Properties of Solubilized UDP-GlcNAc: Dolichyl Phosphate-GlcNAc-1-P-Transferase from Soybean Cultured Cells 1
AUTOR(ES)
Kaushal, Gur P.
RESUMO
The GlcNAc-1-P-transferase was solubilized from microsomal preparations of soybean cultured cells by treatment with 1% Triton X-100. The solubilized enzyme catalyzed the formation of dolichyl pyrophosphoryl-GlcNAc when incubated with UDP-GlcNAc and dolichyl phosphate. The GlcNAc-1-P-transferase activity was stimulated by the addition of phosphatidylglycerol and phosphatidylinositol, but was inhibited by phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The Km value for dolichyl-phosphate was 6.2 micromolar and that determined for UDP-GlcNAc was 0.42 micromolar. The pH optimum for the GlcNAc-1-P reaction was between 7.2 and 7.6; maximum activity occurred at about 10 millimolar Mg2+. The addition of unlabeled GDP-mannose or UDP-glucose considerably inhibited enzyme activity which could be restored to nearly the original value by addition of more dolichyl phosphate to the incubation mixture. On the other hand, the addition of unlabeled ADP-glucose and GDP-glucose enhanced the enzyme activity. This stimulation by these sugar nucleotides was found to be due to the protection of the substrate UDP-[3H]-GlcNAc from pyrophosphatase degradation. The GlcNAc-1-P-transferase reaction was very sensitive to tunicamycin and 50% inhibition required less than 1 microgram of antibiotic per milliliter. Amphomycin, showdomycin, and diumycin also inhibited this reaction but at higher concentrations.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1056202Documentos Relacionados
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