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)

This paper describes a large-scale purification procedure of the amiloride binding component of the epithelium Na+ channel. [3H]Phenamil was used as a labeled ligand to follow the purification. The first two steps are identical with those previously described [Barbry, P., Chassande, O., Vigne, P., Frelin, C., Ellory, C., Cragoe, E. J., Jr., & Lazdunski, M. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4836-4840]. A third step was a hydroxyapatite column. The purified material consisted of a homodimer of two 88-kDa proteins that migrated anomalously in SDS-PAGE to give an apparent Mr of 105,000. Deglycosylation by treatment with neuraminidase and endoglycosidase F or with neuraminidase and glycopeptidase F indicated that less than 5% of the mass of the native receptor was carbohydrate. Sedimentation analysis of the purified Na+ channel in H2O and D2O sucrose gradients and gel filtration experiments led to an estimated molecular weight of the [3H]phenamil receptor protein-detergent-phospholipid complex of 288,000 and of the native [3H]phenamil receptor protein of 158,000. [3H]Br-benzamil is another labeled derivative of amiloride that recognized binding sites that had the same pharmacological properties as [3H]phenamil binding sites and that copurified with them. Upon irradiation of kidney membranes, [3H]Br-benzamil incorporated specifically into a 185-kDa polypeptide chain under nonreducing electrophoretic conditions and a 105-kDa protein under reducing conditions. The same labeling pattern was observed at the different steps of the purification. Reconstitution of the purified phenamil receptor into large unilamellar vesicles was carried out. A low but significant phenamil- and amiloride-sensitive electrogenic Na+ transport was observed.
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PMID:[3H]phenamil binding protein of the renal epithelium Na+ channel. Purification, affinity labeling, and functional reconstitution. 216 Feb 71

Bovine plasma protein C inhibitor was purified; it was then characterized in comparison with human protein C inhibitor. The specific inhibitory activity of the purified inhibitor for bovine activated protein C was 8,500 times that of the inhibitor in plasma. The purified inhibitor showed a single band with Mr 56,000 by SDS-PAGE at pH 7.0, and two bands at pH 8.8, a major one with Mr 56,000 and a minor one with Mr 105,000, under both unreduced and reduced conditions. The pI range of the inhibitor was between 4.4 and 6.1. The Mr of the inhibitor was reduced by treatment with neuraminidase, O-glycanase, and also with glycopeptidase-A, suggesting that the inhibitor has both Asn-linked and Ser/Thr-linked carbohydrate chains. Twenty-seven of the NH2-terminal 49 amino acid residues of the bovine inhibitor, which lacks the first 4 residues from the NH2-terminal amino acid sequence of human inhibitor, were identical to those of the human inhibitor. The bovine inhibitor inhibited bovine and human activated protein C, human thrombin, Factor Xa, Factor XIa, and plasma kallikrein with Ki = 1.0, 5.2, 2.6, 3.0, 1.3 X 10(-8) M, and 4.5 X 10(-9) M, respectively. The inhibitory rates for activated protein C and thrombin were accelerated significantly in the presence of heparin or negatively charged dextran sulfate. However, the acceleration by heparin or dextran sulfate for the inhibition of Factor Xa, Factor XIa, and plasma kallikrein was not significant. The bovine inhibitor did not inhibit human Factor XIIa or plasmin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Bovine plasma protein C inhibitor with structural and functional homologous properties to human plasma protein C inhibitor. 216 Apr 49

The high Mr glycoprotein (gp300) of equine herpesvirus type 1 was found to have an Mr, estimated by SDS-PAGE, of over 400,000 and was confirmed as being a surface glycoprotein by 125I-labelling. In contrast to [3H]glucosamine, gp300 showed very low levels of [3H]glucosamine, gp300 showed very low levels of [3H]mannose incorporation. The Mr of gp300 showed no detectable change upon treatment of purified virus with N-glycanase, and showed only a small change in virus-infected cells treated with tunicamycin. In addition, gp300 failed to bind the lectin concanavalin A. Taken together, these results indicate a lack of N-linked carbohydrate on gp300. The major carbohydrate species were found to be composed primarily of O-linked chains, as indicated by the sensitivity of the protein to monensin, to exoglycanase enzymes specific for sugars present in O-linked chains and to mild alkaline borohydride treatment, which revealed three species of carbohydrate of Mr of greater than 10,000, 2400 and 1100, respectively. Neuraminidase treatment and binding of Helix pomatia lectin indicated the presence of alpha-N-acetylglucosamine and sialic acid as terminal sugars. Immunological cross-reactivity of gp300 with a high Mr protein of equine herpesvirus type 4 was shown and it also exhibited a marked Mr variation in the vaccine strain Rhinomune.
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PMID:Characterization of the high Mr glycoprotein (gP300) of equine herpesvirus type 1 as a novel glycoprotein with extensive O-linked carbohydrate. 217 54

