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)

Human gingival fibroblast procollagenase has been purified to apparent homogeneity from serum-free and serum-supplemented fibroblast culture medium by a combination of ammonium sulfate precipitation, CM-cellulose chromatography, and gel-filtration on Bio-Gel P-150. Sodium dodecylsulfate polyacrylamide gel electrophoretic studies suggests that the purified fibroblast proenzyme is comprised of two closely related zymogens with the estimated Mr of 57,000 and 52,000. Upon densitometric scanning of the gels, the ratio of the two proenzyme forms was about 1 : 4 (57 : 52 kdal). Limited proteolysis of the fibroblast procollagenase with trypsin resulted in the conversion of both proenzyme forms into active enzyme forms of Mr 48,000 and 44,000, respectively. Amino acid analysis of the active enzymes and proenzyme forms revealed that the active enzymes contained fewer basic amino acids than do the proenzyme forms. The purified trypsin-activated fibroblast collagenase hydrolyzed type I collagen fibrils, cleaved tropocollagen in solution at 24 degrees C into TCA and TCB fragments, and cleaved the synthetic peptide substrate, DNP-peptide III, at the Gly-Ile bond. The gingival fibroblast collagenase exhibited a pH optimum of 7.5, was completely inhibited by EDTA or dithioerythritol but was not inhibited by N-ethylmaleimide or phenylmethylsulfonyl fluoride, and appeared to cleave human type III collagen approximately 10-fold faster than homologous type I collagen. In addition, comparison of the biochemical properties of the precursor and active forms of human gingival fibroblast collagenase with the precursor and active forms of human skin fibroblast collagenase, previously characterized by Stricklin and co-workers (Biochemistry 17: 2331-2337, 1978), revealed that they were similar in Mr, amino acid composition, and substrate specificity. Furthermore, the human gingival and skin fibroblast procollagenases were immunologically identical.
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PMID:Human gingival fibroblast collagenase: purification and properties of precursor and active forms. 632 84

Native collagen polypeptides exist in a unique triple helical conformation resistant to most proteinases. In this study, the stability of type I collagen triple helix, employing a mixture of trypsin and alpha-chymotrypsin as a proteolytic probe, was examined. The degradation of type I [3H]collagen was monitored as 3H-labeled peptides soluble in trichloroacetic acid (TCA) or by sodium dodecyl sulfate (SDS)-polyacrylamide slab gel electrophoresis. In one set of experiments, collagen substrates were preincubated at various temperatures for up to 8 h, followed by a 15-min proteolytic treatment at the same temperature. At 43 degrees C, most of the collagen was degraded, while the fraction of the substrate degraded at 40, 38, and 35 degrees C was 53, 41 and 19%, respectively. This fraction was independent of the preincubation time which varied from 10 to 480 min. Thus, at any given temperature, a constant fraction of the collagen substrate was susceptible to proteolysis. Measurement of the midpoint temperature (Tm) of the helix to coil transformation for type I collagen, at neutral pH employing an increasing temperature gradient and brief proteolysis at the individual temperatures, indicated a value of 38.8 degrees C. However, determination of the Tm by employing proteolytic digestions at a constant temperature (30 degrees C) using conditions under which the nonhelical peptides are readily digested to TCA-soluble peptides while native collagen resists such proteolysis, indicated a value of 42.7 degrees C. In further studies, collagen was subjected to continuous proteolysis for up to 24 h. A large fraction of collagen was digested at 30 or 34 degrees C, temperatures well below the Tm of the helix to coil transformation. SDS-polyacrylamide gel electrophoresis of the degradation products obtained at these temperatures revealed multiple cleavage fragments. Finally, temperature double-jump experiments indicated that the destabilization of the triple helix is reversible provided that the Tm of the substrate is not exceeded. The results provide evidence for reversible and local relaxation of the collagen triple helix.
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PMID:Conformational stability of type I collagen triple helix: evidence for temporary and local relaxation of the protein conformation using a proteolytic probe. 634 98

