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)

The experimental modulation of tight junctions (TJ) was studied in the human adenocarcinoma cell line HT 29 by freeze-fracture electron microscopy. The cell line has virtually no TJ when grown in culture. TJ could be induced by mild treatment with a variety of endopeptidases (trypsin, chymotrypsin, collagenase, elastase, plasmin, thrombin, papain, and pronase). Pronase induced the formation of TJ at low (but not at high) concentrations. All exopeptidases studied were unable to induce the formation of TJ. At 0 degree C the trypsin-induced formation of TJ was greatly slowed down although not entirely inhibited. However, when cells were briefly treated with trypsin at 0 degree C and subsequently transferred to 37 degrees C in the presence of protease inhibitors, TJ were rapidly assembled. Thus an induction phase at low temperature and an assembly phase at high temperature could be experimentally separated. When cells were briefly trypsinized at 0 degrees and subsequently kept at 0 degree C without trypsin for several hours, TJ still formed abundantly upon incubation at 37 degrees C. It appears therefore that the effect produced by the protease is retained for long periods in the cold.
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PMID:Formation of tight junctions in epithelial cells. I. Induction by proteases in a human colon carcinoma cell line. 388 Jul 1

The activity of chymase was markedly inhibited by phosphoglycerides such as phosphatidic acid, phosphatidylserine, and phosphatidylinositol, but was not affected by acylglycerides, phosphoglyceroserine, serine, inositol, or glycerol. These results suggest that both the nonpolar hydrophobic hydrocarbon tails and the polar hydrophilic head are essential for the inhibitory effects of phosphoglycerides. Binding of a primary amine to an anionic polar head of phosphatidic acid, such as in phosphatidylserine and phosphatidylethanolamine, slightly decreased the inhibitory effect of phosphatidic acid and, conversely, binding of a strong cation to the head, such as in phosphatidylcholine, resulted in its activation of chymase. Phosphatidic acid containing an unsaturated fatty acid, such as dioleoyl phosphatidic acid, caused the same extent of inhibition as natural phosphatidic acid from bovine brain, but was 20 times more inhibitory than phosphatidic acid containing a saturated fatty acid, such as distearoyl phosphatidic acid. The inhibition by phosphatidylserine was noncompetitive and pseudoirreversible, and the Ki value was 0.54 microM. The inhibition of chymase by phosphatidylserine was pH dependent, being strong at pH 8.5 to 9.5 but weak below pH 7.5. Phosphatidylserine specifically inhibited chymase and elastase; it did not inhibit the other chymotrypsin-type serine endopeptidases tested, trypsin, papain, collagenase, carboxypeptidase A, or cathepsin D.
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PMID:Inhibition of chymase activity by phosphoglycerides. 388 53

Chondroitin sulfate E proteoglycan was extracted in the presence of protease inhibitors from 6 X 10(9) mouse bone marrow-derived, interleukin 3-dependent mast cells, of which 3 X 10(7) had been biosynthetically labeled with [35S]sulfate or [3H]glycine. Chondroitin sulfate E proteoglycan was purified to apparent homogeneity by density-gradient centrifugation, differential molecular weight dialysis, DEAE-52 ion exchange chromatography, and Sepharose CL-4B gel filtration chromatography. Chondroitin sulfate E proteoglycan, radiolabeled with [3H]glycine or [35S]sulfate, filtered as a single peak of radioactivity on Sepharose CL-4B with a Kav of 0.41. When purified [3H]glycine-labeled proteoglycan was digested with chondroitinase ABC and subjected to gel filtration, all of the radioactivity was shifted to a lower molecular weight. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr of the peptide core obtained by chondroitinase ABC treatment was approximately 10,000. The purified proteoglycan was resistant to degradation by collagenase, clostripain, trypsin, chymotrypsin, elastase, chymopapain, V8 protease, proteinase K, and Pronase, as assessed by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the core peptide of the intact proteoglycan revealed that glycine, serine, and glutamic acid/glutamine accounted for 70% of the total amino acids and were present in a molar ratio of 4.3/1.6/1.0. When analyzed for neutral hexose content by gas-liquid chromatography, the proteoglycan contained approximately 2% of its weight as mannose, fucose, galactose, and other sugars, indicating that oligosaccharides were linked to the peptide core. The mouse bone marrow-derived mast cell chondroitin sulfate E proteoglycan, like the rat serosal mast cell heparin proteoglycan, is markedly protease resistant, has highly sulfated glycosaminoglycans, and contains a peptide core that is rich in serine and glycine. These characteristics of the mast cell class of intracellular proteoglycans may contribute to their function in stimulus-induced granule secretion as well as in mediator storage, including retention of cationic neutral proteases.
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PMID:Purification and analysis of the core protein of the protease-resistant intracellular chondroitin sulfate E proteoglycan from the interleukin 3-dependent mouse mast cell. 393 50

