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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A latent form of collagenase had been isolated from crude extracts of the insoluble, fibrous material from Walker tumor homogenates. Purified preparations of this enzyme yielded a major unit of Mr approximately 62000, as determined by gel filtration on AcA 54 Ultrogel. In its activated form collagenase had been purified to apparent homogeneity with an approximate Mr of 42000. The active enzyme cleaved soluble collagen into three-quarter and one-quarter length fragments in the manner of vertebrate collagenases. Latent collagenase from culture media eluted with an apparent Mr of 53000 and was thus slightly larger in size than its activated form that eluted at 42000. Extracted latent collagenase and latent collagenase from culture media could be activated enzymatically by trypsin or chymotrypsin and non-enzymatically by mersalyl, an organic mercurial compound. We suggest that latent collagenase from Walker tumors are complexes of active enzyme with inhibitor(s) of low molecular weight(s) and are not true zymogens.
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PMID:Collagenase in the Walker 256 carcinoma. A study of the latent and active enzyme in vivo and in vitro. 627 77

A new method for the assay of collagenase activity has been developed, whereby the collagen cleavage products, after initial collagenase digestion, are degraded further by a mixture of trypsin and alpha-chymotrypsin. The degradation products are soluble in TCA and can be conveniently separated from the remaining uncleaved collagen substrate by rapid filtration. The enzyme assay is shown to be reproducible and sensitive, and it lends itself to a convenient and rapid determination of collagenase activity in relatively large numbers of samples. The applicability of this method is demonstrated by the detection of increased collagenase activity in skin fibroblast cultures derived from a patient with recessive dystrophic epidermolysis bullosa.
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PMID:Assay of collagenase activity by a rapid, sensitive, and specific method. 628 40

Parietal yolk sacs isolated from 14.5-day rat embryos and incubated in vitro with either [14C]proline, [3H]mannose or 3H-labeled amino acid mixture synthesized and secreted basement membrane collagenous and noncollagenous glycoprotein components with relative molecular weights of 350,000 (350K), 220,000 (220K), 185,000 (185K), 175,000 (175K) and 150,000 (150K). The 185K and 175K components appeared to be similar to the pro-alpha 1 (IV) and pro-alpha 2(IV) chains, respectively, which have been isolated from other sources. These components were completely susceptible to bacterial collagenase, but were only partially susceptible to alpha-chymotrypsin digestion. The 350K and 220K components appeared to be similar to subunits of laminin (or PYS A and PYS B, respectively) which have been characterized by others, while the 150K component may be similar to entactin (or PYS C). These components were completely resistant to bacterial collagenase and completely susceptible to alpha-chymotrypsin digestion. In addition, the basement membrane of the parietal yolk sac (Reichert's membrane) stained intensely with antibodies directed against either rat laminin or mouse basement membrane procollagen. The results of these experiments suggest that the 14.5-day rat embryo parietal yolk sac is a useful system for studying the structure, biosynthesis and deposition of basement membrane components.
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PMID:Partial characterization of collagenous and noncollagenous basement membrane proteins synthesized by the 14.5-day rat embryo parietal yolk sac in vitro. 629 50

A beta 1-serum component, beta 1-anticollagenase, capable of inhibiting various mammalian tissue collagenases, was isolated from human plasma by gel filtration, affinity chromatography and ion-exchange chromatography. The inhibitor contains 1-2 free sulfhydryl groups, which are a prerequiste for inhibitory activity and for binding to the thiol-Sepharose affinity support. Alkylation of beta 1-anticollagenase by iodoacetamide blocks inhibitory activity. The inhibitor was purified to apparent homogeneity and exhibited a Mr = 30500 determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The amino acid and carbohydrate composition was determined. According to its composition and the isoelectric focussing beta 1-anticollagenase is an acidic protein with an isoelectric point of 5.6. Inhibition of human leukocyte collagenase proceeds in a strong 1 : 1 stoichiometric reaction. The mechanism of this association takes place by a disulfide/thiol interchange reaction as has been previously indicated for human leukocyte collagenases in forming the latent enzyme [Macartney, H. W. and Tschesche, H. (1980) FEBS Lett. 119, 327-332]. The beta 1-anticollagenase--leukocyte-collagenase complex (latent enzyme) is activatable by disulfide-containing compounds such as cystine, oxidised glutathione, insulin, relaxin, trypsinogen and others, but not by 179,203-di(S-carboxymethyl)trypsinogen, or its trypsin derivative. Compounds containing inaccessible disulfide bonds, e.g. chymotrypsin, or sulfhydryl groups, e.g. D-penicillamine, do not activate the complex. Activation is, however, easily obtained with the oxidised-glutathione-generating system myeloperoxidase/H2O2/glutathione as was previously demonstrated for the human leukocyte latent collagenase activatable in a phagocytosis-simulated respiratory burst [Tschesche, H. and Macartney, H. W. (1981) Eur. J. Biochem. 120, 183-190].
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PMID:Characterisation of beta 1-anticollagenase from human plasma and its reaction with polymorphonuclear leukocyte collagenase by disulfide/thiol interchange. 629 99