The aim of this study was to isolate the Na(+)-independent bile acid transporter from rat canalicular plasma membranes by affinity chromatography. The affinity matrix used consisted of lysylcholic acid covalently linked to CH-Sepharose 4B, resulting in an anionic ligand essentially identical to glycocholic acid. The protein fraction, adsorbed and eluted from the affinity column, was markedly enriched in a 100-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) compared with the total membrane and membrane extract. The 100-kDa band, further purified by preparative SDS-PAGE, was electroeluted from excised gel fragments and used as an immunogen for antibody production in rabbits. The immune serum, but not preimmune serum, specifically recognized a single, 100-kDa polypeptide on one- and two-dimensional immunoblots of canalicular membranes. In contrast, no reactivity was observed with proteins in liver basolateral or ileal brush-border membranes. The 125I-labeled protein was immunoprecipitated from membrane extracts solubilized in NP-40 and was found to migrate with a pI of 5.3 on two-dimensional electrophoresis. The apparent molecular weight of the protein was reduced by 50% after deglycosylation with N-glycanase. The 100-kDa protein was localized specifically and exclusively by immunocytochemical methods to the bile canalicular domain of the hepatocyte plasma membrane. Moreover, the immunoglobin G fraction prepared from the antiserum significantly inhibited taurocholate transport by canalicular membrane vesicles and decreased the covalent labeling of the 100-kDa protein by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Thus the isolation of a single 100-kDa protein by bile acid-affinity chromatography, as well as the inhibitory effects of antibodies directed against this polypeptide, provide further support for its role in the canalicular transport of bile acids.
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PMID:Isolation and characterization of the canalicular membrane bile acid transport protein of rat liver. 218 68

In this report we describe, using a previously characterised monoclonal antibody (NC-2), the biochemical characteristics of a human leukaemia-associated alloantigen. Two proteins with molecular weights of 50 kDa and 15 kDa were immunoprecipitated from 125I surface labelled HL-60 cells. Both proteins appeared to be sensitive to digestion with trypsin, the 50 kDa protein in particular. Treatment with glycopeptidase F indicated the presence of N-linked oligosaccharides, whereas treatment with neuraminidase had no effect on the mobility of the antigens in SDS-PAGE indicating the absence of detectable sialic acid residues. Sensitivity to glycopeptidase F indicates that the reacting antigens are glycoproteins in nature. The antibody reacts with a range of normal tissues and appears to be associated with cytoplasmic granules in HL-60 cells.
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PMID:Biochemical characterisation studies on a leukocyte alloantigen expressed with high frequency in leukaemia patients. 223 48

Lectin IV of Griffonia simplicifolia (Mr approximately 56,000), which has a strong affinity for both the Lewis b and Y blood-group determinants, is a dimeric protein of two subunits, alpha (29 kDa) and beta (27 kDa), separable by SDS/PAGE and containing covalently linked oligosaccharide. After digestion with N-glycanase, the protein migrates as a single band with a mobility identical with that of the beta-subunit. After cleavage with hydroxylamine of 3H-labelled, but otherwise intact, lectin, the radioactively labelled oligosaccharide was found to be associated with two blocked N-terminal peptides separable by h.p.l.c. and having identical amino acid compositions. One of these had three or four glucosamine residues per molecule, whereas the other had only one or two. Sequence analyses of these, as well as of a 21 kDa hydroxylamine-cleaved fragment and of the intact lectin pretreated with pyroglutamate aminopeptidase, have provided a unique sequence for residues 1-62 of the two subunits. Evidence is presented for two sites of N-linked oligosaccharide attachment at Asn-5 and Asn-18. Whereas the alpha-subunit has oligosaccharide linked to both sites, the beta-subunit has carbohydrate associated with only one (Asn-18). Sugar analyses of the whole lectin reveal a monosaccharide composition of (Xyl)3(Fuc)2(Man)10(GlcNAc)6, representing 6.4% of the mass of the molecule. Taken together with the susceptibility of the Asn-5 linkage (but not of Asn-18) to N-glycanase digestion, the observations indicate that the structures of the oligosaccharides at residues 5 and 18 are different.
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PMID:Molecular-mass heterogeneity of Griffonia simplicifolia lectin IV subunits. Differences in the oligosaccharide moieties in the N-terminal region. 226 64

Two proteins from Leishmania donovani, dp72 and gp70-2, have been previously utilized to specifically serodiagnose patients with visceral leishmaniasis. The proteins were shown by ELISA and Western blotting with monoclonal and polyclonal antibodies to be present in both stages of the parasite. Antibodies to gp70-2 recognize in promastigotes multiple discrete bands of similar m.w. which are common to several isolates of L. donovani. The total amount of Ag and number of bands observed per isolate is not constant. Lectin blots with Con A show gp70-2 to be a glycoprotein. Dp72 shows pronounced microheterogeneity between isolates of L. donovani. The Brazilian isolates examined appear to possess a lower m.w. form (64,000 or 68,000) of this molecule. No reactions were observed with dp72 and lectins in Western blots; and neither tunicamycin, N-glycanase, endoglycosidase H nor F affected the migration of [35S]-methionine-labeled protein on SDS-PAGE. A mAb against dp72 also cross-reacted in Western blots with a 60-kDa protein in Leishmania major, Leishmania aethiopica, and Leishmania tropica. No reaction was observed between the purified promastigote surface protease (gp63) and either monoclonal or polyclonal antibodies produced to dp72 or gp70-2. The ability of the pure proteins to provide protection against a challenge by L. donovani amastigotes was examined. BALB/c mice were immunized with gp70-2 and/or dp72 by using Corynebacterium parvum as an adjuvant. Mice immunized with gp70-2 were not protected; however, mice receiving dp72 showed a 81.1% reduction in the liver parasitemia compared with the adjuvant controls.
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PMID:Characterization of two proteins from Leishmania donovani and their use for vaccination against visceral leishmaniasis. 229 7

