Dolichol kinase activity in the developing rat testis.
Dolichyl phosphate concentrations, a primary factor in regulating the rate of N-glycosidically linked glycoprotein synthesis, are dependent upon a cytidine triphosphate (CTP)-dependent dolichol kinase. This study examines dolichol kinase in rat testicular microsomes and defines assay conditions. As with dolichol kinases from other tissues, addition of 2-mercaptoethanol increased activity 60%. Inclusion of NaF, an inhibitor of testicular dolichyl phosphate phosphatase activity, also resulted in a 38% increase in activity. Triton X-100 was necessary for phosphorylation of both endogenous and exogenous dolichol; however, concentrations of detergent in excess of 0.25-0.35% were inhibitory. A 2- to 5-fold stimulation of kinase activity was obtained by addition of 50-100 microM exogenous dolichol. The high level of nucleoside triphosphatase activity in testicular microsomes mandated the inclusion of high levels of uridine triphosphate (UTP) to protect the [gamma-32 P] CTP. Increasing UTP concentrations up to 50 mM resulted in increased product formation. A clear requirement for divalent cations was observed; 5 mM ethylenediaminetetraacetate (EDTA) abolished activity. The following order of cation effectiveness was observed: Mn greater than or equal to Ca greater than Cd greater than Zn much greater than Mg. Ten mM optima were established for Ca2+ and Mn2+; the presence of UTP, however, results in significantly reduced concentrations of free Ca2+. Ion combination studies demonstrated interactive inhibitory effects between Ca2+ and other stimulatory divalent cations. Addition of 2 microM brain calmodulin, in the presence of 10 mM Ca2+, resulted in a 75-100% stimulation of activity.(ABSTRACT TRUNCATED AT 250 WORDS)
D000367:Age Factors; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D002412:Cations / Q000494:pharmacology; D003570:Cytidine Triphosphate / Q000378:metabolism; D004286:Dolichols / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology; D008297:Male; D008861:Microsomes / Q000201:enzymology; D010770:Phosphotransferases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D051381:Rats; D013737:Testis / Q000201:enzymology* / Q000254:growth & development; D014544:Uridine Triphosphate / Q000494:pharmacology
D002147:Calmodulin; D002412:Cations; D004286:Dolichols; D003570:Cytidine Triphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C020087:dolichol kinase; D005459:Fluorides; D002118:Calcium; D014544:Uridine Triphosphate