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:3.5.1.52 (PNGase F)
1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The essential role of Factor VIII:C (FVIII:C, anti-hemophilia factor A) as a cofactor for Factor IXa-dependent activation of Factor X has been established. In this paper, we describe that capillary endothelial cells from bovine adrenal medulla express active FVIII:C gene. Accumulation of FVIII:C in conditioned media from an 8-day-old culture is approximately twice as high as that stored in the cell when immunoprecipitated FVIII:C was analyzed for its ability to convert Factor X to Factor Xa. Analysis of [35S]methionine-labeled and immunoprecipitated FVIII:C from cells or conditioned media on SDS-PAGE under fully denatured conditions indicated that the newly synthesized FVIII:C consists of heavy chain of M(r) 200,000 and light chain of M(r) 46,000. The secreted FVIII:C in the non-reduced condition however, has a molecular weight of 270,000 which suggests that in native protein, the heavy and light chains are held together by S-S bonds. Furthermore, susceptibility of the immunoprecipitated FVIII:C to N-glycanase digestion establishes that the endothelial cells derived FVIII:C contains asparagine-linked carbohydrate side chains.
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PMID:Expression of blood clotting factor VIII:C gene in capillary endothelial cells. 162 40

Cathepsin B was purified from normal human liver and several human tumour tissues and partially characterized. Three forms of cathepsin B, with molecular masses of 25 kDa, 26 kDa (the two appearing as a doublet) and 30 kDa, were detected in SDS/polyacrylamide gels. The 25-26 kDa doublet was associated with the fractions from tumours and normal liver containing the highest cathepsin B activity. Cathepsin B from both sources showed similar pH optima. Both normal liver and tumour cathepsin B exhibited similar kinetics against selected synthetic substrates. At neutral pH and 24 degrees C, cathepsin B from both normal liver and tumour exhibited a lower Km and a higher kcat./Km than at pH 6.0. Their inhibitory profiles against synthetic inhibitors were also similar. Immunological studies with a monospecific antibody against the mature double-chain form of human liver cathepsin B and an antibody against a cathepsin B-derived synthetic peptide established the immunological similarity of liver and tumour enzymes. The N-terminal sequences of the 25 kDa and 26 kDa forms were identical with that of the heavy chain of the mature double-chain form of human cathepsin B, whereas the N-terminal sequence of the 30 kDa species was identical with that of the single-chain form of human cathepsin B. Treatment of the double-chain form of cathepsin B from normal liver and tumours with the endoglycosidase peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase converted the 26 kDa form into 25 kDa in SDS/polyacrylamide gels, suggesting that cathepsin B may exist as both glycosylated and unglycosylated forms. Our results, in contrast with those reported earlier for mouse cathepsin B, indicate that human liver and tumour cathepsin B are similar.
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PMID:Human tumour cathepsin B. Comparison with normal liver cathepsin B. 163 35

We isolated 7.4 mg of pure renin from 2 kg of rat kidneys using affinity chromatography on pepstatin-aminohexyl-Sepharose and an octapeptide renin inhibitor, H-77-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that renin consists of two polypeptide chains linked by a disulfide bond, one of Mr = 36,000 (heavy chain) and the other of Mr = 3,000 (light chain). The amino-terminal 10-amino acid sequences of the heavy and the light chains were identical to the sequences beginning at Ser72 and Asp355, respectively, of the amino acid sequence of preprorenin deduced from the renin cDNA sequence. Amino acid sequencing of the carboxyl-terminal peptide of the heavy chain, generated by digestion with lysyl endopeptidase, showed that the carboxyl-terminal residue of the heavy chain is Phe. Thus, the propeptide of prorenin is cleaved after Thr71, followed by removal of two amino acids, Arg353 and Asn354, the result being formation of the heavy and light chains. Thus, the site of cleavage of rat prorenin is after a nonbasic amino acid, in contrast to the cleavage of the propeptide after a pair of basic amino acids in mouse submaxillary renin, human renal renin, and many secretory proteins. Treatment of renin with neuraminidase or glycopeptidase F had no apparent effect on the charge heterogeneity of renin. Glycosylation probably does not contribute to charge heterogeneity.
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PMID:Amino-terminal amino acid sequence and heterogeneity in glycosylation of rat renal renin. 201 14

A 56K protein co-purified with bovine milk fat globule membrane (MFGM) proteins bound to Wisteria floribunda agglutinin (WFA) like most MFGM glycoproteins. Treatment with N-glycanase or beta-N-acetylhexosaminidase abolished the lectin binding to the protein. Amino acid sequence and immunoblot analyses revealed that the 56K protein is an IgG heavy chain. Lectin column chromatography of the oligosaccharides released by hydrazinolysis from the purified IgG heavy chains revealed that 0.08% of the total N-linked sugar chains bind to a WFA-agarose column, suggesting that they contain the beta-N-acetylgalactosaminylated structure.
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PMID:A bovine IgG heavy chain contains N-acetylgalactosaminylated N-linked sugar chains. 775 1

