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:3.2.1.24 (alpha-mannosidase)
1,509 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The localization of acid hydrolases was examined in Chinese hamster ovary cells with defective mannose 6-phosphate receptors; these mutants had been shown to exhibit reduced uptake and altered binding of exogenously added acid hydrolase (Robbins, A. R., Myerowitz, R., Youle, R. J., Murray, G. J., and Neville, D. M., Jr. (1981) J. Biol. Chem. 256, 10618-10622). Cells were grown in the presence of [3H]mannose, alpha-L-iduronidase and beta-hexosaminidase were immunoprecipitated sequentially, electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate, and detected by fluorography. About 55% of the alpha-L-iduronidase and beta-hexosaminidase synthesized by the mutants in 12 h was found in the growth medium; parental cells secreted only approximately 15%. The mutants also secreted 2 to 6 times more alpha-mannosidase, beta-glucuronidase, and alpha-L-fucosidase than the parent as determined by measurements of enzyme activity. Intracellular levels of these enzymes were reduced in the mutants. The mutants secreted acid hydrolases in the precursor forms, within the cells these enzymes resided in lysosomes and were processed normally; thus, the mutants appeared aberrant only with respect to distribution of hydrolases between intracellular and extracellular compartments. [35S]methionine-labeled beta-hexosaminidase and alpha-L-iduronidase secreted by the mutants were taken up normally by both human fibroblasts and wild type CHO cells, and this uptake was inhibited by mannose 6-phosphate. Thus, the elevated secretion of acid hydrolases was not due to alteration of the mannose 6-phosphate recognition marker on the enzymes, but appears to result from alterations in the mannose 6-phosphate receptor.
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PMID:The mannose 6-phosphate receptor of Chinese hamster ovary cells. Compartmentalization of acid hydrolases in mutants with altered receptors. 627 Jan 23

Acid sphingomyelinase (sphingomyelin phosphodiesterase, EC 3.1.4.12) was purified from human brain by extraction with 0.1% Triton X-100, followed by sequential chromatography on Concanavalin A-Sepharose, octyl-Sepharose, hydroxylapatite, DEAE-cellulose, red A-agarose, Sephadex G-200, and DEAE-cellulose with ampholyte elution. Sphingomyelinase activity was purified more than 20,000-fold from the starting homogenate with a 1% yield. Specific activity of up to 800 mumol/h/mg protein could be achieved. Gel electrophoresis with 6% polyacrylamide containing sodium dodecyl sulfate gave a single protein band with a molecular weight of 70,000, in good agreement with the molecular weight previously estimated from sucrose density gradient centrifugation in 0.1% Triton X-100. Triton X-100 could be readily removed from the enzyme by sucrose density gradient centrifugation. The Triton-free enzyme showed the same Km and pH optimum. Heat stability of the enzyme was reversibly affected by Triton X-100, in that removal of the detergent made the enzyme more heat labile. The Km of purified enzyme for sphingomyelin was 36 microM. It was unaffected by sulfhydryl reagents, but was inhibited by dithiothreitol at high concentrations. The preparation was free of all lysosomal hydrolase activities tested, including galactosylceramidase and alpha-mannosidase, which tended to copurify in our previous procedure. The enzyme was inactive toward sphingosylphosphorylcholine. It was active with bis[p-nitrophenyl]- and bis[4-methylumbelliferyl]phosphate and the chromogenic and fluorogenic sphingomyelin analogues.
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PMID:Acid sphingomyelinase of human brain: purification to homogeneity. 628 87

