Purification and properties of UDP-GlcNAc:dolichyl-phosphate GlcNAc-1-phosphate transferase. Activation and inhibition of the enzyme

Abstract

The GlcNAc-1-P transferase was solubilized from pig aorta microsomal fractions using 0.5% Nonidet P-40. The activity of the solubilized enzyme was stimulated by exogeneously added phospholipids in the order phosphatidylglycerol greater than phosphatidylinositol greater than phosphatidylserine. When the enzyme was stored in 20% glycerol containing 20 micrograms of phosphatidylglycerol/mg of protein, more than 80% of the activity remained after storage for 6 days at 0-4 degrees C. On the other hand, in the absence of the stabilizers, the enzyme lost most of its activity within 24 h. The transferase was purified about 68-fold using ammonium sulfate and DEAE-cellulose fractionation. The DEAE-cellulose chromatography separated a heat-stable factor from the enzyme, which when added back to the partially purified enzyme stimulated about 5-fold. With this partially purified enzyme, the Km for UDP-GlcNAc was found to be 1 X 10(-7) M, and that for dolichyl-P about 1 X 10(-6) M. The stimulatory factor increased the Vmax for both UDP-GlcNAc and dolichyl-P 5-10-fold, but the Km values remained the same. The pH optimum for the enzyme was between 7.4 and 7.6, and either Mn2+ (1 mM) or Mg2+ (10 mM) was required for optimum activity. The GlcNAc-1-P transferase was also stimulated by the addition of GDP-mannose (or other purine sugar nucleotides) or dolichyl-phosphoryl-mannose to the incubation mixtures. These two compounds acted in different ways on the enzyme since their stimulatory effects were additive. The effect of GDP-mannose was found to be due to protection of the substrate, UDP-GlcNAc, from degradation, but the effect of dolichyl-P-mannose remains to be established. In addition, the stimulations shown by phosphatidylglycerol, GDP-mannose, and factor, or phosphatidylglycerol, dolichyl-P-mannose, and factor, were all additive, indicating that they were acting at different sites on the enzyme. The transferase was quite sensitive to the action of sulfhydryl reagents such as N-ethylmaleimide or p-chloromercuribenzene sulfonate, and was rapidly inactivated in their presence. The enzyme could be protected to the extent of about 50% when all of the substrates (UDP-GlcNAc, dolichyl-P, Mn2+) were added before the addition of the sulfhydryl reagents.