RESUMO
Two arylmannosidases (signified as A and B) were purified to homogeneity from soluble and microsomal fractions of mung bean seedlings. Arylmannosidase A from the microsomes appeared the same on native gels and on SDS gels as soluble arylmannosidase A, the same was true for arylmannosidase B. Sedimentation velocity studies indicated that both enzymes were homogeneous, and that arylmannosidase A had a molecular mass of 237 kd while B had a molecular mass of 243 kd. Arylmannosidase A showed two major protein bands on SDS gels with molecular masses of 60 and 55 kd, and minor bands of 79, 39 and 35 kd. All of these bands were N-linked since they were susceptible to digestion by endoglucosaminidase H. In addition, at least the major bands could be detected by Western blots with antibody raised against the xylose moiety of N-linked plant oligosaccharides, and they could also be labeled in soybean suspension cells with [2-3H]mannose. Arylmannosidase B showed three major bands with molecular masses of 72, 55 and 45 kd, and minor bands of 42 and 39 kd. With the possible exception of the 45 and 42 kd bands, all of these bands are glycoproteins. Arylmannosidases A and B showed somewhat different kinetics in terms of mannose release from high-mannose oligosaccharides, but they were equally susceptible to inhibition by swainsonine and mannostatin A. Polyclonal antibody raised against the arylmannosidase B cross-reacted equally well with arylmannosidase A from mung bean seedlings and with arylmannosidase from soybean cells. However, monoclonal antibody against mung bean arylmannosidase A was much less effective against arylmannosidase B. Antibody was used to examine the biosynthesis and structure of the carbohydrate chains of arylmannosidase in soybean cells grown in [2-3H]mannose. Treatment of the purified enzyme with Endo H released approximately 50% of the radioactivity, and these labeled oligosaccharides were of the high-mannose type, i.e. mostly Man9GlcNAc. The precipitated protein isolated from the Endo H treatment still contained 50% of the radioactivity, and this was present in modified structures that probably contain xylose residues.
Assuntos
Fabaceae/enzimologia , Glycine max/enzimologia , Glicoproteínas/isolamento & purificação , Isoenzimas/isolamento & purificação , Manosidases/isolamento & purificação , Plantas Medicinais , Western Blotting , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Glicopeptídeos/análise , Glicoproteínas/metabolismo , Isoenzimas/metabolismo , Cinética , Manosidases/metabolismo , Peso Molecular , Oligossacarídeos/análiseRESUMO
Glucosidase II was purified approximately 1700-fold to homogeneity from Triton X-100 extracts of mung bean microsomes. A single band with a molecular mass of 110 kDa was seen on sodium dodecyl sulfate gels. This band was susceptible to digestion by endoglucosaminidase H or peptide glycosidase F, and the change in mobility of the treated protein indicated the loss of one or two oligosaccharide chains. By gel filtration, the native enzyme was estimated to have a molecular mass of about 220 kDa, suggesting it was composed of two identical subunits. Glucosidase II showed a broad pH optima between 6.8 and 7.5 with reasonable activity even at 8.5, but there was almost no activity below pH 6.0. The purified enzyme could use p-nitrophenyl-alpha-D-glucopyranoside as a substrate but was also active with a number of glucose-containing high-mannose oligosaccharides. Glc2Man9GlcNAc was the best substrate while activity was significantly reduced when several mannose residues were removed, i.e. Glc2Man7-GlcNAc. The rate of activity was lowest with Glc1Man9GlcNAc, demonstrating that the innermost glucose is released the slowest. Evidence that the enzyme is specific for alpha 1,3-glucosidic linkages is shown by the fact that its activity on Glc2Man9GlcNAc was inhibited by nigerose, an alpha 1,3-linked glucose disaccharide, but not by alpha 1,2 (kojibiose)-, alpha 1,4(maltose)-, or alpha 1,6 (isomaltose)-linked glucose disaccharides. Glucosidase II was strongly inhibited by the glucosidase processing inhibitors deoxynojirimycin and 2,6-dideoxy-2,6-imino-7-O-(beta-D- glucopyranosyl)-D-glycero-L-guloheptitol, but less strongly by castanospermine and not at all by australine. Polyclonal antibodies prepared against the mung bean glucosidase II reacted with a 95-kDa protein from suspension-cultured soybean cells that also showed glucosidase II activity. Soybean cells were labeled with either [2-3H]mannose or [6-3H]galactose, and the glucosidase II was isolated by immunoprecipitation. Essentially all of the radioactive mannose was released from the protein by treatment with endoglucosaminidase H. The labeled oligosaccharide(s) released by endoglucosaminidase H was isolated and characterized by gel filtration and by treatment with various enzymes. The major oligosaccharide chain on the soybean glucosidase II appeared to be a Man9(GlcNAc)2 with small amounts of Glc1Man9(GlcNAc)2.