Characterization of monoclonal antibodies (MAbs) produced for therapeutic or diagnostic purposes increasingly includes an assessment of their carbohydrate content. Using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC/PAD), we have analyzed the PNGase F released oligosaccharides of several IgG preparations including human polyclonal IgG, a humanized monoclonal IgG (MAb M115), and a murine monoclonal IgG (MAb MY9-6) derived respectively from serum, hybridoma cultures, and ascites fluid. The N-linked oligosaccharides released by PNGase F treatment of the above IgGs were found to consist mainly of neutral, fucosylated, biantennary species. Comparison of glycosylation of human polyclonal IgG, MAb M115, and MAb MY9-6 revealed differences in the levels of galactosylation and in the levels as well as the form of sialic acid present. HPAEC/PAD oligosaccharide profiling, combined with the use of enzymes (PNGase F, endoglycosidase F2, endoglycosidase H, neuraminidase, beta-galactosidase, and beta-N-acetylhexosaminidase), and monosaccharide analysis allowed making of tentative structural assignments. By performing monosaccharide analysis directly on PVDF electroblotted heavy and light chain bands separated by SDS-PAGE, it was verified that IgGs used in this study were glycosylated predominantly in their heavy chain.
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PMID:Analysis of carbohydrates on IgG preparations. 789 Dec 93

We have previously showed that factor X activator of Russell's viper venom (RVV-X) contains six N-linked oligosaccharide chains: four in the heavy chain and one in each of the two light chains [Gowda, D.C., Jackson, C.M., Hensley, P., & Davidson, E.A. (1994) J. Biol. Chem. 269, 10644-10650]. In the present study, we have investigated the role of the carbohydrate moieties in the structure and functional activity of RVV-X. Sequential removal of sugar residues from the terminal ends by exoglycosidases, up to 50% of total carbohydrates, did not significantly alter the activity of RVV-X, demonstrating that the peripheral carbohydrate moieties are not involved in interactions with factor X. However, removal of whole oligosaccharide chains by N-glycanase caused an almost total loss of the ability of RVV-X to activate factor X to factor Xa. In parallel with these observations, circular dichroism spectroscopy showed that complete deglycosylation, but not the removal of peripheral sugars, caused a significant change in the secondary structure. Together, these data demonstrate that the oligosaccharide chains are necessary for the functional activity, and that the trimannosylchitobiose core residues are sufficient for the maintenance of the native polypeptide structure.
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PMID:Core sugar residues of the N-linked oligosaccharides of Russell's viper venom factor X-activator maintain functionally active polypeptide structure. 863 44

As4.1, a renin-expressing cell line isolated from a mouse renal tumor, was characterized for synthesis, processing, storage and secretion of renin polypeptides. Metabolic labeling, immunoprecipitation and SDS/PAGE analysis revealed that renin was secreted into the culture supernatant predominantly in the form of prorenin which migrated as products of 42-47 kDa. The predominant intracellular renin was processed into two chains, of 33-34 and 5 kDa. N-glycanase treatment removed N-linked oligosaccharides and yielded products of 41 kDa for prorenin and 31-32 kDa for the heavier chain of two-chain renin. The N-terminus of the constitutively secreted prorenin was determined by automated Edman degradation to be Leu22 while the N-terminus of the heavy chain was Ser72. Renin polypeptides constituted 3.1 +/- 1.4% (mean percentage of total precipitable radioactivity +/- SD) of de-novo-synthesized protein secreted into the medium and 0.2 +/- 0.17% retained intracellularly. Extrapolation of renin activity assays suggest that a single cell stores approximately 680 fg of active renin. A slow incremental release into the medium of processed renin heavy chain was detected by immunoprecipitation and SDS/PAGE. Renin activity assays confirmed the release of approximately 4 fg prorenin and 0.32 fg active renin cell(-1) h(-1). Indirect immunofluorescence demonstrated intracellular renin to be distributed in a punctate pattern. Renin was found to be colocalized with the lysosomal marker, beta-glucuronidase, by double-fluorescent labeling. These cells have enabled characterization of glycosylated mouse renin-1 and may prove a valuable tool for studying intracellular trafficing of renin and associated processing enzymes.
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PMID:Biosynthesis of renin in mouse kidney tumor As4.1 cells. 903 Jul 38

