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)

High resolution segregation of erythrocyte membrane polypeptides achieved by isoelectric focusing in 8 M urea was employed in conjunction with surface-restricted radioiodination to analyze the disposition of polypeptides within the human erythrocyte membrane. Several membrane polypeptides showed significant uptake of radioiodine, with the principal labeled component migrating between pH values of 3.0 and 3.5. Two approaches were taken in examining membrane polypeptide disposition on both faces of the erythrocyte membrane. Saturation labeling of the outer face of the membrane with one iodine isotope followed by cell lysis and reiodination with a second iodine isotope did not prove feasible and another procedure based on surface iodination with 125-I, formation of sealed inside-out vesicles and re-iodination with 131-I was adopted. Studies of sialic acid release from the membrane surface and trypsin cleavage of radioiodinated peptides indicated that selectively labeled, sealed inside-out vesicles had been formed. The ratio of 125-I to 131-I in membrane polypeptides separated by isoelectric focusing confirmed the existence of externally disposed, internally disposed and spanning proteins.
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PMID:Analysis of polypeptide disposition in human erythrocyte membranes employing membrane inversion. 23 86

The properties of big renin, a relatively inactive form of renin isolated from human plasma, were examined following partial purification by gel filtration. Exposure of big renin to pH 3.0-3.6, or brief incubation with trypsin or pepsin, resulted in a ten-fold increase in enzymatic activity. Activation was not effected by 4M NaCl, 6M urea, or incubation with neuraminidase. Both before and after inactivation, big renin eluted from Sephadex gel more rapidly than normal plasma renin. During polyacrylamide gel disc electrophoresis, inactive big renin migrated more slowly than either normal renin or big renin previously activated. Using sheep substrate, the enzyme kinetics of normal renin and previously activated big renin were identical, while inactive big renin possessed a higher Michaelis constant. These data indicate that big renin is closely related biochemically to normal plasma renin. As the activation of big renin results in the formation of the substance even more similar to normal renin, the possibility exists that big renin may prove to be a precursor form of normal renin.
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PMID:Biochemical properties of big renin extracted from human plasma. 23 15

The major form of the trypsin-like proteinases from the larvae of the webbing clothes moth Tineola bisselliella has been further purified and some of its properties investigated. It differs from bovine trypsin in several respects. It is anionic at neutral pH, is very stable at alkaline pH, has no requirement for calcium ions for this stability and is very sensitive to urea. It resembles vertebrate trypsins in its complete inhibition by diisopropylfluorophosphate, its pH optimum of 8.5 for hydrolysis of benzoyl-arginine p-nitroanilide and its cleavage specificity against glucagon and the beta-chain of S-carboxymethyl insulin.
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PMID:Properties and specificity of the major anionic trypsin-like enzyme in the keratinolytic larvae of the webbing clothes moth. 23 56

1. Isoionic chemical modification of amino groups of trypsin (EC 3.4.21.4) was studied for the purpose of obtaining a well-defined modified trypsin with minimum changes in physicochemical properties and with sufficient stability at neutral pH. Acetamidination with methyl acetimidate hydrochloride proceeded very rapidly at pH9.8 and 5degrees C and all 14 epsilon-amino groups were modified in 2h. The reaction was limited to epsilon-amino groups. The alpha-amino group of N-terminal isoleucine was modified only by repeated reactions in the presence of 5.5 M-guanidine or 8 M-urea. 2. The epsilon-acetamidinated derivative of beta-trypsin retained enzymic activity at values comparable with those of native enzyme tested with alpha-N-benzoyl-L-arginine ethyl ester and alpha-N-benzoyl-L-arginine p-nitroanilide as substrates; it also showed substrate activation comparable with that of native enzyme. The acetamidination of alpha-trypsin resulted in approx. 50% decrease in its esterolytic activity. 3. The epsilon-acetamidinated beta-trypsin was very stable at pH8 and 25degrees C in the absence of Ca2+. The activity of 0.04% (W/V) enzyme solution remained practically unchanged for 10h, and after 24h 90% of the activity was still retained. Possible autolytic cleavage of peptide bonds of acetamidinated enzymes was followed by N-terminal analysis by using automated Edman degradation. Only the Arg(105)-Val(106) bond was found to be cleaved to an appreciable extent. Thus beta-trypsin can be stabilized simply by complete acetamidination of epsilon-amino groups without modifying guanidino groups of arginine residues. Acetamidinated alpha-trypsin was unstable, but its inactivation at a neutral pH could not be attributed to the cleavage of a single specific peptide bond. 4. The acetamidination of the alpha-amino group of the N-terminal isoleucine results in the inactivation of esterolytic activity. However, this enzyme retained the ability to react with p-nitrophenyl p'-guanidinobenzoate. 5. It was concluded that acetamidination of beta-trypsin is a convenient method for preparing a well-defined stable and soluble trypsin derivative without appreciable change in its physical properties.
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PMID:Chemical modification of amino groups and guanidino groups of trypsin. Preparation of stable and soluble derivatives. 23 4

