Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.26 (invertase)
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Saccharomyces cerevisiae Man9-alpha-mannosidase, responsible for trimming Man9GlcNAc2 in the endoplasmic reticulum to Man8GlcNAc2, the substrate for oligosaccharide elongation, has been purified to homogeneity from stabilized microsomal membranes without employing autolytic digestion. The activity was solubilized by the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulphonate (CHAPS), whose presence was necessary for maximal activity. Purification included Q-Sepharose ion-exchange chromatography, preparative isoelectric focusing and HPLC gel filtration on TSK 3000 matrix. Overall purification from post-nuclear supernatants was estimated to be 110,000-fold with a 50% recovery of activity. The purified enzyme hydrolysed Man9GlcNAc1,2 from thyroglobulin or oligosaccharide-lipid, but not invertase Man9GlcNAc, Man1 alpha 2Man1 alpha OCH3 or p-nitrophenyl-alpha-D-mannopyranoside. Conversion of thyroglobulin Man9GlcNAc to Man8GlcNAc was linear with time and enzyme concentration, with an apparent Km of 0.2 mM and a specific activity of 220 IU/mg. Glc3Man9GlcNAc2 from oligosaccharide-lipid was as good a substrate as Man9GlcNAc, but the lipid-linked Man7GlcNAc2 isomer was hydrolysed at only 10% of this rate. Hydrolysis of defined isomers of IgM and bovine thyroglobulin Man6,7,8GlcNAc indicated that, for maximal alpha 1,2-mannosidase activity, only the alpha 1,2-linked terminal mannoses on the alpha 3 branch of the Man9GlcNAc precursor were dispensable. Isomers lacking the terminal alpha 1,2-linked mannose on the alpha 6 branch were hydrolysed at only approximately 10% of the maximal rate. The enzyme exhibited a pI of 5.3 and a pH optimum at 6.5. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the absence of reducing agents gave a single sharp band at 66 kDa, while in the presence of beta-mercaptoethanol equimolar amounts of two peptides, one of 44 kDa and one of 23 kDa, were obtained. Sizing on Sephacryl SF300, Superose 12 and TSK 3000 provided a holoenzyme mol. wt of 60-68 kDa, indicating that the isolated active form of the Man9-alpha-mannosidase was composed of one each of the sulphydryl-bonded dissimilar peptides. The enzyme bound to concanavalin A (ConA)-Sepharose and was eluted with alpha-methylmannoside, indicating the presence of high-mannose oligosaccharides. The Man9-alpha-mannosidase required low levels of Ca2+, which could be removed by EGTA. Activity was restored by Ca2+ or Zn2+, but not by Mg2+ or Mn2+.
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PMID:Glycoprotein biosynthesis in yeast: purification and characterization of the endoplasmic reticulum Man9 processing alpha-mannosidase. 182 40

A scheme has been developed for isolation and purification of the enzyme with alpha-N-acetylgalactosaminidase and alpha-galactosidase activities which included fractionation by ammonium sulphate and chromatography on TSK-gels Toyopearl HW-60 and Fractogel DEAE-650-s and Sepharose 6B. The enzyme was purified 600 times with the yield of 28%. The enzyme preparation did not contain fucosidase, invertase and proteolytic activities. Molecular mass of the enzyme from the data of gel-filtration on Sepharose 6B was 430 kDa, according to the data of electrophoresis in DS-PAAG--70 kDa. It is shown that acidic and hydrophobic aminoacids prevail in the enzyme molecule, the carbohydrate component containing galactose, mannose, glucosamine and two nonidentified hexosamines is also present there. The enzyme preparation is stable during 48 hours at 20 degrees C; its pH-optimum is at pH 3.5-4.1. Michaelis constants concerning n-nitrophenyl-alpha-N-acetylgalactopyranoside and n-nitrophenyl-alpha-D-galactopyranoside were 1.18 and 1.25 mM, respectively.
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PMID:[Purification and physico-chemical properties of glycosidase of Aspergillus niger 185sh]. 1507 44