The arachidonic acid pathway plays an important role in many inflammatory reactions. Current evidence suggests that platelets can play a central part in host inflammation. Since microfilariae are mobilized into the bloodstream following diethylcarbamazine (DEC) treatment, we have studied the effects of Onchocerca cervicalis cuticle preparations on equine platelet aggregation. The authors have found that O cervicalis cuticular preparations can induce platelet aggregation in vitro. Furthermore, this activity was abrogated by treatment with collagenase and not hyaluronidase, elastase, or alpha-chymotrypsin. When this evidence is viewed collectively with the evidence for in vivo parasite cuticular damage following DEC treatment, it becomes entirely plausible that the cuticular damage may indeed reveal a platelet-reactive surface, thus permitting platelet-parasite binding to occur. This binding would result in platelet aggregation and the generation and release of platelet-derived arachidonate metabolites. These metabolites may play a very critical role in the development of the described pathologic sequelae observed following DEC treatment. Field studies using cyclooxygenase and lipoxygenase inhibitors might therefore be very efficacious in decreasing the frequency of side effects due to DEC or other potentially effective drug regimens.
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PMID:Aggregation of equine platelets by Onchocerca cervicalis collagen. 629 6

Induction of the neutral proteinase, collagenase, is a marker for a specific switch in gene expression observed in rabbit synovial fibroblasts. A variety of agents, including 12-O-tetradecanoylphorbol-13-acetate, cytochalasins B and D, trypsin, chymotrypsin, poly(2-hydroxyethylmethacrylate), and trifluoperazine induced this change in gene expression. Induction of collagenase by these agents was always correlated with a marked alteration in cell morphology, although the cells remained adherent to the culture dishes. The amount of collagenase induced was positively correlated with the degree of shape change produced by a given concentration and, to some extent, with the duration of treatment. Altered cell morphology was required only during the first few hours of treatment with inducing agents; after this time collagenase synthesis continued for up to 6 d even when agents were removed and normal flattened cell morphology was regained. All agents that altered cell morphology also produced a characteristic switch in protein secretion phenotype, characterized by the induction of procollagenase (Mr 53,000 and 57,000) and a neutral metalloproteinase (Mr 51,000), which accounted for approximately 25% and 15% of the protein secreted, respectively. Secretion of another neutral proteinase, plasminogen activator, did not correlate with increased collagenase secretion. In contrast, synthesis and secretion of a number of other polypeptides, including the extracellular matrix proteins, collagen and fibronectin, were concomitantly decreased. That changes in cell shape correlated with a program of gene expression manifested by both degradation and synthesis of extracellular macromolecules may have broad implications in development, repair, and pathologic conditions.
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PMID:Changes in cell shape correlate with collagenase gene expression in rabbit synovial fibroblasts. 632 18

A 140 000 D glycoprotein (140 kD gp), labelled radioactively with surface-specific techniques, remained as the major cell surface glycoprotein in the detergent-resistant cytoskeletal preparations of cultured human fibroblasts. The 140 kD gp was present also in trypsinized cells and was not affected by treatment of the cells either with collagenase, chymotrypsin or thrombin. In density gradient fractionation of whole cells the 140 kD gp was recovered in the plasma membrane fraction together with small amounts of cytoskeletal components. In fractionation of cytoskeletal preparations, on the other hand, the 140 kD gp could not be dissociated from cytoskeletal proteins and together with vimentin it formed the major component of the oligomeric polypeptide complex generated by treating the surface-labelled cytoskeletal preparations with bifunctional cross-linking reagent, dithiobis succinimidyl propionate (DTPS). Moreover, the 140 kD gp seemed to copurify with vimentin upon reconstitution of intermediate filaments from urea-solubilized cytoskeletal preparations. On the other hand, low ionic-induced degradation of vimentin led to a decrease in the amount of the detergent-resistant 140 kD gp on the cell surface. In electron microscopy, a close apposition between bilayer-like plasma membrane remnants of the adherent cytoskeletons and cytoskeletal elements could be seen. The results indicate that the 140 kD gp is a plasma membrane glycoprotein which closely interacts with the detergent-resistant cytoskeleton of cultured human fibroblast. Possible mechanisms of the association are discussed.
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PMID:140 000 Dalton surface glycoprotein. A plasma membrane component of the detergent-resistant cytoskeletal preparations of cultured human fibroblasts. 633 55

