preliminary characterization of poly(ethylene glycol)-pepstatin conjugate. 820 68

Parabiosis and cross-circulation experiments with spontaneously hypertensive and normotensive rats gave indications for a previously unidentified circulating hypertensive agent. In this study, plasma from normotensive and hypertensive rats was fractionated and the vasopressor action of the corresponding fractions was measured in the isolated perfused rat kidney. One of three vasoactive fractions obtained by gel filtration (Biol-Gel P2) from hypertensive rats showed a significantly higher activity (increase in perfusion pressure by 1502.9 +/- 438.9 Pa) than that from normotensive rats (increase in perfusion pressure by 505.4 +/- 186.2 Pa, P < 0.01). Further chromatographic separations of this fraction revealed that the hypertensive factor is hydrophilic and has no ionic groups or vicinal diol groups. The molecular mass was estimated by dialysis and the matrix-assisted laser desorption/ionization mass spectrometry to be in the range of 1 kDa. The vasopressor is heat resistant and not degradable with trypsin or carboxypeptidase Y. The vasopressor action was not inhibited with the angiotensin-II-receptor antagonist saralasin, the alpha-receptor antagonist phentolamine, the thromboxane-receptor antagonist carbocyclic thromboxane A2 or the serotonin antagonist ketanserin. The results confirm the existence of a vasopressor factor in the plasma of hypertensive rats and, in a lower concentration, of normotensive rats, which is possibly related to the pathogenesis of essential hypertension. The chromatographic behavior suggests that this factor is different from the parathyroid hypertensive factor described recently.
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PMID:Partial purification and characterization of a circulating hypertensive factor in spontaneously hypertensive rats. 824 78

The major proteinase in maize (Zea mays) roots behaves as a serine endopeptidase. A possible physiological role of this enzyme could be in the turnover of nitrate reductase (NR) and, as such, it could be of great importance in regulating the assimilation of nitrate. The objective of this research was to elucidate the specificity and uniqueness of maize root proteinase. When bovine serum albumin and an NR purified from Chlorella vulgaris were used as substrates, the maize root proteinase exhibited a preference for cleavages such that the amino acid on the amino side of the scissile bond was alanine. This information was established by microsequence analysis of the N termini of proteolytic fragments, and carboxypeptidase Y analysis of the C termini of proteolytic fragments of substrates hydrolyzed by the proteinase. Cleavage occurred at the sequence Ala/Ala-Ala-Ala-Pro-Glu in Chlorella NR, and at the sequence Ala-Asp-Glu-Ser-His-Ala-Gln in bovine serum albumin. When bovine serum albumin was the substrate, the maize root proteinase yielded a peptide map that is unique relative to those created with the other serine endopeptidases elastase, trypsin, or chymotrypsin. Based on our data, the maize root proteinase appears to cleave peptide bonds at the carboxy side of alanine. Because of its specificity, it should have useful applications in protein chemistry.
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PMID:Characterization of a maize root proteinase. 827 5

Incubation of cytosolic extracts of bovine brain with S-adenosyl[methyl-3H]methionine results in the predominant [3H]methyl esterification of a 36-kDa polypeptide. This reaction appears to be distinct from any of the three known types of protein carboxyl methylation reactions previously established. We show here that the methylated 36-kDa polypeptide is a component of a cytosolic protein with a native molecular mass estimated at 178 kDa by gel filtration chromatography. The methyl group is not stable on the protein and is lost as [3H]methanol with a half-life of about 180 min at pH 7.0, 37 degrees C. The methyltransferase responsible for this reaction is a cytosolic protein with a native molecular mass of about 40 kDa that is readily separated from the well described protein-L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1.77). The methyl ester linkage is cleaved by carboxypeptidase Y, suggesting that the 36-kDa polypeptide is methylated on its C-terminal carboxyl group. Extensive digestion of gel-purified 3H-methylated 36-kDa polypeptide with trypsin and leucine aminopeptidase results in a radioactive product that co-chromatographs with authentic L-leucine methyl ester in reverse phase high performance liquid chromatography (HPLC), thin layer chromatography, thin layer electrophoresis, and high resolution-sulfonated polystyrene cation-exchange chromatography. Additionally, the o-phthalaldehyde/beta-mercaptoethanol-derived isoindole derivative of the 3H digestion product co-migrates on HPLC with the corresponding isoindole for L-leucine methyl ester. We demonstrate that a similar methylation system is present in yeast Saccharomyces cerevisiae but not in the bacterium Escherichia coli. These results provide evidence for a new type of reversible posttranslational modification reaction that may function to modulate the activities of its methyl-accepting substrates.
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PMID:Methyl esterification of C-terminal leucine residues in cytosolic 36-kDa polypeptides of bovine brain. A novel eucaryotic protein carboxyl methylation reaction. 851 74


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