Properties of a soluble polyprenyl phosphate. UDP-D-N-acetylglucosamine N-acetylglucosamine-1-phosphate transferase.

Abstract

A soluble enzyme that catalyzes the transfer of D-Nacetylglucosamine 1-phosphate from UDP-a-D-N-acetylglucosamine (UDP-GlcNAc) to dolichyl phosphate has been prepared by sonic oscillation of encysting cultures of Acanthamoeba castellanii The product of catalysis is dolichyl a-D-N-acetylglucosamine pyrophosphate.The enzyme requires 8 m Mg"', pH 7.5, 30°C, and the presence of a reducing agent for maximum activity.The approximate K, is 4.5 PM for dolichyl phosphate and 0.5 PM for UDP-GlcNAc.The enzyme retains full activity for at least an hour at 30"C, for at least 2 months at 4"C, and can be stored indefinitely at -20°C or below.Typical yields of product are around 40%.The enzyme solution includes stable enzymes that catalyze the production of what appears to be ( a ) dolichyl a-D-N,N"diacetylchitobiosyl pyrophosphate from UDP-GlcNAc and dolichyl a-D-GlcNAc pyrophosphate, but not from the glucosamine lipid alone, and ( 6 ) dolichyl a-D-N, N'-diacetylchitobiosylmannosyl pyrophosphate from GDP-mannose and chitobiosyl lipid, but not from dolichyl fl-mmannosyl phosphate and the chitobiosyl lipid.At 50 pg/ml of diumycin, the transfer of GlcNAc from UDP-GlcNAc to dolichyl a-D-GlcNAc pyrophosphate is inhibited 90%.Much lower inhibitions of polyprenyl phosphate: sugar nucleotide transglycosylases utilizing UDP-glucose, GDP-mannose, and UDP-GlcNAc are observed at 50 pg/ml concentrations of diumycin.At 250 pg/ml of diumycin the GlcNAc-1phosphate and mannosyl transferases are almost completely inhibited, but the polyprenyl phosphate:UDPglucose transglucosylase is only slightly inhibited.The glycosylation of proteins has become the subject of extensive research within recent years.One of the reasons for this concentration of effort is the central position that glycoproteins hold in such important biological phenomena as secretory mechanisms, cell recognition processes, cell surface reception, and in physiological processes such as lung function and defenses against invasion by foreign organisms (for review of glycoprotein structure and function see Refs. 1 to 4).In addition to classification by function, glycoproteins can be categorized by the structure of attached oligosaccharides (5).A large number of glycoproteins which carry asparaginelinked sugars have oligosaccharides of common structure for the portions contiguous to the peptide chain and, apparently, common initial glycosylation reactions involving lipid-linked oligosaccharides (see Refs. 6 and 7 ) .Little is known about the physical properties, specificity, and possible mechanisms of