Characterization of dolichol kinase from soybean microsomes

Abstract

Microsomes from germinating soybeans contain a dolichol kinase with some unique and interesting properties. Kinase activity was linear with time, protein concentration and the concentration of one of the substrates, dolichol. The second substrate for all known dolichol kinases, CTP, was not required for maximal activity. CTP and other nucleoside tri- and diphosphates, particularly CDP, were actually inhibitory. In addition, 80% of maximal activity was observed in the absence of divalent cations, although a low level of stimulation was noted with Mg 2+. The kinase reaction had pH optima at 7–8, and 9.5. Addition of nonlabeled dolichol to assays caused a proportional decrease in the incorporation of [l- 14C]dolichol into [1- 14C]dolichyl phosphate (Dol- p). Addition of nonradioactive Dol- p to incubations greatly increased [ l- 14 C]Dol- p formation. Despite the unusual properties of the soybean enzyme, analysis by TLC and HPLC clearly indicated that Dol- p is the reaction product. The inhibitory and slight stimulatory effects of CDP and Mg 2+, respectively, suggest that endogenous CTP and Mg 2+ are preferentially utilized in the dolichol kinase reaction. These observations together with our recent study concerning developmental changes in concentrations of dolichol intermediates and changes in both dolichol kinase and dolichyl phosphate phosphatase activity (accompanying paper, Ravi K., Rip J.W. and Carroll K.K. (1986) Biochim. Biophys. Acta 875, 626–632) suggest that increased Dol- p synthesis is required for the formation of N-linked glycoproteins during germination.