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 amino acid sequence for vitamin D-dependent bovine intestinal calcium binding protein has been established. It contains 85 amino acids in a single chain and lacks cysteine, tryptophan, methionine, histidine, and arginine. The NH2-terminal lysine is blocked by an N-acetyl group. Enzymatic digestion with trypsin, chymotrypsin, and pepsin yielded a number of peptides which were purified by two-dimensional high voltage paper electrophoresis. These peptides were examined by end group analysis and sequenced by the dansyl procedure. The absence of tryptophan permitted by a single cleavage of the molecule by N-bromosuccinimide at the tyrosine residue at position 8 and the larger fragment was subjected to automated Edman degradation. By these means, the following sequence was established: N-Ac-Lys-Gln-Ser-Pro-Leu-Glu-Tyr-Ala-Ala-Glu-Lys-Ser-Ile-Gln-Lys-Glu-Ile-Glu-Lys-Gly-Phe-Phe-Lys-Gln-Leu-Leu-Val-Ser-Val-Gln-Lys-Ala-Gly-Asp-Lys-Glu-Ser-Leu-Gln-Pro-Leu-Phe-Thr-Leu-Leu-Lys-Ser-Gly-Pro-Glu-Glu-Asn-Leu-Lys-Glu-Ser-Gln-Asn-Gly-Pro-Asp-Leu-Ls7-Ser-Gly-Pro-Gly-Asn-Asp-Leu-Glu-Glu-Lys-Gly-Thr-Asp-Val-Phe-Ser-Leu-Lys-Gln. Microheterogeneity may exist in the molecule at residue 76 in which position threonine may be replaced by serine. Comparison of the sequence of calcium-binding protein to the "test" sequence of Tufty and Kretsinger ((1975) Science 187, 167-169) proposed to identify E-F hands in muscle proteins suggests that intestinal calcium-binding protein may likewise contain one or possibly two E-F hands which could account for calcium-binding property. Dayhoff alignment scores, however, calculated for calcium-binding protein against nine E-F hands in muscle proteins parvalbumin, troponin and alkali light chains do not indicate that intestinal calcium-binding protein is homologous to these muscle protein chains.
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PMID:Calcium-binding protein of bovine intestine. The complete amino acid sequence. 117 41

Spectroscopic measurements of virgin bovine trypsin-kallikrein inhibitor and its modified species (in which the reactive-site peptide bond Lys-15--Ala-16 is split) indicate a conformational difference between both proteins. The inhibitor contains four tyrosines but no tryptophan. In the modified inhibitor a tyrosyl blue shift is seen in the difference absorption spectrum of modified against virgin inhibitor. The solvent perturbation spectra show an increase of the fraction of exposed tyrosyls from 0.45 in the virgin inhibitor to 0.59 in the modified form. Comparison of the circular dichroism spectra of the modified and virgin inhibitors reveals a decrease of the mean residue ellipticity in the tyrosine and peptide bond region of the modified inhibitor. In the fluorescence spectra a 50% increase in the quantum yield of the tyrosine fluorescence is observed in the modified inhibitor. All these spectroscopic data support the idea, which is also evidenced by the X-ray crystallographic model, that in the modified inhibitor up to five residues from Ala-16 to Arg-20 gain rotational freedom.
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PMID:The effect of cleaving the reactive-site peptide bond Lys-15--Ala-16 on the conformation of bovine trypsin-kallikrein inhibitor (K unitz) as revealed by solvent-perturbation spectra, circular dichroism and fluorescence. 124 86

The geometry of the binary and ternary complexes of two black-eyed pea inhibitors with trypsin and chymotrypsin has been established by distance measurements using the technique of singlet-singlet energy transfer. Triangulation of measured distances in the ternary double-headed complex of the trypsin-chymotrypsin inhibitor (BEPCI) with trypsin and chymotrypsin limits the possible structural models for this complex to those in which the center to center distance between trypsin and chymotrypsin is about 64 A, the distance from the center of trypsin to the single fluorescently labeled tyrosyl residue of the BEPCI dimer is about 33 A, and the distance between the chymotrypsin center and the labeled tyrosine of the inhibitor is about 43 A. Energy transfer results for the trypsin inhibitor (BEPTI) complexes show conclusively that the weak trypsin site is structurally analogous to the strong chymotrypsin binding site of BEPCI. The weak chymotrypsin binding site of BEPTI is structurally analogous to the strong trypsin sites of BEPCI and BEPTI. Corresponding distances in binary and ternary complexes are the same, indicating that little or no structural rearrangement occurs when the ternary complexes are formed. Complex formation was shown to involve tryptophan and tryosine residues of both trypsin and chymotrypsin as judged by absorption and circular dichroism difference spectroscopy. In addition, circular dichroism difference spectra revealed some disulfide contributions.
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PMID:Double-headed protease inhibitors from black-eyed peas. VI. Singlet-singlet energy transfer and other optical studies on the structure of trypsin and chymotrypsin complexes. 124 53

