Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The chemical nature of anionic sites located on both fronts of the endothelial cells (ECs) and in the basement membrane (BM) of mouse brain capillaries was studied using tissue sections embedded in Lowicryl K4M and cationic colloidal gold. Before labelling with cationic probe, the sections were digested with the following enzymes: trypsin, papain, pronase E, proteinase K, collagenase, chondroitinase ABC, hyaluronidase, heparinase, heparitinase, neuraminidase and endoglycosidase H. The results indicate that the negatively charged surface layer on the luminal front differs in chemical nature from that on the abluminal front of the EC. Anionic sites located on the luminal surface of the plasmalemma of the ECs are mainly contributed by sialic acid residues of acidic glycoproteins. On the contrary, the anionic domains on the abluminal front of the EC represent mixed proteoglycan and acid glycopeptides containing hydrophobic amino acids, sialic acid residues, and are rich in heparan sulphate-bearing glycosaminoglycans. The anionic sites of the BM are contributed in a substantial degree by chondroitin and heparan sulphate-rich glycosaminoglycans. The effect of endoglycosidase H suggests that glycopeptides containing oligomannosyl residues linked to N-acetylglucosamine contribute in small degree in maintenance of the negative charge in the BM, but not on the surfaces of the EC. These results show that brain endothelium bears surface anionic domains differing chemically from those described for some fenestrated and continuous endothelia. The distribution of anionic sites indicates that the discrimination against various negatively charged molecules takes place on both fronts of the ECs as well as in the BM of brain micro-blood vessels. The exact role of these domains in the function of the blood-brain barrier remains to be established.
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PMID:Ultracytochemical characterization of anionic sites in the wall of brain capillaries. 274 7

A procedure for purification of surfactant-associated glycoproteins A from canine surfactant was established utilizing preparative isoelectric focusing as a major purification step in absence of detergents. The proteins migrated as charge trains, isoelectric points 4.2-5.0. Unglycosylated forms of surfactant-associated protein A1 (26 kDa) and glycoproteins A2 and A3 (32-36 kDa) were identified by silver-staining and immunoblot analysis. These forms were demonstrated to be identical polypeptides by fingerprint analysis of 125I-labeled peptides generated by tryptic-chymotryptic digests of the iodinated proteins. Size and charge heterogeneity were related to varying amounts of N-linked complex carbohydrates, including sialic acid, which were sensitive to endoglycosidase F and neuraminidase but resistant to endoglycosidase H. A collagenase-sensitive region was demonstrated which was required for sulfhydryl-dependent oligomerization of the molecule. Collagenase treatment resulted in removal of approx. 10 kDa from the glycoprotein molecule. Collagenase-resistant fragments of 21-23 kDa migrated with carbohydrate-dependent size and charge heterogeneity and were reduced to 16 kDa by endoglycosidase F. Amino acid composition of the surfactant glycoproteins demonstrated high glycine content which was diminished after digestion with collagenase. Several glycine-rich tryptic peptides were isolated by reverse-phase chromatography. Partial sequence information shows Gly-X-Y repeat sequences containing hydroxyproline residues. The major canine surfactant-associated proteins, glycoproteins A contain complex-type N-linked carbohydrate. In addition, a separate collagen-like peptide domain is present and is required for sulfhydryl-dependent oligomerization.
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PMID:Purification of canine surfactant-associated glycoproteins A. Identification of a collagenase-resistant domain. 300 81