Pre-alpha-inhibitor (P alpha I) is a serine proteinase inhibitor from human plasma. It comprises bikunin (BK) responsible for antiprotease activity, covalently linked to a heavy chain H3. Here we describe its isolation from a side fraction of an industrial preparation of plasma clotting factors. By using a highly specific polyclonal antiserum prepared from rabbit immunized with a H3P polypeptide obtained in a bacterial expression system, we were able to identify the fractions containing P alpha I. Then, taking advantage of the differential affinity of the members of the inter-alpha-inhibitor family (I alpha I) for heparin-Sepharose and blue-Sepharose, we isolated P alpha I. Its specific antitryptic activity was 580 IU/g, higher than that of I alpha I: 420 IU/g. Its M(r), determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with or without prior reduction, was 130,000. Its peptide chains were identified by N-terminal sequencing. The H3 heavy chain was isolated from P alpha I by alkaline dissociation and anion-exchange chromatography. Its electrophoretic mobility was compared to that of the HI and H2 heavy chains of I alpha I. In reducing conditions, it was quite similar to that of H2 (M(r) 85,000) but clearly different from that of H1 (M[r] 78,000). Thus, the so-determined apparent M(r) of H3 was overestimated since its molecular mass determined by MALDI-TOF was 74,100. This result agrees with the proposed structure for H3. Indeed, by carbohydrate analysis and PNGase F digestion, we demonstrate that the two potential N-glycosylation sites present in the core-protein (theoretical mass: 69,454) are really occupied by two N-glycans, probably of biantennary type.
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PMID:Human pre-alpha-inhibitor: isolation from a by-product of industrial scale plasma fractionation and structural analysis of its H3 heavy chain. 918 16

An important risk factor for thrombosis is the polymorphism R506Q in factor V that causes resistance of factor Va to proteolytic inactivation by activated protein C (APC). To study the potential influence of the carbohydrate moieties of factor Va on its inactivation by APC, factor V was subjected to mild deglycosylation (neuraminidase plus N-glycanase) under nondenaturing conditions. The APC resistance ratio values (ratio of activated partial thromboplastin time [APTT] clotting times with and without APC) of the treated factor V were increased (2.4 to 3.4) as measured in APTT assays. O-glycanase treatment of factor V did not change the APC resistance ratio. The procoagulant activity of factor V as well as its activation by thrombin was not affected by mild deglycosylation. Treatment of factor V with neuraminidase and N-glycanase mainly altered the electrophoretic mobility of the factor Va heavy chain, whereas treatment with O-glycanase changed the mobility of the connecting region. This suggests that the removal of the N-linked carbohydrates from the heavy chain of factor Va, which is the substrate for APC, is responsible for the increase in susceptibility to inactivation by APC. Thus, variability in carbohydrate could account for some of the known variability in APC resistance ratios, including the presence of borderline or low APC resistance ratios among patients who lack the R506Q mutation.
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PMID:The carbohydrate moiety of factor V modulates inactivation by activated protein C. 919 57

Western blots, enzyme assays, protein glycosylation studies, and immunohistochemical staining were used to characterize cathepsin B expression at successive stages of colorectal tumor progression. In normal colon mucosa and premalignant adenomas, cathepsin B expression was predominantly due to mature two-chain protein detected on Western blots as the nonglycosylated 27-kDa form, with overexpression of this protein occurring in only 4 of 18 adenomas. Overexpression increased significantly in Dukes A and B carcinomas (26 of 37 cases), with cathepsin B protein generally detectable in carcinomas as a combination of both 27-kDa nonglycosylated and 28-kDa glycosylated mature two-chain forms. Glycosylated cathepsin B protein in carcinoma extracts was sensitive to PNGase F but resistant to Endo H, indicating a pattern consistent with complex rather than high mannose type glycosylation. When sorted by advancing tumor stage, peak expression of cathepsin B protein occurred in carcinomas involved in local invasion compared with adenomas or metastatic cancers. At all stages, cathepsin B activity correlated significantly with the levels of heavy chain mature cathepsin B protein (r = 0.6682, p < 0.0001) irrespective of glycosylation. Immunohistochemical staining of cathepsin B protein revealed fine diffuse cytoplasmic staining in both adenomas and carcinomas compared with coarse granular cytoplasmic staining (typical of lysosomes) seen in matched normal mucosa. Our results demonstrate several sequential, apparently independent changes in cathepsin B expression during colorectal tumor progression including early changes in subcellular localization, up-regulation of cathepsin B protein and activity in invasive cancers, and altered protein glycosylation detected in malignant tumors at all stages.
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PMID:Elevations in cathepsin B protein content and enzyme activity occur independently of glycosylation during colorectal tumor progression. 936 Sep 97


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