The purification and properties of an estradiol-sensitive hydrolytic activity from mouse uterus which fits several criteria for being an induced protein are described. The activity in the uteri of immature animals can be stimulated 2--4-fold by estradiol to that approaching the adult level. Stimulation is blocked by puromycin. The enzyme which we have designated hydrolase II, was purified approx. 400-fold to apparent homogeneity by chromatography on Affigel Blue, DEAE-cellulose and octyl-Sepharose. Hydrolase II is a single chain polypeptide with an estimated mol. wt = 65,000 daltons and has an N-terminal serine residue. A variety of N-blocked L-amino acid nitrophenyl esters are cleaved by the enzyme. Km's at pH 7.2 were all approx. 40 microns. Of substrates tested, phenylalanine nitrophenyl ester had the highest Vmax. Cbz-beta-alanine nitrophenyl ester, which is not a normal protease substrate was cleaved with a Km of 145 microM. The enzyme had no detectable activity against peptide nitroanilide substrates for trypsin-, chymotrypsin- or elastase-like enzymes. It is inhibited by ZPCK and DIFP but not by TLCK and Ala-Ala-Pro-Ala chloromethyl ketone, a potent inhibitor of elastase-like enzymes. Mouse plasma protein protease inhibitors were without effect as was SBTI. Our results rule out hydrolase II being a carnosinase, non-serine esterase, plasminogen activator, collagenase or collagenase activator and suggest that it is a chymotrypsin-like protease.
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PMID:Properties of an estrogen-induced hydrolytic enzyme from mouse uterus. 635 Jul 23

A technique is described to detect the activity of protease inhibitors present in sodium dodecyl sulfate (SDS)-polyacrylamide gels (PAG) containing a copolymerized enzyme substrate. The method involved (1) incorporation of substrate (gelatin or casein) into the SDS-PAG at the time of casting; (2) electrophoresis of the protease inhibitors in the presence of SDS; (3) removal of SDS by washing the gel in 2.5% (w/v) Triton X-100; (4) incubation of the gels in a solution containing the proteolytic enzyme at 37 degrees C for 16 h; and (5) staining undigested substrate with amido black. Standard inhibitors such as bovine pancreatic trypsin inhibitor (BPTI), soybean trypsin inhibitor (SBTI), alpha 1-antitrypsin inhibitor, and a protease inhibitor derived from human articular cartilage have been examined by this method and displayed sharp inhibition bands when the gels were treated with bovine trypsin, chymotrypsin, or other enzymes. The technique cannot be used for precise quantification of protease inhibitors. However, there is a relationship between the concentration of inhibitor used and the intensity of staining. By this means, it was possible to estimate the smallest amount of inhibitor that could be detected (against a particular enzyme) under a given set of conditions. Inhibition was detected when 10 ng of SBTI or 20 ng of BPTI were applied to the gels; human alpha 1-protease inhibitor could be detected at a level of 2-3 micrograms. The technique was used to investigate the effectiveness of the human cartilage inhibitor against a variety of proteolytic enzymes, including thermolysin, Pronase, neutral protease, elastase, protease VII, pepsin, bacterial collagenase, protease IV, and papain.
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PMID:Detection of protease inhibitors using substrate-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 635 99

These studies analyze the effects of various enzymes on the terminal, killer cell-independent (KCIL) stages of the human natural killer (NK) cytolytic mechanism. The addition of trypsin (T), chymotrypsin (CT), or papain (P) to standard NK reaction mixtures (PBL or LGL and K562 cells) completely ablated cytolytic activity, whereas collagenase was ineffective. Inhibition by T was reversed by preincubation with soybean trypsin inhibitor (SBTI) or fetal calf serum, indicating that the inhibition was indeed due to T. Kinetic analysis with the Ca++ pulse experiment indicated that T, CT, and P inhibited lysis well beyond the Ca++-dependent (EDTA-sensitive) stages and essentially stopped further 51Cr release at the time of addition. This observation was confirmed by the ability of T, CT, and P to block lysis during KCIL of programmed K562 targets that were detached from NK cells by EDTA and were suspended in dextran-containing media. The lysis of K562 cells by natural killer cell-derived cytotoxic factors (NKCF) was also blocked by T and CT but not by P. Inhibition of NKCF activity by T could be reversed by SBTI or fetal calf serum. The ability of T, CT, or P to inhibit the lysis of "programmed" K562 targets during KCIL indicates that the NK lethal hit is an active process mediated by protease-sensitive structures, possibly NKCF, delivered to the target cell by the NK cell during the Ca++-dependent programming steps.
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PMID:Studies on the mechanism of the human natural killer cell lethal hit: evidence for transfer of protease-sensitive structures requisite for target cell lysis. 635 53


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