The AP1 protein, a unique aspermatogenic protein localized in the sperm acrosome, exists as a single polypeptide chain of 136 amino acids, as shown by a single band on gel electrophoresis in sodium dodecyl sulfate and the recovery of the expected 21 to 22 tryptic peptides on peptide mapping. The AP1 protein appears to exist in a compact, highly stable conformation, as shown by its resistance to trypsin hydrolysis. Its aspermatogenic acitivity is not affected by trypsin treatment, by heating at 99 degrees C for 1 h, by 8 M urea, or by acid conditions. After reduction and alkylation, however, the molecule appears to open up, since it becomes hydrolyzable by trypsin and migrates more slowly on gel electrophoresis at pH 2.7 and 8.6. After alkylation, the AP1 protein still migrates as a single band at pH 2.7. The AP1 protein shows microheterogeneity near its isolectric point at pH 8.6; each of five bands shows the same amino acid analysis. Aggregation was not observed following treatment with dimethylsuberimidate. The molecular weight of 15 000, obtained from gel electrophoresis consists of 136 amino acids with a relatively high content of proline, half cystine, glycine, histidine and tryptophan. No galactose, mannose, fucose, glucose, or hexosamines were found; the AP1 protein is thus not a glycoprotein.
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PMID:Experimental allergic aspermatogenic orchitis. II. Some chemical properties of the AP1 protein of the sperm acrosome. 126

The trpB8 mutation of Escherichia coli tryptophan synthase is unique in that the cells bearing this lesion are not only capable of utilizing indole for growth, but they also accumulate indole, under conditions of tryptophan limitation. The lesion was shown by DNA sequencing to be a G to C transversion at nucleotide 5528 of the trp operon, resulting in a Gly to Arg switch at codon 281. Gly-281, within the trypsin-sensitive "hinge" region, is invariant among all known beta polypeptides. The catalytic activity of the mutant beta 2(B8) protein is dramatically stimulated by alpha subunit, both in vivo and in vitro. In the absence of alpha subunit, ammonium ion effectively stimulated the activity in an apparently cooperative manner. The pH optimum for the mutant subunit was 9.8, which is 2 units higher than that of wild type. In contrast to the wild-type subunit, beta(B8) partially aggregated within cells upon overexpression. At the optimal concentration of ammonium ions (2.25 M), the beta 2(B8) mutant enzyme displayed lower affinity than wild-type enzyme toward indole and L-serine, but the Vmax was almost unchanged. The physicochemical behavior of beta 2(B8) is supported by computer graphic modeling studies. An open versus closed model of conformational change within the beta 2 protein is proposed. A plausible role for the hinge region is discussed.
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PMID:Genetic and biochemical characterization of the trpB8 mutation of Escherichia coli tryptophan synthase. An amino acid switch at the sharp turn of the trypsin-sensitive "hinge" region diminishes substrate binding and alters solubility. 130 52

The trypsin-inhibiting activity of human serum is lowered upon addition of formaldehyde or acetaldehyde thereto. Acetaldehyde reacts with alpha-1-proteinase inhibitor (alpha 1-PI) to decrease its trypsin-inhibiting ability. Acetaldehyde has only a slight effect on the tryptic hydrolysis of benzoyl-DL-arginine-p-nitroanilide. It did not decrease the inhibitory activity of the Kunitz inhibitor (Aprotinin) or soybean trypsin inhibitor. Since aldehydes form covalent products with primary amines, primary amides, arginine, tyrosine, and tryptophan in protein, as well as methylene bridges thereby crosslinking functional groups, it is proposed that one or more such interactions affect alpha 1-PI activity. It is further suggested that chronically high levels of acetaldehyde, as a metabolic produce of ethanol, may be a contributory factor to the generation of pancreatitis in alcoholics by possibly lowering the effective alpha 1-PI level which is a natural protective element from proteolysis by trypsin.
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PMID:Acetaldehyde decreases the antitryptic activity of alpha 1-proteinase inhibitor. 131 22

