Stimulation by dolichol phosphate-mannose and phospholipids of the biosynthesis of N-acetylglucosaminylpyrophosphoryl dolichol.
Dolichol phosphate-mannose (dol-P-mannose) has been shown previously to stimulate the reaction: dolichol phosphate + UDP-[3H]GlcNAc----[3H]GlcNAc-P-P-polyprenols (Kean, E. L. (1982) J. Biol. Chem. 257, 7952-7954). Further studies on this phenomenon are described, using microsomes from the retina of the embryonic chick as the major source of enzyme. Neither dolichol-P-glucose nor retinyl-P-mannose showed this stimulatory activity. Phosphatidylglycerol also stimulated this same process and was most active among a variety of phospholipids which were tested, in accord with previous reports. The presence of GDP-2-deoxy-2-fluoro-D-mannose or GTP had no effect on the reaction. The apparent activation constant for dolichol-P-mannose was 2.2 microM, and for phosphatidylglycerol, 401 microM. The major product (90% or greater) obtained under basal and stimulatory conditions was GlcNAc-P-P-dolichol and the site of the stimulatory effect was the glucosaminyltransferase catalyzing the formation of this compound. The effects of stimulation on the kinetic properties were similar for both activators: increases in the Vmax of the reactions of 7-10-fold; increases in apparent Km for UDP-GlcNAc of 5-7-fold; a 3-fold decrease in apparent Km for dolichol-phosphate. When present together, a mutual inhibition of stimulation was observed compared to the additive effect from dol-P-mannose or phosphatidylglycerol alone. Although a substrate for the reaction, dolichol phosphate repressed the stimulation by dolichol-P-mannose but not that by phosphatidylglycerol. Dol-P-glucose, while not an activator of the reaction, acted as a negative modifier of the stimulation by dol-P-mannose by acting as a competitive inhibitor of the stimulation. The stimulatory phenomenon was observed in microsomes prepared from a variety of tissues from the embryonic chick and from postnatal tissue after partial delipidation. The addition of pyrophosphatase inhibitors did not bring about stimulation of GlcNAc-lipid synthesis, but did enhance the effect. These studies extend the previous observations of the participation of dolichol-P-mannose and phosphatidylglycerol as allosteric activators of GlcNAc-lipid synthesis and indicate additional aspects of metabolic regulation of the dolichol pathway.
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D000818:Animals; D001921:Brain / Q000648:ultrastructure; D002642:Chick Embryo; D004287:Dolichol Monophosphate Mannose / Q000494:pharmacology*; D004288:Dolichol Phosphates / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006155:Guanosine Diphosphate Mannose / Q000494:pharmacology; D007700:Kinetics; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology*; D011103:Polyisoprenyl Phosphate Monosaccharides / Q000096:biosynthesis* / Q000494:pharmacology; D011105:Polyisoprenyl Phosphate Sugars / Q000096:biosynthesis* / Q000494:pharmacology*; D051381:Rats; D012160:Retina / Q000196:embryology / Q000648:ultrastructure; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000494:pharmacology
D004288:Dolichol Phosphates; D010715:Phosphatidylglycerols; D010743:Phospholipids; D011103:Polyisoprenyl Phosphate Monosaccharides; D011105:Polyisoprenyl Phosphate Sugars; D006155:Guanosine Diphosphate Mannose; D014537:Uridine Diphosphate N-Acetylglucosamine; D004287:Dolichol Monophosphate Mannose; C002968:dolichol-D-glucosylmonophosphate; C018439:N-acetylglucosaminylpyrophosphoryldolichol