Characterization of muscarinic acetylcholine receptors in acinar cells from rat pancreas and lacrimal and parotid glands was achieved by binding of the reversible muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) and the specific alkylating reagent [3H]propylbenzilylcholine mustard (PrBCM) to intact acini or dispersed acinar cells. Binding studies with [3H]QNB showed that acinar cells from pancreas contain 26,400, from parotid 21,400, and from lacrimal gland 25,700 binding sites/cell. To assess molecular size of the receptor in each gland, acini were prepared by digestion with purified collagenase and singly dispersed acinar cells were prepared by a combination of digestion with crude collagenase, hyaluronidase, and alpha-chymotrypsin and divalent cation chelation using EDTA. Muscarinic receptors on acini or dispersed cells were covalently labeled with 5 nM [3H]PrBCM, solubilized directly in hot sodium dodecyl sulfate buffer, and resolved by polyacrylamide gel electrophoresis. When solubilized acini were electrophoresed, a major labeled peak was observed on gels along with a smaller peak of lower apparent molecular weight. For pancreatic acini, the apparent molecular weights of these peaks were 117,600 and 85,700; for parotid acini, 104,800 and 74,500; and for lacrimal acini, 87,200 and 63,100. Addition of muscarinic antagonists to the labeling medium abolished both peaks. When dispersed acinar cells were labeled, the larger peak was eliminated, and all radioactivity was concentrated in a single peak: 87,600 for pancreas, 78,000 for parotid gland, and 62,800 for lacrimal gland. Digestion of prelabeled acini with the mixture of enzymes used to produce dispersed acinar cells similarly shifted all radioactivity into this second peak. Limited digestion of acini or dispersed cells with 1 mg/ml of papain resulted in the disappearance of these higher molecular weight peaks and the appearance of a broad peak at Mr = 40,000. Cells of nonepithelial origin, IM-9 lymphocytes and NG108 neuroblastoma X glioma hybrids, also were labeled with [3H]PrBCM and electrophoresed.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Muscarinic acetylcholine receptor structure in acinar cells of mammalian exocrine glands. 398 Apr 74

Cultured skin fibroblasts from a variant of osteogenesis imperfecta were previously shown to synthesize a type I procollagen which was a homotrimer of pro alpha 1(I) chains. Trimers of alpha 1(I) collagen were isolated by pepsin digestion of culture medium from these fibroblasts. The amino acid composition of the isolated protein indicated that it contained an increased amount of hydroxylysine, apparently because of post-translational over-modification. The thermal stability of the alpha 1(I) trimers was examined by circular dichroism. We found no consistent difference in the melting curve of the alpha 1(I) trimers compared to control type I collagen. We next examined the thermal stability of the alpha 1(I) trimers using digestion with a combination of trypsin and alpha-chymotrypsin as an alternative probe of helical stability. When enzymatic digestions were carried out at 36 degrees to 40 degrees C, the alpha 1(I) chains in the trimers were cleaved to polypeptides which were shortened by approximately 100 amino acids. Vertebrate collagenase digestion of the shortened molecules indicated that the 100 amino acid segment removed from each alpha 1(I) chain was located at the carboxyl-terminus. The decreased thermal stability of the alpha 1(I) trimers was probably explained by the absence of alpha 2(I) chains in the molecules. The results, however, did not exclude the possibility that the post-translational over-modification of the alpha 1(I) chains contributed to the altered helical structure.
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PMID:Altered helical structure of a homotrimer of alpha 1(I)chains synthesized by fibroblasts from a variant of osteogenesis imperfecta. 405 61

Calf lymph smallpox vaccines contain too much extraneous debris for an accurate assessment of their virus particle content. The process of partial purification of the vaccine utilizing enzymatic digestion by chymotrypsin, subtilisin, and collagenase solubilized enough debris to permit electron microscopic virus particle count. Enzyme treatment did not degrade or destroy the virus nor did it reduce the infective titer. Commercial vaccines studied ranged in virus content from 1.89 x 10(9) to 1.09 x 10(11) virus particles/ml. The pocking efficiencies on the chorioallantoic membrane of some of these vaccines varied from 200 to 1,200 virus particles per pock-forming unit.
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PMID:Virus particle content of smallpox vaccines. 431 62