Assuntos
Fabaceae/enzimologia , Glycine max/enzimologia , Plantas Medicinais , alfa-Glucosidases/isolamento & purificação , Sequência de Carboidratos , Cromatografia , Cromatografia de Afinidade , Cromatografia DEAE-Celulose , Cromatografia em Gel , Detergentes , Durapatita , Eletroforese em Gel de Poliacrilamida , Inibidores de Glicosídeo Hidrolases , Hidroxiapatitas , Cinética , Dados de Sequência Molecular , Peso Molecular , Octoxinol , Oligossacarídeos/metabolismo , Polietilenoglicóis , Especificidade por Substrato , alfa-Glucosidases/metabolismoRESUMO
Mannosidase II was purified from mung bean seedlings to apparent homogeneity by using a combination of techniques including DEAE-cellulose and hydroxyapatite chromatography, gel filtration, lectin affinity chromatography, and preparative gel electrophoresis. The release of radioactive mannose from GlcNAc[3H]Man5GlcNAc was linear with time and protein concentration with the purified protein, did not show any metal ion requirement, and had a pH optimum of 6.0. The purified enzyme showed a single band on SDS gels that migrated with the Mr 125K standard. The enzyme was very active on GlcNAcMan5GlcNAc but had no activity toward Man5GlcNAc, Man9GlcNAc, Glc3Man9GlcNAc, or other high-mannose oligosaccharides. It did show slight activity toward Man3GlcNAc. The first product of the reaction of enzyme with GlcNAcMan5GlcNAc, i.e., GlcNAcMan4GlcNAc, was isolated by gel filtration and subjected to digestion with endoglucosaminidase H to determine which mannose residue had been removed. This GlcNAcMan4GlcNAc was about 60% susceptible to Endo H indicating that the mannosidase II preferred to remove the alpha 1,6-linked mannose first, but 40% of the time removed the alpha 1,3-linked mannose first. The final product of the reaction, GlcNAcMan3GlcNAc, was characterized by gel filtration and various enzymatic digestions. Mannosidase II was very strongly inhibited by swainsonine and less strongly by 1,4-dideoxy-1,4-imino-D-mannitol. It was not inhibited by deoxymannojirimycin.
Assuntos
Fabaceae/enzimologia , Manosidases/isolamento & purificação , Plantas Medicinais , Sementes/enzimologia , 1-Desoxinojirimicina , Alcaloides/farmacologia , Arabinose , Membrana Celular/enzimologia , Cromatografia DEAE-Celulose , Estabilidade Enzimática , Glucosamina/análogos & derivados , Glucosamina/farmacologia , Concentração de Íons de Hidrogênio , Imino Furanoses , Manosidases/antagonistas & inibidores , Oligossacarídeos/análise , Solubilidade , Especificidade por Substrato , Álcoois Açúcares/farmacologia , SwainsoninaRESUMO
When the purified plant glucosidase II was incubated with [3H]Glc2Man9GlcNAc in the presence of glycerol and the products were analyzed by gel filtration, a large peak of radioactivity emerged just before the glucose standard. The formation of this peak was dependent on both the presence of Glc2Man9GlcNAc and the presence of glycerol, and the amount of this product increased with time of incubation and amount of glucosidase II in the incubation. When the incubation was performed with [3H]Glc2Man9GlcNAc plus [14C]glycerol, the product contained both 14C and 3H. Strong acid hydrolysis of the purified product gave rise to [14C]glycerol and [3H]glucose. Various other chemical treatments and chromatographic techniques showed that the product was glucosyl----glycerol. Since the glucose was released by alpha-glucosidase, the product must be glucosyl-alpha-glycerol. This study demonstrates that the processing glucosidase II catalyzes a trans-glycosylation reaction in the presence of acceptors like glycerol. Since this transglycosylation reaction may give rise to unexpected products, investigators should be aware of its possible occurrence.