Alterations of the structure of EF-Tu have been investigated by using the rate of EF-Tu cleavage by trypsin as a conformational probe. The presence of EF-Ts bound to EF-Tu results in a 10-fold increase in the cleavage rate. The antibiotic kirromycin, which inhibits protein synthesis by virtue of its interaction with EF-Tu, mimics this effect of EF-Ts. Both kirromycin and EF-Ts also facilitate the exchange of free GDP with GDP bound to EF-Tu. The results suggest that EF-Ts and kirromycin induce a similar conformational change in EF-Tu, thereby "opening" the guanine nucleotide binding site. The trypsin-cleaved EF-Tu still can bind GDP and EF-Ts and can function in Qbeta replicase, but it no longer spontaneously renatures following denaturation in urea.
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PMID:Conformational alteration of protein synthesis elongation factor EF-Tu by EF-Ts and by kirromycin. 26 89

Human milk contains a mitogenic factor that stimulates DNA synthesis and cell division in mouse and human fibroblasts in vitro. Milk at a concentration of 1% (vol/vol) is as active in stimulating DNA synthesis as is 5% (vol/vol) human serum and 10% (vol/vol) calf serum. The mitogenic activity of human milk is destroyed by incubation with trypsin and chymotrypsin. However, neither urea, guanidine hydrochloride-dithiothreitol, nor exposure to pH 1 will inactivate the milk-derived growth factor. Gel filtration and isoelectric focusing indicate that the mitogenic activity of human milk has a molecular weight between 14,000 and 18,000 and an isoelectric point between 4.4 and 4.7.
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PMID:Human milk stimulates DNA synthesis and cellular proliferation in cultured fibroblasts. 28 15

The formation of a highly organized vascular and corneal endothelial cell monolayer is associated with the appearance of a 60,000-dalton cell surface protein (CSP-60) (30,000 daltons after reduction with dithiothreitol) which is not detectable in rapidly growing endothelial cells and in subconfluent cultures that do not yet exhibit the strict morphology of a confluent monolayer. It is also absent from vascular smooth muscle cells and from endothelial cultures that are maintained in the absence of fibroblast growth factor and grow on top of each other at confluence. After disorganization of cells in a confluent endothelial monolayer by urea, EDTA, or trypsin, CPS-60 is no longer exposed on the cell surface, but it reappears as soon as the cells readopt their characteristic two-dimensional configuration. This reorganization can be achieved in the presence of cycloheximide and despite removal of fibronectin by urea, EDTA, or trypsin. Maximal amounts of fibronectin and no CSP-60 are detected in subconfluent, but not yet organized, endothelial cultures or in endothelial cells that no longer form a monolayer of nonoverlapping cells at confluence. Likewise, cultures of vascular smooth muscle cells contain fibronectin but no CSP-60. These results suggest that CSP-60, rather than fibronectin, could be involved in the adoption of a monolayer configuration by confluent endothelial cells.
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PMID:Appearance in confluent vascular endothelial cell monolayers of a specific cell surface protein (CSP-60) not detected in actively growing endothelial cells or in cell types growing in multiple layers. 28 73