Chondrocytes produce large pericellular coats in vitro that can be visualized by the exclusion of particles, e.g., fixed erythrocytes, and that are removed by treatment with Streptomyces hyaluronidase, which is specific for hyaluronate. In this study, we examined the kinetics of formation of these coats and the relationship of hyaluronate and proteoglycan to coat structure. Chondrocytes were isolated from chick tibia cartilage by collagenase-trypsin digestion and were characterized by their morphology and by their synthesis of both type II collagen and high molecular weight proteoglycans. The degree of spreading of the chondrocytes and the size of the coats were quantitated at various times subsequent to seeding by tracing phase-contrast photomicrographs of the cultures. After seeding, the chondrocytes attached themselves to the tissue culture dish and exhibited coats within 4 h. The coats reached a maximum size after 3-4 d and subsequently decreased over the next 2-3 d. Subcultured chondrocytes produced a large coat only if passaged before 4 d. Both primary and first passage cells, with or without coats, produced type II collagen but not type I collagen as determined by enzyme-linked immunosorbent assay. Treatment with Streptomyces hyaluronidase (1.0 mU/ml, 15 min), which completely removed the coat, released 58% of the chondroitin sulfate but only 9% of the proteins associated with the cell surface. The proteins released by hyaluronidase were not digestible by bacterial collagenase. Monensin and cycloheximide (0.01-10 microM, 48 h) caused a dose-dependent decrease in coat size that was linearly correlated to synthesis of cell surface hyaluronate (r = 0.98) but not chondroitin sulfate (r = 0.2). We conclude that the coat surrounding chondrocytes is dependent on hyaluronate for its structure and that hyaluronate retains a large proportion of the proteoglycan in the coat.
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PMID:Pericellular coat of chick embryo chondrocytes: structural role of hyaluronate. 650 14

Pieces of trypsin-isolated 14-day embryonic mouse epidermis were recombined with various living or non-living dermal or non-dermal substrates, in order to analyse the reconstruction of the dermal-epidermal junction. The constitution and ultrastructure of the epidermal basement membrane were characterized by immunolabelling of laminin, type IV collagen and bullous pemphigoid antigen, and by transmission electron microscopy. Trypsin treatment of dorsal skin followed by dermal-epidermal separation does not visibly damage the epidermal basement membrane, which remains attached to the lower face of epidermis. When freshly isolated epidermis is reassociated with dermis, the basement membrane is first degraded during the first 4 h of culture, then reconstituted within 24 h. When epidermis is cultured in isolation the basement membrane disappears within 4 h and is not reconstructed. Epidermis, precultured for 4 h and thus deprived of its basement membrane prior to reassociation, is able to reconstruct an antigenically and ultrastructurally normal basement membrane, when recombined with living or frozen-killed (-20 degrees C) dermis, with muscle tissue, or with a film of fibrous type I collagen. No basement membrane is reconstituted when the epidermis is recombined with heat (100 degrees C) killed dermis. It is concluded that, in the reconstituted epidermal basement membrane, laminin, type IV collagen, bullous pemphigoid antigen, and lamina densa are of exclusive epidermal origin.
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PMID:Reconstitution of the epidermal basement membrane after enzymatic dermal-epidermal separation of embryonic mouse skin. 653 37

Collagen chains separated on 5% SDS-acrylamide gels were stained with a 0.1% Sirius F3BA solution in saturated aqueous picric acid. After destaining the gels with methanol: acetic acid: water (30: 7 : 63), they were scanned at 540 nm and the area under each peak was determined. After that, the bands were sliced and the slices incubated overnight with trypsin at 37 degrees C. The color of the slices was eluted completely when the denatured collagen was ingested with trypsin. The absorbance of the color eluted was determined at 540 nm. The results obtained demonstrated that both procedures are reproducible and linear from 10 to at least 120 micrograms of protein. The correlation coefficient between both procedures was greater than 95%. The color is stable and the same end-point is obtained after destaining. In order to test the usefulness of the procedure in the typing of collagens from parenchymatous tissues, liver biomatrix was prepared from normal and cirrhotic human specimens obtained at autopsy. Over 95% of the collagen originally present in each liver was recovered in the corresponding biomatrix . We also showed that over 80-85% of biomatrix collagen could be solubilized with pepsin. These extracts were neutralized to pH 7.0 to inactivate pepsin and were applied onto the acrylamide gels. After electrophoresis and staining with Sirius red the types of collagen were determined by densitometric analysis. Our findings confirmed the results obtained by others using more complex and time consuming methodologies, mainly that type I and type III collagens are present in the normal liver in equal concentrations and that the ratio of I/III is 1. In all the cirrhotic livers investigated, the ratio of type I/type III collagen was greater than 1 due to an increase in type I collagen.
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PMID:A simple quantitative method for collagen typing in tissue samples: its application to human liver with schistosomiasis. 672 51

