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)

Earlier studies have shown that native tryptophanyl-tRNA synthetase from beef pancreas is composed of two apparently identical subunits having a molecular weight of 60000 plus or minus 2000 each. Incubation of the pruified enzyme with trypsin under restrictive conditions results in splitting of each subunit to form an enzymatically inactive polypeptide chain of mol. wt 24500 plus or minus 1500. During proteolysis, two distinct intermediate forms of mol. wt 51000 plus or minus 2000 and 40000 plus or minus 2000 and fragments of mol. wt 14000 plus or minus 2500 are formed. The presence of substrates, viz. ATP, tryptophan or tryptophanyl adenylate, decreases the rate of proteolysis. However, a band pattern monitored by acrylamide gel electrophoresis is qualitatively indistinguishable from that obtained in the absence of substrates. Native and trypsin-modified subunits (the latter having a molecular weight of 24500) have been maleylated, reduced, carbosymethylated and subjected to exhaustive digestion by trypsin followed by peptide mapping. Comparison of the finger prints has shown that the trypsin-modified subunit represents a polypeptide with lowered content of dicarboxylic amino acids. That the number of peptides revealed after complete proteolysis of native and trypsin-modified subunits does not favour the presence of long repetitive sequences in each subunit, is at variance with some bacterial aminoacyl-tRNA synthetases. Study of the fluorescence polarisation of 1-anilino-8-napthalene sulphonate adsorbed on the dimeric tryptophanyl-tRNA synthetase, indicates that the molecule behaves as a complete entity in Brownian rotation. The trypsin-resistant end products, composed of two types of polypeptides (mol. wts 24500 and 14000), remain associated with each other. From the mol. wt of this associate it follows that each fragment is present in the associate in duplicate. When the purification procedure was carried out in the absence of a protease inhibitor, the active modified enzyme form was obtained. As judged from the molecular weight values, it is composed of two equal subunits corresponding to one of the products of limited proteolysis. The data presented are compatible with compact three-dimensional structure of tryptophanyl-tRNA synthetase having very limited regions exposed to exogenous or endogenous proteolysis.
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PMID:Limited proteolysis of the tryptophanyl-tRNA synthetase. 116 77

The ability of aromatic tryptophyl and tyrosyl side-chain donors to form charge-transfer (CT) complexes with the acceptor 1-methyl-3-carbamidopyridinium chloride has been used to investigate the degree of exposure of these aromatic residues in denaturated proteins. The coplanar geometry of the CT complexes requires that virtually a full ring face of the donor be available for interaction with the acceptor, and the aromatic donor residues of lysozyme, trypsin, chymotrypsin, and the zymogens of the latter two enzymes do not appear to be wholly "exposed" in 6 M guanidine hydrochloride. Comparison of the CT proerties of the proteins with the corresponding properties of model complexes suggests that the incomplete exposure is due at least in part to statistical fluctuations in the continuously mobile, randomly coiled polypeptide chain which result in residues being alternately fully exposed and partly covered. Reduction and alkylation of the disulfide cross-links increase the apparent availability of the aromatic residues but the exposure is still less than that expected from a comparable mixture of tryptophan and tyrosine residues. Previous studies on the exposure of the aromatic residues of lysozyme and trypsin in aqueous salt solutions, when taken together with the present results, further suggest that there are two distinct kinds of surface environment possible on native proteins in solution. Some residues appear to be located in areas of the protein surface which are characterized by relatively fixed or stable local conformations, and have apparent CT association constants closely resembling these of comparable model complexes. Other residues may be located in a region where the protein conformation is flexible or continuously mobile, as evidenced by their smaller apparent association constants. It is probably significant that Trp-62 of lysozyme and Trp-215 of trypsin, both specificity site residues, appear to belong to the class of residues which can be considered as being in a flexible environment on the protein surface.
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PMID:Charge-transfer studies of the availability of aromatic side chains of proteins in guanidine hydrochloride. 117 11

Four isoperoxidases of turnip root and isoperoxidase C of horseradish root were digested with trypsin, and their peptide maps, prepared by high-voltage paper electrophoresis, were compared. All five tryptic digests were completely soluble at pH 8. The maps were developed with a variety of general and specific reagents: ninhydrin, histidine, tyrosine, tryptophan and arginine reagents. Cystine peptides and cysteic acid derivatives have also been characterized. All detected half-cystine residues seemed engaged in disulfide bridges. For each individual peroxidase the number of specifically staining peptides agreed very well with the amino acid composition. The two most acidic peroxidases of turnip, P1 and P2, only differ significantly in one peptide. The P2 gene is tentatively proposed to have developed from the P1 gene by a single base mutation, changing an asparagine residue to alysine residue. A less acidic turnip peroxidase, P3, is distinct, although related to peroxidases P1 and P2. Horseradish isoperoxidase C also belongs to this group which appears to be closely related in the amino acid sequences around four disulfide bridges. Peroxidase P7 differs from this group, at least around two of its disulfide bridges, and therefore, may differ from the other four in parts of its three dimensional structure. Sequences of particular importance to peroxidase function must be present in all peroxidases. From the peptide mapping studies we only find two highly homologous sequences present in all five examined peroxidases. Both contain histidine. This finding corroborates previous suggestions of two histidine sequences near the peroxidase heme prosthetic group. The rules applied in relating peptides of different proteins are outlined, and the sources of errors in mapping of glycoproteins of high carbohydrate content (about 20%) are discussed in detail.
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PMID:Similarities and differences of five peroxidases from turnip and horseradish. Peptide mapping studies on glycoproteins. 117 50

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


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