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)

Saccharomyces cerevisiae contains an amphiphilic cAMP-binding glycoprotein at the outer face of the plasma membrane (M(r) = 54,000). It is converted to a hydrophilic form by treatment with glycosyl-phosphatidylinositol-specific phospholipases C and D (GPI-PLC/D), suggesting membrane anchorage by a covalently bound glycolipid. Determination of the constituents of the purified anchor by gas-liquid chromatography and amino acid analysis reveals the presence of glycerol, myo-inositol, glucosamine, galactose, mannose, ethanolamine, and asparagine (as the carboxyl-terminal amino acid of the Pronase-digested protein to which the anchor is attached). Complementary results are obtained by metabolic labeling, indicating that fatty acids and phosphorus are additional anchor constituents. The phosphorus is resistant to alkaline phosphatase, whereas approximately half is lost from the protein after treatment with GPI-PLD or nitrous acid, and all is removed by aqueous HF indicating the presence of two phosphodiester bonds. Inhibition of N-glycosylation by tunicamycin or removal of protein-bound glycan chains by N-glycanase or Pronase does not abolish radiolabeling of the anchor structure by any of the above compounds. Analysis of the products obtained after sequential enzymic and chemical degradation of the anchor agrees with the arrangement of constituents in GPIs from higher eucaryotes. Evidence for anchorage of the yeast cAMP-binding protein by a GPI anchor is strengthened additionally by the reactivity of the GPI-PLC-cleaved anchor with antibodies directed against the cross-reacting determinant of trypanosomal variant surface glycoproteins.
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PMID:The cAMP-binding ectoprotein from Saccharomyces cerevisiae is membrane-anchored by glycosyl-phosphatidylinositol. 133 92

Purified plasma membranes from the yeast Saccharomyces cerevisiae bind about 1.2 pmol of cAMP/mg of protein with high affinity (Kd = 6 nM). By using photoaffinity labeling with 8-N3-[32P]cAMP, we have identified in plasma membrane vesicles a cAMP-binding protein (Mr = 54,000) that is present also in bcy1 disruption mutants, lacking the cytoplasmic R subunit of protein kinase A (PKA). This argues that it is genetically unrelated to PKA. Neither high salt, nor alkaline carbonate, nor cAMP extract the protein from the membrane, suggesting that it is not peripherally bound. The observation that (glycosyl)phosphatidylinositol-specific phospholipases (or nitrous acid) release the amphiphilic protein from the membrane, thereby converting it to a hydrophilic form, indicates anchorage by a glycolipidic membrane anchor. Treatment with N-glycanase reduces the Mr to 44,000-46,000 indicative of a modification by N-linked carbohydrate side chain(s). In addition to the action of a phospholipase, the efficient release from the membrane requires the removal of the carbohydrate side chain(s) or the presence of high salt or methyl alpha-mannopyranoside, suggesting complex interactions with the membrane involving not only the glycolipidic anchor but also the glycan side chain(s). Topological studies show that the protein is exposed to the periplasmic space, raising intriguing questions for the function of this protein.
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PMID:A cAMP-binding ectoprotein in the yeast Saccharomyces cerevisiae. 165 42

Mo3 is an activation Ag expressed on the surface of human mononuclear phagocytes stimulated in vitro or in vivo by various activating factors. Mo3 is obtained by immunoprecipitation with anti-Mo3 mAb from lysates of PMA-stimulated U-937 cells. The Ag is a heterogeneous glycoprotein with a molecular mass range of 42 to 66 kDa (nonreducing conditions) containing N-linked carbohydrate chains. When the cells are treated with phosphatidylinositol-specific phospholipase C, greater than 60% of total precipitable gp42-66 Ag is released in the supernatant. This phosphatidylinositol-specific phospholipase C-sensitive linkage to the plasma membrane has provided a means for the one-step purification of Mo3 by immunoaffinity chromatography. The eluted soluble Mo3 (sMo3) was greater than 90% pure as documented by the appearance of a single major protein peak on reverse phase HPLC and SDS-PAGE. The average yield was 12.1 micrograms/10(8) cells. Sufficient quantities of sMo3 have been purified to permit the determination of amino acid and carbohydrate composition. Complex N-linked carbohydrates make up nearly 50% of the glycoprotein content and contribute to its heterogeneity. An anti-Mo3 polyclonal antiserum generated from sMo3 was used to immunoprecipitate Mo3 and its precursor from biosynthetically labeled, PMA-stimulated U-937 cells or LPS-stimulated monocytes. These 35S-methionine "pulse-chase" experiments demonstrated the existence of a 40- to 42-kDa endo-beta-N-acetylglucosaminidase-sensitive precursor, which over a period of 4 to 5 h gave rise to an endo-beta-N-acetylglucosaminidase-resistant, but N-glycanase-sensitive 42- to 66-kDa mature form.
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PMID:Purification, biochemical composition, and biosynthesis of the Mo3 activation antigen expressed on the plasma membrane of human mononuclear phagocytes. 186 26