In human fibroblasts, the receptor for low density lipoprotein (LDL) is synthesized as a precursor of apparent Mr = 120,000 which is converted to a mature form of apparent Mr = 160,000, as determined by migration in sodium dodecyl sulfate (SDS)-polyacrylamide gels (Tolleshaug, H., Goldstein, J. L., Schneider, W. J., and Brown, M. S. (1982) Cell 30, 715-724). The current paper describes the relationship of N- and O-glycosylation to this post-translational modification. Oligosaccharides were analyzed from precursor and mature forms of LDL receptors that had been immunoprecipitated from cells grown in media containing radioactive sugars. In human epidermoid carcinoma A-431 cells, the receptor precursor appears to contain one N-linked high mannose oligosaccharide and approximately 6-9 N-acetylgalactosamine residues linked O-glycosidically to Ser/Thr residues. In the mature receptor, the O-linked oligosaccharides are mono- and disialylated species having the core structure of galactose leads to N-acetylgalactosamine leads to Ser/Thr. The single N-linked oligosaccharide of the mature receptor can either be a tri- or tetraantennary complex-type species. Similar results were obtained with normal human fibroblast receptor except that the O-linked oligosaccharides on the precursor are neutral disaccharides, of which one component is GalNAc and the N-linked complex type unit on the mature receptor is less branched. Since the addition of GalNAc residues to Ser/Thr residues precedes the conversion of N-linked high mannose-type oligosaccharides to complex-type structures, the transfer of N-acetylgalactosamine must occur prior to the entry of glycoproteins into the region of the Golgi containing the processing enzyme alpha-mannosidase I. We also studied the receptor from tunicamycin-treated cells and after treatment with neuraminidase. In addition, we analyzed the receptor synthesized by a lectin-resistant clone of Chinese hamster ovary cells that is deficient in adding galactose residues to both N- and O-linked oligosaccharides. These studies suggest that the apparent differences in molecular weight between the precursor and mature forms of the LDL receptor are largely, if not entirely, due to the addition of sialic acid and galactose residues to the O-linked GalNAc residues.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Biosynthesis of N- and O-linked oligosaccharides of the low density lipoprotein receptor. 631 91

Mammalian beta-adrenergic receptor binding peptides can be visualized by covalently labeling them with the photoaffinity reagent p-azido-m-[125I]iodobenzylcarazolol followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The receptor peptides migrate as broad bands of Mr approximately equal to 62,000. In the present study, we examined the carbohydrate composition of the mammalian beta receptor through the use of specific exo- and endoglycosidases and lectin affinity chromatography. Treatment of p-azido-m-[125I]iodobenzylcarazolol-labeled beta2-adrenergic receptors from hamster lung or rat erythrocyte with the exoglycosidases neuraminidase and alpha-mannosidase provided evidence for the existence of both high mannose and complex type carbohydrate chains on beta 2-adrenergic receptors. The nonadditivity of the effect of sequential treatments with these enzymes suggested discrete populations of beta-adrenergic receptors containing either complex or high mannose type chains. Deglycosylation of receptor with endoglycosidase F results in a single labeled polypeptide at Mr = 49,000 for both systems. The same two populations of the beta receptors (high mannose or complex type chain) could also be fractionated by lectin affinity chromatography of solubilized p-azido-m-[125I]iodobenzylcarazolol-labeled receptors. The high mannose-containing receptors could be absorbed to and specifically eluted from concanavalin A-agarose. Those containing complex type carbohydrates could be adsorbed to and eluted from wheat germ agglutinin-agarose. Taken together, these data suggest that mammalian beta-adrenergic receptors contain both complex and high mannose type carbohydrate chains and that microheterogeneity of these chains likely explains the broad band pattern typically obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
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PMID:Mammalian beta-adrenergic receptors. Distinct glycoprotein populations containing high mannose or complex type carbohydrate chains. 633 Jan 18

Yeast- and mycelial-form cells of the dimorphic fungus Mucor rouxii incubated with [U-14C]glucose were found to synthesize Man-P-dolichol, Glc-P-dolichol, and Glc3Man9GlcNAc2-P-P-dolichol. The structure of the oligosaccharide moiety of the latter was similar to that of the same compound isolated from other eucaryotic cells. Oligosaccharides that migrated on paper chromatography as Man6-30GlcNAc standards were obtained upon treatment of delipidated proteins with a protease and endo-beta-N-acetylglucosaminidase H. The oligosaccharides that migrated apparently as single substances on paper chromatography could be separated into three different populations by paper electrophoresis in sodium borate buffer. The fastest migrating substances contained only mannose and N-acetylglucosamine residues, whereas the other two contained, in addition, different proportions of 3-O-methylmannose units. The oligosaccharides with the highest content of 3-O-methylmannose residues appeared to be completely resistant to alpha-mannosidase degradation; they were, however, cleaved by endo-beta-N-acetylglucosaminidase H. Mycelial cells synthesized a much higher proportion of 3-O-methylmannose-containing oligosaccharides than yeast cells. Cells incubated with [methyl-14C]methionine were found to label only the N-linked oligosaccharides containing 3-O-methylmannose residues. It is concluded that transfer of Glc3Man9GlcNAc2 to protein is followed by excision of glucose and probably one or two mannose residues, followed by further mannosylation and in some cases also methylation of oligosaccharides. This represents a novel reaction in the processing of N-linked oligosaccharides.
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PMID:3-O-methylation of mannose residues. A novel reaction in the processing of N-linked oligosaccharides occurring in Mucor rouxii. 649 Jun 28