The monoclonal antibody (MAb) 5T4 defines a human trophoblast antigen marker with a restricted pattern of expression in normal adult tissues but this antigen is expressed on a variety of carcinomas. The purification of 5T4 antigenic molecules is described from term syncytiotrophoblast by a combination of lectin- and immunoaffinity chromatography and gel filtration giving up to 10,000-fold purification with 70% yield. The antigen is carried by non-associated glycoprotein molecules with an apparent molecular weight of 72 kDa on SDS-PAGE and a neutral pI. Removal of N-linked sugars by N-glycanase reveals a core protein of 42 kDa. Treatment with enzymes that cleave O-linked sugars does not substantially alter the molecular size. The native 5T4 molecules are very resistant to proteolysis until the N-linked sugars are removed or the glycoprotein is denatured and reduced. Glycopeptides generated by these approaches will be suitable for amino acid sequencing.
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PMID:Isolation and characterization of 5T4, a tumour-associated antigen. 229 3

IL-5 is a T cell-derived lymphokine that induces B cell growth and differentiation in murine systems. In this study, we examined the role of carbohydrate moiety of IL-5 in the expression of biological function. IL-5 polypeptides translated in Xenopus oocytes were heterogeneous in terms of isoelectric point (pI 4.7 to 8.0) and m.w. (45,000 to 60,000 under nonreducing conditions) and yielded m.w. of 25,000 to 30,000 under reducing conditions. Treatment of rIL-5 with N-glycanase under reducing conditions yielded an IL-5 monomer of m.w. 12,000 to 14,000. Furthermore, deglycosylated rIL-5 that had been translated in the presence of tunicamycin showed very limited heterogeneity by two-dimensional gel electrophoresis (first dimension, nonequilibrium pH gradient electrophoresis; second dimension, SDS-PAGE). The m.w. was 27,000 to 28,000 under non-reducing conditions and migrated to m.w. 13,000 to 14,000 under reducing conditions. These results indicate that IL-5 is a glycoprotein carrying the N-glycosidically-linked carbohydrates. Treatment of IL-5 with sialidase caused the decrease in the heterogeneity in isoelectric point of IL-5. Deglycosylated rIL-5 that had been obtained from tunicamycin-treated oocytes could bind to IL-5-responding cells (T88-M), which express both high- and low-affinity IL-5 receptors, as efficient as intact rIL-5 under high-affinity conditions. Scatchard plot analysis of equilibrium binding of 35S-labeled rIL-5 to T88-M cells revealed that the dissociation constants (Kd) of glycosylated rIL-5 and deglycosylated rIL-5 were 127 pM and 110 pM, respectively. IL-5 activities determined by both B cell growth and differentiation assays were not affected by deglycosylation. These results indicate that N-linked glycoside moiety of IL-5 molecules may not play an essential role in the expression of its activity.
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PMID:Role of carbohydrate moiety of IL-5. Effect of tunicamycin on the glycosylation of IL-5 and the biologic activity of deglycosylated IL-5. 230 8

Nicotinic ACh receptors were immunoaffinity-purified from chick ciliary ganglia, radioiodinated, and examined by SDS-PAGE. Components with Mr's of 49, 52, and 60 kDa were obtained. Limited proteolysis produced different peptide maps from the components, confirming the 3 as distinct species. All are glycoproteins since treatment with glycopeptidase F altered their migration during electrophoresis. The 60 kDa component appears to be encoded by the AChR alpha 3 gene since it was selectively immunoprecipitated by an antiserum to a fusion protein containing a putative cytoplasmic region of the predicted alpha 3 gene product. The 49 kDa component selectively cross-reacted on immunoblots with 4 monoclonal antibodies that recognize a component of similar size in AChR preparations from chicken brain. The 52 kDa component is a novel species not previously identified in preparations of brain AChRs. If all 3 components represent integral AChR subunits, they may compose 2 receptor subtypes in the ganglion, e.g., one containing 49 and 60 kDa subunits and another containing 52 and 60 kDa subunits. This is supported by the finding that a receptor preparation can be obtained containing only the 49 and 60 kDa components and is consistent with reports of brain AChRs having only 2 types of subunits. Alternatively, ganglionic AChRs may contain 3 or more types of subunits, with at least one being selectively lost under certain conditions.
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PMID:Subunit composition of nicotinic acetylcholine receptors from chick ciliary ganglia. 235 45


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