Mo3 is an activation Ag expressed by human monocytic cells after stimulation in vitro by PMA, LPS, certain cytokines, and muramyl dipeptide. The structural characterization of Mo3 has been made possible by the development of a mAb (anti-Mo3f) that immunoprecipitates Mo3 from Nonidet P-40 lysates of radiolabeled PMA-stimulated U-937 cells and LPS-activated monocytes. On SDS-PAGE (nonreducing conditions) of anti-Mo3f immunoprecipitates, U-937 Mo3 is a single broad band of 39 to 66 kDa, whereas monocyte Mo3 is smaller with an apparent molecular mass of 32 to 56 kDa. Under reducing conditions, there is an increase in the m.w. of both species of Mo3 suggesting the existence of internal disulfide bonds. Mo3 is a glycoprotein with carbohydrate of the N-linked complex type as evidence by a reduction in m.w. by 40 to 50% after treatment with endoglycosidase F or N-glycanase; neuraminidase treatment produces a 3-kDa reduction in m.w. Deglycosylated Mo3 isolated from U-937 and monocytes have similar m.w. suggesting that the molecular heterogeneity of the native Mo3 may be due to differences in glycosylation. Mo3 is sensitive to phosphatidylinositol-specific phospholipase C with the release of native Mo3 from the surface of PMA-stimulated U-937 cells. These results indicate that Mo3 is a member of the glycosylphosphatidylinositol-linked family of surface glycoproteins.
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PMID:A structural characterization of the Mo3 activation antigen expressed on the plasma membrane of human mononuclear phagocytes. 213 44

Human Fc gamma RIII (CD16), a low-affinity receptor expressed on several different cell types, has a polymorphism on polymorphonuclear cells (Fc gamma RIIIPMN) identified by the NA1 and NA2 markers. Inasmuch as this polymorphism has functional consequences, an understanding of the structural biology of Fc gamma RIII may provide important insight into receptor function. We analyzed Fc gamma RIIIPMN by SDS-PAGE and found that receptor from individuals allotyped for either NA1 or NA2 contained only one protein after removal of N-linked glycosylations (19 and 21 kDa respectively) whereas receptor from NA1/2 individuals contained both bands. Because some reports indicate that digestion of Fc gamma RIII on NK cells (Fc gamma RIIINK) with N-glycanase also results in two bands on SDS-PAGE, we investigated Fc gamma RIIINK to explore the possibility of a corresponding allelic polymorphism in this receptor. Contrary to expectation, Fc gamma RIIINK from all donors irrespective of their NA allotype contained two bands (20 and 24 kDa) on SDS-PAGE after deglycosylation. In addition, those distinct epitopes on the extracellular domain of Fc gamma RIIIPMN found with mAb B73.1 and CLB gran 11 in association with the NA allotypic differences are expressed (or not expressed) on Fc gamma RIIINK independent of donor NA allotype. Fc gamma RIIIPMN and Fc gamma RIIINK differ at the protein level as they have different m.w. (glycosylated and deglycosylated), different epitopes in the extracellular domain (not attributable to tissue-specific glycosylation), and differential expression of the NA allelic protein polymorphism. Although the membrane anchor of Fc gamma RIIIPMN is a phosphatidylinositol-specific phospholipase C sensitive glycosyl-phosphatidylinositol linkage, Fc gamma RIIINK is insensitive to phosphatidylinositol-specific phospholipase C. However, a form of Fc gamma RIIINK is released from NK cells upon incubation at 37 degrees C. Thus, the basis for the two bands in Fc gamma RIIINK after N-linked deglycosylation is neither coexpression of two molecular isoforms with different membrane anchors nor an identifiable allelic polymorphism in m.w. restricted to Fc gamma RIIINK (p less than 10(-6)). The differences between the two receptors indicate that, independent of cell anchor type, PMN and mononuclear cells must have different molecular isoforms. The allelic variants, different isoforms, alternative anchor mechanisms and release processes provide for an extensive genetic and regulatory diversity in Fc gamma RIII function.
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PMID:Human Fc gamma RIII (CD16). Isoforms with distinct allelic expression, extracellular domains, and membrane linkages on polymorphonuclear and natural killer cells. 247 7

