RESUMO
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
A second indolizidine alkaloid, epimeric with castanospermine, has been isolated from seeds of the Australian tree Castanospermum australe. The structure was established as 6-epicastanospermine by proton and carbon-13 nuclear magnetic resonance spectroscopy and mass spectrometry. 6-Epicastanospermine was found to be a potent inhibitor of amyloglucosidase, (an exo-1,4-alpha-glucosidase), a weak inhibitor of beta-galactosidase, and not to inhibit beta-glucosidase and alpha-mannosidase. These results indicate that glycosidase inhibitory activity cannot be predicted by comparison of the structure and stereochemistry with the appropriate sugars, since 6-epicastanospermine is an analog of mannose and not of glucose. The inhibition of amyloglucosidase was found to be competitive and to be more effective at higher pH values. Castanospermine and 6-epicastanospermine differed in their effect upon the mung bean processing enzymes, glucosidase I and II, in that the former is a potent inhibitor whereas the latter is a very poor inhibitor. Subtle alterations in stereochemistry of these alkaloids can therefore produce significant changes in their biological activity.
Assuntos
Alcaloides/isolamento & purificação , Fabaceae/análise , Inibidores de Glicosídeo Hidrolases , Indolizinas , Plantas Medicinais , Alcaloides/farmacologia , Glucana 1,4-alfa-Glucosidase/antagonistas & inibidores , Glicosídeo Hidrolases/antagonistas & inibidores , Concentração de Íons de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The microsomal enzyme fraction from mung bean seedlings was found to contain glucosidase activity capable of releasing [3H]glucose from the glucose-labeled Glc3Man9GlcNAc. The enzymatic activity could be released in a soluble form by treating the microsomal particles with 1.5% Triton X-100. When the solubilized enzyme fraction was chromatographed on DE-52, it was possible to resolve glucosidase I activity (measured by the release of [3H]glucose from Glc3Man9GlcNAc) from glucosidase II (measured by release of [3H]glucose from Glc2Man9GlcNAc). The glucosidase I was purified about 200-fold by chromatography on hydroxylapatite, Sephadex G-200, dextran-Sepharose, and concanavalin A-Sepharose. The purified enzyme was free of glucosidase II and aryl-glucosidase activities. Only a single glucose residue could be released from the Glc3Man9GlcNAc by this purified enzyme and the other product was the Glc2Man9GlcNAc. Furthermore, this enzyme was inhibited in a dose-dependent manner by kojibiose, an alpha-1,2-linked glucose disaccharide, but not by other alpha-linked glucose disaccharides. These data indicate that this glucosidase is a specific alpha-1,2-glucosidase. The pH optimum for the glucosidase I was about 6.3 to 6.5, and no requirements for divalent cations were observed. The enzyme was inhibited strongly by the glucosidase processing inhibitors, castanospermine and deoxynojirimycin, and less strongly by the plant pyrrolidine alkaloid, 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine. However, the enzyme was not inhibited by the mannosidase processing inhibitors, swainsonine, deoxymannojirimycin or 1,4-dideoxy-1,4-imino-D-mannitol. The stability of the enzyme under various conditions and other properties of the enzyme were determined.
Assuntos
Fabaceae/enzimologia , Glucosidases/isolamento & purificação , Plantas Medicinais , alfa-Glucosidases/isolamento & purificação , Cromatografia/métodos , Dissacarídeos/farmacologia , Glucose/metabolismo , Inibidores de Glicosídeo Hidrolases , Concentração de Íons de Hidrogênio , Octoxinol , Polietilenoglicóis , Sementes/enzimologia , Temperatura , alfa-Glucosidases/metabolismoRESUMO
A solubilized enzyme preparation from mung bean seedlings catalyzed the transfer of GlcNAc from UDP-GlcNAc to the Man5GlcNAc acceptor to form GlcNAc-Man5GlcNAc. In the presence of the mannosidase inhibitor, swainsonine, this oligosaccharide accumulated, but in the absence of this inhibitor, the oligosaccharide was processed further to smaller sized oligosaccharides with the release of radioactive mannose. The formation of GlcNAc-Man5GlcNAc required the presence of Man5GlcNAc, UDP-GlcNAc, Mn++ and swainsonine. The product, GlcNAc-Man5GlcNAc was characterized by chromatography on calibrated columns of Biogel P-4, and by various enzymatic digestions. These data indicate the presence of GlcNAc transferase I and mannosidase II in plants.