Thyroid-stimulating hormone (TSH) subunit glycosylation was compared to that of total cell glycoproteins in mouse thyrotropic tumors. Lipid-linked oligosaccharides, total cell glycoproteins, and TSH subunits were labeled with either [3H]mannose, [3H]galactose, or [3H]glucose in pulse and pulse-chase experiments. The various oligosaccharides were isolated respectively by lipid extraction and mild acid hydrolysis, by selective immunoprecipitation, or by acid precipitation followed by trypsin and endoglycosidase H treatment. The nature of the oligosaccharides was assessed by their migration in paper chromatography, their relative incorporation of different precursors, and also their resistance to alpha-mannosidase. At 60 min, lipid-linked oligosaccharides were found to be composed of Glc3-2Man9GlcNAc2, Man9-8GlcNAc2, and Man5GlcNAc2. At 10 or 60 min of labeling, total cell proteins contained Glc3Man9GlcNAc2, Glc1Man9GlcNAc2, Man9GlcNAc2, Glc1Man8GlcNAc2, Man8GlcNAc2, and Man7GlcNAc2. The largest oligosaccharide, Glc3Man9GlcNAc2, had an unusually long half-life of about 2 h. In contrast, no Glc3Man9GlcNAc2 was found either on TSH + alpha subunits or on free beta subunits isolated either by immunoprecipitation or by sodium dodecyl sulfate gel electrophoresis. Instead, primarily Man9GlcNAc2 was found after a 10-min pulse both on TSH + alpha subunits and on beta subunits. When the pulse was followed by a chase up to 2 h, there was a progressive increase in Man8GlcNAc2 in higher amounts on TSH + alpha-subunit carbohydrate chains than on beta subunits.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Glycosylation and processing of high-mannose oligosaccharides of thyroid-stimulating hormone subunits: comparison to nonsecretory cell glycoproteins. 649 54

Studies have been carried out on the activities and properties of the isozymes of alpha-mannosidase, alpha-glucosidase and beta-glucosidase in granulocytes, monocytes, lymphocytes and platelts from peripheral blood of heatlhy adult donors. The findings reveal the differences in activities as well as a characteristic distribution of the different molecular forms of these lysosomal hydrolases in specific cell types. Therefore, the results obtained with unfractionated total leukocyte smples from different subjects may vary according to the distribution of cell types in the circulation. Granulocytes and monocytes show only the acid alpha-mannosidase activity whereas lymphocytes and platelets show both acid and neutral activities. The specific activity of acid alpha-mannosidase in granulocytes and monocytes is higher than in lymphocytes and platelets. By DEAE-cellulose chromatography, the acid alpha-mannosidase in granulocyte and monocyte extracts elutes as two peaks, but only one peak is seen in lymphocytes. All cell types show both acid and neutral alpha-glucosidase activities. The specific activities of both isozymes are higher in granulocytes and monocytes than in lymphocytes and platelets. Monocytes show a higher acid than neutral activity. All other cell types show a higher neutral activity. Beta-Glucosidase in all cell types is mainly membrane-bound and it can be released by Triton X-100 and sodium taurocholate. Taurocholate also stimulates the beta-glucosidase activity of granulocytes, monocytes and lymphocytes whereas it inhibits the activity of this enzyme in platelets. These results indicate that variations in the total number of leukocytes and in the relative proportion of the various cell types in health and disease may yield inconsistent or unreliable values for enzyme activity in the diagnosis of lysosomal storage disease and in carrier detection.
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PMID:Studies on the activities and properties of lysosomal hydrolases in fractionated populations of human peripheral blood cells. 676 26