Early steps in the biosynthesis of chondroitin sulfate proteoglycan (CSPG) and collagenous cartilage matrix molecules were examined by the comparison of products translated in mRNA-directed cell-free reactions and those synthesized by intact cartilage cells. RNA isolated from embryonic chicken sterna was used to direct cell-free translation reactions. Chicken sternal chondrocytes in culture were pulse-labeled with [35S]-methionine. The CSPG core protein was identified by immunoprecipitation. The Mr of the cartilage cell-synthetized core protein was determined to be 370K, approximately 10-15K greater than that of the comparable cell-free translation product. Experimental results strongly support the view that the observed difference in Mr reflects the cotranslational addition of mannose-rich, N-asparagine-linked oligosaccharides to the cell-synthesized core protein: 1) the cell-synthesized product was labeled with [3H]-mannose and precipitated by concanavalin A-sepharose beads; 2) the incorporated [3H]-mannose could be subsequently removed by digestion with endoglycosidase H (Endo H); 3) the Mr of the cell-synthesized core protein was reduced by Endo H digestion to that of the comparable cell-free translation product; 4) the core protein synthesized by tunicamycin-treated chondrocytes (inhibited in their ability to add N-asparagine-linked mannose-rich oligosaccharides to proteins) was comparable in electrophoretic mobility to that of the core protein cell-free translation product; and 5) the core protein translated in microsome-coupled cell-free reactions had an Mr 8-10K greater than that of the core protein translated in the absence of microsomes. For the purpose of examining biosynthetic intermediates, chondrocytes were labeled continuously or pulse-chase labeled for varying times. No biosynthetic CSPG intermediates migrating between the core protein and the CSPG monomer were detected. However, a band of 355Kdal appeared to share certain characteristics with the 307Kdal core protein (including its immunoprecipitability with CSPG antibodies), and a 340Kdal band was noted. Type II procollagen and other collagenase-sensitive products of 205Kdal and 110Kdal were observed among translation and chondrocyte-synthesized products. In chondrocytes, all three products exhibited labeling or chase time-dependent increases in Mr which were accelerated by ascorbate supplements and inhibited by the addition of alpha, alpha'-dipyridyl. These results suggest that the observed time-dependent increases in Mr are a consequence of collagen hydroxylation. The 110Kdal and 205Kdal collagenous proteins may be related to the minor collagens recently described in cartilage.
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PMID:Biosynthetic precursors of cartilage chondroitin sulfate proteoglycan. 330 Nov 84

The Heymann nephritis antigenic complex (HNAC) consists of two components, i.e., 1) gp330, a large glycoprotein localized in coated pits of the proximal tubule and glomerular epithelium, and 2) a 44-kDa protein which is homologous to the human alpha 2-macroglobulin receptor-associated protein (RAP). To examine the biosynthesis and assembly of HNAC, tissue fragments prepared from collagenase-digested 1-day-old rat kidneys were radiolabeled, and gp330 and RAP were immunoprecipitated with specific antibodies. By electron microscopy the tubule organization was seen to be largely intact. Results obtained on the biosynthesis of a control brush border protein, dipeptidylpeptidase IV (DPPIV), showed that tubules prepared in this manner are capable of synthesis and posttranslational processing of brush border membrane proteins and thus are suitable for short-term (< 3 h) biosynthetic experiments in vitro. Results of pulse chase and digestion with endoglycosidase H (Endo H) indicated that the time required for newly synthesized gp330 to mature in the endoplasmic reticulum (ER) and transit the middle Golgi compartments [half time (t1/2) = 90 min] was significantly longer than that of DPPIV (t1/2 = 20 min). Coprecipitation and cosedimentation (sucrose velocity gradient centrifugation) experiments showed that gp330 associates with RAP very early after synthesis and that the 44-kDa protein remains associated with gp330 during its subsequent folding, oligomerization, and transport to the Golgi. These findings demonstrate that HNAC assembles in at least two steps. The first step is the association of gp330 with RAP forming a large (19.3S) heterodimer, which sediments with the thyroglobulin (mol wt = 669,000) standard. This step begins within 30 min of synthesis and is Ca2+ dependent. The second step, which occurs > 60 min after synthesis, is the formation of a larger heterooligomer, which results in a shift in size of the complex from 19.3 to 38.6S. Both steps occur before acquisition of Endo H resistance. These results indicate that HNAC consists of a large multimeric complex that is assembled in the rough ER.
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PMID:Biosynthesis of the gp330/44-kDa Heymann nephritis antigenic complex: assembly takes place in the ER. 832 89