A well-characterized line of rat Schwann cells has been examined for its ability to produce collagen. About 14% of the [(3)H]proline in the proteins that were secreted into the culture medium by the cells was hydroxylated, while only 1% of the labeled proteins in the cell layer contained [(3)H]hydroxyproline. Three sizes of procollagen polypeptides, of molecular weights about 105,000, 120,000, and 155,000, were present in the medium, as well as tropocollagen molecules that contained the usual alpha1 and alpha2 chains. Subsequently, the Schwann cells ceased producing the smaller collagenous polypeptides, although the total [(3)H]hydroxyproline content of the medium was unchanged. The [(3)H]hydroxyproline was almost entirely accounted for by the polypeptide of 155,000 daltons; this peptide was rapidly digested by collagenase or pepsin or chymotrypsin. The destruction by pepsin and chymotrypsin indicates that the large polypeptide, in contrast to procollagen and tropocollagen, is not in the collagenous (helical) conformation. Possibly, this substance is a very early form of procollagen that does not fold into the collagen conformation. The data show that cells of neuroectodermal origin can synthesize collagen, and also suggest that Schwann cells may be responsible for a large proportion of the collagen seen in peripheral neurinomas in vivo.
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PMID:Collagen and procollagen production by a clonal line of Schwann cells. 435 64

A procedure for dissociation of the guinea pig pancreas into individual cells is described which employs enzymatic digestion with pure collagenase, chymotrypsin, and hyaluronidase, utilizes an interposed chelation of divalent cations by EDTA, and is terminated by gentle shearing. Yields of cells are 50-60%, based on DNA recovered. The population comprises approximately 95% exocrine cells, the remainder consisting of endocrine, duct, and vascular endothelial cells. The exocrine cells, though spherical, retain the structural attributes of their in situ counterparts, including differentiation of the plasmalemma into zones corresponding to the former apical and basal plasmalemma, polarized distribution of organelles indicated by fields of zymogen granules in the cytoplasm underlying the former apex, central location of the Golgi complex, and placement of the rough endoplasmic reticulum and nucleus in the former basal pole of the cell. Electron microscope study of the effects of individual treatments used during dissociation indicates that digestion of basement membrane and collagen is solely due to collagenase activity and that separation of desmosomes (and possibly of zonulae adherentes) results only from exposure to low [Ca(++)] and EDTA and is not effected by the enzymes used. Gap junctions are resistant to enzymes and EDTA; tight junctions resist enzyme treatment but undergo rearrangement upon exposure to EDTA. Both junctions require mechanical shear for complete cell separation. Neither chymotrypsin nor hyaluronidase produces visible alterations in stromal or junctional elements. Dissociation requires the concerted action of enzymes, chelation of divalent cations, and mechanical shear, since the individual treatments are alone ineffective.
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PMID:Studies on dispersed pancreatic exocrine cells. I. Dissociation technique and morphologic characteristics of separated cells. 437 77

Latent and active collagenase were demonstrated following direct extraction from normal skin homogenates with 0.1M calcium chloride at 60 degrees C. 83% of the collagenase activity was in latent form and could be maximally activated with trypsin. Partial activation of the latent enzyme could also be demonstrated by incubation of the skin extract without added trypsin. This endogenous activation was inhibited by the addition of soya bean trypsin inhibitor, trasylol, di-isopropylphosphofluoridate and phenylmethanesulphonylfluoride, none of which inhibited collagenase directly. This suggests that the skin extracts contain a collagenase activating enzyme with the inhibition profile of a serine proteinase. A chymotryptic proteinase with a similar inhibition profile was extracted from normal human skin and partially purified. This enzyme activated fibroblast procollagenase derived from tissue culture of normal skin. The procollagenase was also partially activated by plasmin and chymotrypsin. This is the first demonstration of a collagenase activating enzyme in human skin and raises the possibility that collagenase activation by this mechanism may be responsible for collagen degradation in some disease processes.
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PMID:Serine proteinase activation of latent human skin collagenase. 609 38

Three collagenases from Clostridium histolyticum, designated C1, C2, and C3, with apparent molecular weights of 96 000, 92 000, and 76 000 were purified. Peptide maps of the enzymes prepared by digestion with Staphylococcus aureus V-8 protease were found to be similar. Cleavage of native C1 with alpha-chymotrypsin or V-8 protease yielded C2 and C3. This suggested that proteolysis of the Mr 96 000 collagenase may have occurred in vivo, producing the other two lower molecular weight enzymes. Previously prepared antiserum directed against a form of the bacterial enzyme similar by molecular weight and charge to collagenase C3 and Fab' fragments generated from this antiserum inhibited the collagenolytic activity. C1, C2, and C3 were immunologically identical by Ouchterlony double diffusion, and C3 was able to compete with C1 for the antiserum binding site. The ability of each enzyme to bind to antiserum raised against the bacterial collagenase supported the hypothesis that these three proteins were closely related. Zinc analyses of C1 and C3 resulted in a value of 1.14 mol of zinc/mol of C1 and 0.82 mol of zinc/mol of C3. C1 did not contain carbohydrate as measured by gas-liquid chromatography or periodic acid-Schiff staining.
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PMID:Purification and characterization of three forms of collagenase from Clostridium histolyticum. 609 92


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