During histochemical studies of connective tissue in the early 1960's, striking differences were observed between basement membranes and collagen fibers. A third histochemically distinct collagen was identified in premature infants. At that time, these findings could not be correlated with chemical data. However, during the last decade chemists described several types of collagen. Correlation of chemical, histochemical and immunofluorescence data indicated that the tendon-type collagen seen in coarse collagen fibers, e.g. in skin and adventitia of adults, contains type I collagen. The distribution of embryonic-type or pseudo-elastic collagen was similar to that of type III collagen; the major exception was reticulum fibers. Since antibodies and dyes are bound at different sites, it seems possible that fibers with the immunofluorescence characteristics of type III collagen may differ in their binding sites for other reagents. The trypsin-lresistant protein of basement membranes corresponded to type IV collagens. The collagen formed in glomerulosclerosis was histochemically indistinguishable from tendon-type, i.e. type I, collagen. A review or early literature since 1850 showed that histologists repeatedly described distinct chemical and histochemical differences between various collagens. These long ignored findings can easily be fitted into the framework of current collagen chemistry.
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PMID:Histochemical investigations of different types of collagen. 680 64

This review deals with some structural features of the collagen molecules involved in the adhesion of platelets representing the initial step of hemostasis, thrombosis, and (partly) atherosclerosis. The adhesion occurs at the level of a vascular lesion or deendothelialized area, whatever the genetic type of collagen. In vitro experiments with purified collagens have shown that vascular interstitial collagens (types I and III, the latter present in subendothelium) as well as basement membrane-derived collagens (types IV and V) induce an adhesion of platelets, provided that an ordered arrangement linked to the quaternary and tertiary structures of their molecule is preserved. Whatever the quaternary structure, the important point seems to be the size of the fibers and more precisely the availability of an optimal number of adhesion sites on multimerized fibers. Various direct or indirect proofs (for example, the occurrence of the impairment of collagen multimerization on platelet adhesion/aggregation) are reviewed. Our recent studies on interstitial collagens have shown the involvement of certain specific amino-acid sequences obtained after cyanogen bromide cleavage of collagen. These are the C-terminal alpha1 (I) CB6 peptide of the alpha 1 chains of type I collagen (216 amino acids) and the central alpha1 (III) CB4 peptide from type III collagen (149 amino acids) Cleavage of this last peptide by chymotrypsin, hydroxylamine, and trypsin has suggested the possibility that a nonapeptide (sequence gly-lys-hyp-gly-glu-hyp-gly-pro-lys) is a minimum site of adhesion for platelets. This assumption has been reinforced by the fact that a synthetic nonapeptide with this sequence specifically inhibits the aggregation of platelets to collagen in vitro. The adhesion of platelets may consequently be due to the repetitive staggering of short amino acid sequences (such as this nonapeptide from type III collagen) along the rigid structure formed by a multimerized collagen fiber.
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PMID:[The adhesion of blood platelets to collagen: molecular features of collagen (author's transl)]. 702 81