Isolation of two membrane-bound alkaline phosphatase (AP) species from avian growth plate cartilage matrix vesicle (MV) fractions is described. AP was first released from the membranes by phosphatidylinositol-specific phospholipase C (PIase C), followed by chromatography on DEAE-Bio-Gel A and Reactive-Red agarose. Two AP species having apparent Mr of 81.5 and 77 kDa by SDS-PAGE were purified in high yield and specific activity by this simple method. Treatment with neuraminidase to remove sialic acid residues reduced their size slightly, but did not diminish the difference in Mr between the two species. Digestion with N-glycanase, however, decreased both AP species to a common size of 59 kDa. This reveals that both enzymes are highly glycosylated and suggests that the two forms may result from differences in degree of glycation. The amino acid compositions of the two avian enzyme forms are very similar, but are markedly enriched in serine, glycine and glutamate when compared to those reported for mammalian liver-kidney-bone AP. Possible differences in amino acid sequence between the two avian forms have not been excluded. The cross-reactivity of polyclonal antibodies to these enzymes with bovine kidney, but not intestinal AP, indicate that the avian cartilage APs are of the liver-kidney-bone isozyme type.
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PMID:Isolation of two glycosylated forms of membrane-bound alkaline phosphatase from avian growth plate cartilage matrix vesicle-enriched microsomes. 280 49

Carbonic anhydrase (CA) was purified from the gills of the shore crab Carcinus maenas using affinity chromatography and HPLC. The predominantly membrane-bound CA was found to share several features with mammalian CA IV. Its apparent molecular weight of 36 kDa was reduced to 33 kDa by treatment with PNGase F, suggesting that crab CA is a glycoprotein with one N-linked oligosaccharide chain. More than half of the membrane-bound crab CA was released from membranes by treatment with a phosphatidylinositol-specific phospholipase C, indicating that the branchial CA is anchored to membrane surfaces by a phosphatidylinositol-glycan linkage. The enzyme also resembles mammalian CA IV in its relative sensitivity to inhibition by sulfonamides and the resistance to inhibition by halide ions. Amino acid composition of the HPLC-purified crab CA was examined and CNBr cleavage was carried out followed by N-terminal amino acid sequencing. Amino-terminal sequence of the native enzyme differed considerably from those of mammalian isozymes (human CA I and CA II, bovine CA III, human and rat CA IV). However, antisera raised against rat CA IV, CA II, and CA I all cross-reacted weakly with crab CA. Unlike mammalian CA IVs, crab gill CA was sensitive to 0.2% sodium dodecyl sulfate, suggesting that although crab gill CA is like mammalian CA IVs in many ways, it is less stabilized by intramolecular disulfide bonds.
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PMID:Membrane-associated carbonic anhydrase from the crab gill: purification, characterization, and comparison with mammalian CAs. 803 56