Immobilized artificial membrane (IAM) HPLC supports have been used to immobilize the enzymes alpha-chymotrypsin and trypsin. The enzymes were trapped in hydrophobic cavities on the support and were not covalently attached to the IAM surface. The resulting IAM-enzyme supports retained the hydrolytic activity of the immobilized enzymes: the IAM-trypsin support catalyzed the hydrolysis of N alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA), and the IAM-alpha-chymotrypsin support (IAM-ACHT) catalyzed the hydrolysis of a number of substrates, including tryptophan methyl ester. The activities of both supports were decreased by known enzyme inhibitors and the activity of the IAM-ACHT was affected by changes in pH and temperature. When a substrate was chromatographed on an IAM-ACHT HPLC, the hydrolytic activity of the immobilized enzyme could be determined from the resulting substrate/product ratios. These data were obtained either directly from the IAM-ACHT chromatogram or from the chromatogram produced by a coupled column system. The results of this study indicate that IAM-immobilized alpha-chymotrypsin and trypsin can be used as chromatographic probes for the qualitative determination of enzyme/substrate and enzyme/inhibitor interactions.
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PMID:Enzyme-based high-performance liquid chromatography supports as probes of enzyme activity and inhibition: the immobilization of trypsin and alpha-chymotrypsin on an immobilized artificial membrane high-performance liquid chromatography support. 132 67

Oxidative damage to bovine serum albumin (BSA) was induced by hydroxyl radical (HO.) generating systems of xanthine oxidase (XO) + EDTA-Fe3+ and ascorbate + EDTA-Fe3+. Formation of bityrosine and loss of tryptophan were observed in the ascorbate + EDTA-Fe3+ system and carbonyl formation was induced by both systems. Mannitol and ethanol very strongly inhibited the carbonyl and/or bityrosine formation, indicating that the oxidative damage to BSA was due to HO(.). The sulfhydryl (SH) groups of BSA were very sensitive to the XO + EDTA-Fe3+ but not to the ascorbate + EDTA-Fe3+ system. Catalase but not hydroxyl radical scavengers or superoxide dismutase strongly inhibited the loss of SH groups, indicating that H2O2 is involved in their oxidation. Fragmentation of BSA was observed during exposure to the XO + EDTA-Fe3+ and ascorbate + EDTA-Fe3+ systems and the products presented a broad band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Little formation of amine groups was observed in these systems, indicating that little peptide bond cleavage occurred. BSA exposed to the ascorbate + EDTA-Fe3+ system was more readily degraded by trypsin than that exposed to the XO + EDTA-Fe3+ system. Elastase degraded BSA exposed to the ascorbate + EDTA-Fe3+ system but not to the XO + EDTA-Fe3+ system.
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PMID:Oxidative damage to bovine serum albumin induced by hydroxyl radical generating systems of xanthine oxidase + EDTA-Fe3+ and ascorbate + EDTA-Fe3+. 133 12

Somatostatin 28 (S-28), originating in gastrointestinal cells, is secreted into the circulation and increases in humans after ingestion of a mixed meal. To evaluate the possibility that the increased levels of S-28 post cibum might modulate the release of enzymes and bicarbonate from the exocrine pancreas, S-28 was infused intravenously into healthy volunteers to levels seen after food intake. During S-28 infusion, the output of lipase, trypsin, amylase, and bicarbonate stimulated by either exogenous cholecystokinin octapeptide or endogenous signals from intraduodenal administration of tryptophan or a mixture of amino acids was significantly reduced. It is concluded that S-28 released from the gut during food intake modulates pancreatic exocrine function in humans.
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PMID:Evidence for hormonal inhibition of exocrine pancreatic function by somatostatin 28 in humans. 135 58

Efficient formation of the cpn60-rhodanese complex can be achieved by mixing unfolded rhodanese with excess cpn60 at low temperature. By employing these conditions, a stable and highly reactivatable complex is formed. The complex is not formed when native enzyme is used. Concentrations of NaCl, as high as 0.75 M, do not have any effect on the formation or disruption of the binary complex. cpn60-bound rhodanese contains an exposed hydrophobic surface, as detected by the binding of the fluorescent reporter, 1-anilinonaphthalene-8-sulfonic acid. The intrinsic fluorescence of cpn60-bound rhodanese reports that the average tryptophan is in an intermediate environment between that found in unfolded and native states. This form of rhodanese has an accessibility to quenching by acrylamide or iodide that is intermediate between the unfolded and native forms of the enzyme. Protease susceptibility studies show that rhodanese bound to cpn60 exhibits a trypsin digestion pattern similar to the native enzyme, although it is more rapidly proteolyzed. The results suggest that the conformation of cpn60-bound rhodanese resembles a native-like conformation, but with increased flexibility. Further, only intact rhodanese or enzyme lacking its N-terminal sequence can interact with cpn60 and form a stable binary complex. The protein fragment corresponding to the rhodanese N-terminal sequence did not form part of a stable complex with cpn60.
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PMID:Characterization of a stable, reactivatable complex between chaperonin 60 and mitochondrial rhodanese. 136 12


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