The enzymatic mechanism of proteoglycan breakdown is of major interest, since it has been proposed that osteoarthritis involves increased proteolytic breakdown of proteoglycans. This paper describes the properties of the proteoglycan-degrading enzymes released into the extracellular milieu by chondrocyte cultures that produce cartilage-specific type II collagen but no detectable type I collagen. Attention has been focused on enzymes active at neutral pH, since the pH of the extracellular matrix is around neutrality. Biogel P-60 chromatography of concentrated culture medium showed a major peak of enzyme activity on proteoglycan monomer entrapped in polyacrylamide beads as well as on native proteoglycan aggregates. The enzyme yields a specific limit digestion peptide from the aggregate of approximately 55,000 daltons (in the presence of SDS). This limit peptide is probably derived from the hyaluronic acid-binding region of proteoglycan. The proteolytic enzyme is latent but can be activated by aminophenylmercuric acetate or trypsin. The molecular weight of both the active and latent forms, determined by gel filtration, is approximately 33,000. The activity is not inhibited by phenylmethylsulfonyl fluoride or pepstatin but is completely inhibited by o-phenanthroline; the activity is restored by Zn or Co ions in the presence of calcium chloride. Removal of calcium by dialysis results in a reversible loss of activity. The release of such a metalloproteinase by chondrocytes into the extracellular milieu, its activity at physiological pH and its ability to degrade native proteoglycans are consistent with a role of the enzyme in proteoglycan metabolism.
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PMID:The properties of the neutral proteinase released by primary chondrocyte cultures and its action on proteoglycan aggregate. 705 34

We have previously demonstrated that chick-skin type I collagen and the alpha 1(I) chain mediate platelet aggregation. Aggregation was associated with specific binding of these substances by platelet membranes. We now describe the isolation and purification of the receptor from isolated human platelet membrane. The receptor can be solubilized from platelet membranes with 0.5% Triton or 0.5% sodium deoxycholate. Using the combination of gel filtration, affinity column chromatography on alpha 1-Sepharose 4B, and preparative slab gel electrophoreses, the receptor protein can be purified to a single band as judged on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its activity sulfate-polyacrylamide gel electrophoresis. Its activity was destroyed by incubation with trypsin or Pronase. The apparent molecular weight was estimated to be 65,000 by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Bound [14C]glycine-labeled alpha 1(I) is displaced by unlabeled alpha 1 chain. The binding of [14C]glycine-labeled alpha 1(I) by the purified alpha 1(I) receptor can also be inhibited by the receptor isolated from type I fibrillar collage-Sepharose affinity chromatography. The data suggest that the alpha 1(I) binding site is identical with the type I fibrillar collagen binding site.
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PMID:Isolation and purification of collagen alpha 1(I) receptor from human platelet membrane. 708 40

A comprehensive approach for the structural microanalysis of collagen based of collagen based on high-performance liquid chromatography (h.p.l.c.) has been developed using calf skin type I collagen as a model system. The alpha, beta and gamma components were separated, after heat denaturation, on a TSK 4000 SW gel permeation column, using a nonvolatile buffer. Monitoring at 210 nm permits the detection of 1 microgram of a single chain. The alpha 1(I) and alpha 2(I) chains were completely resolved using a large-pore reversed-phase column (Vydac 201 TP 4.6) eluted by an aqueous acetonitrile gradient (24-48%) containing 0.01 M heptafluorabutyric acid as an ion-pairing agent. The purified alpha 1(I) chain was digested with CNBr and the resulting fragments separated in the same chromatography system with a gradient containing a 12.8-44.8% acetonitrile gradient. The purified alpha 1(I)CB 3 peptide was further cleaved with trypsin and the resulting peptides separated first by a similar chromatography with a 4-32% acetonitrile gradient. Resolution of some poorly separated peptides was obtained by a rechromatography using trifluoroacetic acid as counterion. The isolated peptides were hydrolyzed and identified by their amino-acid composition. Sequencing of h.p.l.c.-purified alpha 1(I)CB 3 was also performed to demonstrate the suitability of the technique for the preparation of peptides for amino-acid sequencing. This study demonstrates that detailed structural analysis can be performed on 3 mg of a purified collagen.
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PMID:A comprehensive approach to the study of collagen primary structure based on high-performance liquid chromatography. 711 47


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