alpha-Mannosidase of Medicago sativa (alfalfa) was purified 1340-fold. The purification method included dialysis of the crude extract against a citrate/phosphate buffer, pH 3.9, (NH4)SO4 precipitation, hydroxyapatite chromatography, chromatography on Sephadex G-200 and finally a preparatory electrophoresis on polyacrylamide-gel gradient by Doly & Petek's [(1977) J. Chromatogr. 137. 69--81] method. Each step of purification was checked by polyacrylamide-gel disc electrophoresis. The purified enzyme showed a single band, corresponding to alpha-mannosidase activity. alpha-Mannosidase has a mol.wt. 230 000 as estimated by Hedrick & Smith's [(1968) Arch. Biochem. Biophys. 126, 155--164] method and also by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate by Weber & Osborn [(1969) J. Biol. Chem. 244, 4406--4412]. The enzyme comprises four subunits of different molecular weight. Optimum pH and Km values were determined with p-nitrophenyl alpha-D-mannoside as substrate. When incubated at a temperature between 20 and 62 degrees C before assay, alpha-mannosidase initially shows an increase in activity. alpha-Mannosidase is stable when the pH is about neutrality. It can be inactivated by several metal ions, including Zn2+. At a pH below 5 the enzyme undergoes irreversible inactivation. The presence of EDTA at acid pH considerably enhances the inactivation of the enzyme. This inactivation due to EDTA can be specifically reversed by incubation with Zn2+.
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PMID:Purification and properties of alpha-D-mannosidase from the germinated seeds of Medicago sativa (alfalfa). 677 49

Swainsonine, an indolizidine alkaloid, inhibits the alpha-mannosidase that is involved in glycoprotein processing. Thus, in cultured animal cells, this alkaloid causes an increase in the surface content of high mannose glycoproteins and a decrease in the amount of complex type glycoproteins (Elbein, A. D., Solf, R., Dorling, P. R., and Vosbeck, K. (1982) Proc. Natl. Acad. Sci. U. S. A., 78, 7393-7397). In this report, the effect of swainsonine on the synthesis virus hemagglutinins was examined. Primary calf kidney cultures were infected with influenza virus and viral replication was allowed to proceed in the absence or presence of swainsonine. Several hours after the addition of swainsonine, [2-3H]mannose or [6-3H]glucosamine were added to label the hemagglutinins and the mature virus particles were isolated. Virus particles raised in the presence of this alkaloid had the same infectivity and hemagglutination titer as virus particles from control cells. However, when the hemagglutinins were examined on sodium dodecyl sulfate gels, the major hemagglutinin (HA0) and its subunits, HA1 and HA2, from swainsonine-treated cells, migrated faster, indicating that they were of lower molecular weights. The labeled hemagglutinins were digested with pronase and the resulting glycopeptides were chromatographed on Bio-Gel P-4. Both the mannose-labeled and glucosamine-labeled glycopeptides from swainsonine-treated virus migrated more slowly on these columns than those of controls cells, suggesting that they were altered in structure. Furthermore, when the glycopeptides were digested with endoglucosaminidase H, 90% of the glycopeptides from swainsonine-treated cells were susceptible to this enzyme, whereas only 30% of those from control cells were digested. The major oligosaccharide released from inhibited cells by endoglucosaminidase H was digestible with alpha-mannosidase, whereas that of control cells was resistant to this enzyme. However, the control cell glycopeptide was digested by a combination of neuraminidase, beta-galactosidase, beta-N-acetylhexosaminidase, and alpha-mannosidase. These data show that swainsonine prevents the formation of complex glycoproteins and gives rise to increased amounts of high-mannose glycoproteins.
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PMID:Swainsonine prevents the processing of the oligosaccharide chains of influenza virus hemagglutinin. 679 7

The glycolipoprotein (GLP) extracted from the surface slime of Pseudomonas aeruginosa produces effects in mice similar to those of the viable cell. The lethal activity has been located in the lipid moiety; however, degradation of the carbohydrate moiety with sodium metaperiodate reduced the antigenicity and abolished the lethality of the GLP. Similar degradation with a phage-induced polysaccharide depolymerase reduced the antigenicity only slightly but reduced the lethality over 60%. The neutral sugar composition of the isolated polysaccharide moiety was shown to be that of the parental GLP. Of the component neutral sugars, mannose and its derivatives were capable of inhibiting the agglutination of erythrocytes coated with GLP. Inhibition also occurred with a soluble mannose polymer from the cell walls of yeast. Antiserum to GLP and to its isolated polysaccharide moiety agglutinated yeast cells, whereas antiserum to a glycolipid fragment of the GLP lacking mannose did not. The lethality of the GLP was reduced by degradation with alpha-mannosidase or by blocking the mannose residues with concanavalin A, and the glycolipid fragment showed less lethality than the native GLP. We conclude that mannose, in addition to being an immunodominant sugar, is an effector sugar in the expression of GLP lethality.
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PMID:Carbohydrate mediation of the biological activities of the glycolipoprotein of Pseudomonas aeruginosa. 681 28


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