Because of the limited information available about the synthesis of N-linked glycoproteins in nerve cells, in regard to both processing steps and enzyme characterization, the biosynthetic processing of Thy-1 of the rat neuronal tumor cell line BN-1010-1 has been investigated using several inhibitors of biosynthesis and transport. (i) Tunicamycin completely inhibited mannose incorporation into Thy-1. Unglycosylated Thy-1 was not transported to the cell surface and was probably degraded rapidly following synthesis. (ii) Brefeldin A completely inhibited the transport of all [3H]mannose-labeled proteins releasable by phosphatidylinositol-specific phospholipase C, including Thy-1, to the surface of BN-1010-1 cells. Removal of the inhibitor led to rapid reversal of the inhibition. Pulse-chase experiments demonstrated that approximately 50% of Thy-1 was degraded after 4 h in the presence of brefeldin A. (iii) Castanospermine treatment slowed the appearance of Thy-1 on the cell surface. The surface Thy-1 contained mainly normal Man5, Man6, and Man7 oligosaccharides, suggesting that Golgi endo-alpha-D-mannosidase effected the removal of glucose. (iv) Treatment with deoxymannojirimycin resulted in the synthesis of Thy-1 that contained Man8 and Man9 oligosaccharides compared to Man5, Man6, and Man7 in the control. Neither the rate of appearance nor the level of surface expression was affected by the drug. (v) Swainsonine did not affect either the rate of appearance or the level of surface expression of Thy-1. The HPLC elution profile of neutral oligosaccharides resulting from Endo-H digestion of Thy-1 synthesized in the presence of swainsonine was indistinguishable from controls. The lack of an effect of swainsonine is explained by the unexpected absence of a complex type oligosaccharide in Thy-1 of BN-1010-1 cells, as shown in experiments with a variety of lectins as well as digestions with Endo-H or glycopeptidase F, or digestions with both enzymes in sequence. The fact that, after [3H]fucose-labeling, Endo-H digestion produced Thy-1 still labeled with fucose indicates that hybrid oligosaccharide is present in Thy-1.
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PMID:Biosynthetic and structural studies on Thy-1 in a rat neuronal tumor cell line. 809 81

We have recently reported the cloning of the rat zymogen granule membrane glycoprotein GP-3 and the related pancreatic secretory lipase (Wishart, M. J., Andrews, P. C., Nichols, R., Blevins, G. T., Logsdon, C.D., and Williams, J. A. (1993) J. Biol. Chem. 268, 10303-10311). Specific antipeptide antibodies were generated against both GP-3 and secretory lipase and used for the biochemical and physiological characterization of GP-3. Western blotting confirmed that GP-3 was found exclusively in zymogen granule membranes and was absent from zymogen granule content which contains the majority of secretory lipase. Extraction of zymogen granule membranes with Triton X-114 showed GP-3 to be significantly more hydrophobic than lipase. The GP-3 amino acid sequence contains one potential N-linked glycosylation site at Asn-336. The loss of concanavalin A labeling after both chemical deglycosylation with trifluoromethanesulfonic acid and enzymatic deglycosylation with N-glycanase showed GP-3 to possess a small N-linked oligosaccharide side chain. Digestion of intact and permeabilized zymogen granules with the nonspecific protease Pronase localized GP-3 to the inner surface of zymogen granule membranes. Since GP-3 is resident on the inner surface of the zymogen granule membrane, it should appear on the outer cellular surface after exocytosis. Although membrane attachment of GP-3 was resistant to treatment with phosphatidylinositol-specific phospholipase C, we observed that GP-3 is released into the pancreatic juice and that secretion of GP-3 was greatly enhanced by cholecystokinin.
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PMID:GP-3, a newly characterized glycoprotein on the inner surface of the zymogen granule membrane, undergoes regulated secretion. 813 47

An arginine-specific ADP-ribosyltransferase activity was detected in chicken spleen membrane fraction using a capillary electrophoresis assay and the activity was extracted by phosphatidylinositol-specific phospholipase C but not by 1 M NaCl or 1% Triton X-100. The enzyme protein was purified from chicken spleen membrane fraction to apparent homogeneity with a six-step method containing phosphatidylinositol-specific phospholipase C treatment, ammonium sulfate precipitation and conventional column chromatographies. Apparent molecular mass of the purified enzyme estimated with SDS/PAGE was 44 kDa. N-glycanase treatment of the enzyme reduced the apparent molecular size on SDS/PAGE. The enzyme was recognized by anti-cross reacting determinant antibodies. Partial amino acid sequence of the purified enzyme protein showed high homologies with primary structures of previously reported chicken arginine-specific ADP-ribosyltransferases.
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PMID:A newly identified glycosylphosphatidylinositol-anchored arginine-specific ADP-ribosyltransferase in chicken spleen. 919 60


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