This communication presents evidence for the existence in the ovine testis of proteinaceous factors which suppress LH as well as FSH. Isolation of these factors has been achieved by using three different procedures: cytosol preparation, metaphosphoric acid extraction and ultrafiltration. Chromatography of cytosol or metaphosphoric acid extract on Sephadex G-75 resulted in separation into three protein fractions designated as G-75-I, II and III in order of their elution. When administered to castrated male rats, Fraction G-75-I suppressed circulatory levels of LH (53% inhibition, P less than 0.05) without altering FSH. The most retarded fraction, G-75-III, suppressed FSH (29% inhibition, P less than 0.001) without any concomitant change in LH. When fraction G-75-III was further fractionated on Sephadex G-25, three components were found and two, G-25-II and G-25-III, were biologically active. These fractions were homogeneous on polyacrylamide disc-gel electrophoresis. The FSH-suppressing factor (inhibin) was heat labile and susceptible to trypsin digestion, indicating that it is proteinaceous. Treatment with urea did not reveal any subunits. The molecular weight of this factor, as determined by gel filtration and SDS-urea gel electrophoresis was estimated to be around 1400-1500. The absence of sialic acid and the molecular weight data suggested that the isolated material was a simple protein and probably a small peptide. Gel filtration on Sephadex G-75 of the metaphosphoric acid extracts of liver, kidney, testis and ovary revealed an identical elution pattern for ovarian and testicular inhibin.
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PMID:Characterization of a gonadal factor involved in the control of FSH secretion. 29 12

The LAF produced by the mouse macrophage cell line, P388D1, is a single polypeptide chain of m.w. 12,000 to 16,000 daltons. Native LAF was destroyed by Streptomyces griseus protease, but not by trypsin, chymotrypsin, and papain, although in the presence of 8 M urea, papain completely destroyed LAF activity. LAF did not bind to concanavalin A-Sepharose, suggesting that LAF does not contain significant amounts of mannosyl or glycosyl residues. Since LAF activity was not inactivated by a treatment of reduction and alkylation the active conformation of LAF does not appear to be dependent on disulfide linkages. LAF was not irreversibly denatured by 8 M urea or 0.1 to 0.5% SDS. On SDS-polyacrylamide gels, the m.w. of LAF was 12,000 daltons, as compared to a value of 16,000 daltons, as determined by gel filtration. The isoelectric point of LAF was 5.0 to 5.4 as determined on 7.5% acrylamide gels (pH 3 to 10). On the basis of these results it appears that the P388D1 cell line-derived LAF is a relatively stable molecule that shares several physicochemical properties with normal human and mouse macrophage-derived LAF.
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PMID:Physicochemical characterization of lymphocyte-activating factor (LAF). 31 60

Limited proteolysis of native elongation factor Tu (Mr 44 000) by trypsin occurs in at least three distinct steps. The first intermediate arises through cleavage at a site about 65 residues from the amino-terminal end of the protein. It is functionally active [Jacobson, G. R. & Rosenbusch, J. P. (1976) Biochemistry, 15, 5105-5110] and is partially protected from further degradation by the antibiotic kirromycin. The second step converts this intermediate to one of similar size (Mr 37 000) which now is partially inactivated. It is likely to be identical with the intermediate described by Arai et al. [(1976) J. Biochem. Tokyo, 79, 69-83]. In the third step, the partially inactive intermediate is cleaved without any apparent change in the functional properties tested. The resulting two trypsin-resistant fragments have molecular weights of 24 000 and 14 000, and remain associated under nondenaturing conditions. When either of these polypeptides, after isolation in 8 M urea, is allowed to renature, no significant reactivation of GDP binding is observed unless the isolated fragments are mixed before renaturation. These results show that the two fragments are structurally and functionally interdependent.
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PMID:Limited proteolysis of elongation factor Tu from Escherichia coli, Multiple intermediates. 33 Jan 67


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