Enzymatic dephosphorylation of endogenous and exogenous dolichyl monophosphate by calf brain membranes

Abstract

Calf brain membranes have been shown to enzymatically dephosphorylate endogenous and partially purified, exogenous dolichyl [ 32P]monophosphate. The properties and specificity of the dolichyl monophosphatase activity have been studied by following the release of [ 32P]phosphate from exogenous dolichyl [ 32P]monophosphate added in a dispersion with Triton X-100. The calf brain phosphatase (1) is inhibited by Mn 2+, Mg 2+, Ca 2+, fluoride, and phosphate; (2) exhibits a neutral pH optimum; and (3) has an apparent K m of 200 μ m for dolichyl monophosphate. Dolichyl monophosphatase activity can be distinguished from phosphatidate phosphatase on the basis of their responses to fluoride and phosphate. Based on differential thermolability and the effects of divalent cations and EDTA, the calf brain dolichyl monophosphatase can also be discriminated from the general phosphatase activity assayed with p-nitrophenyl phosphate. Dolichyl monophosphatase activity can be solubilized by treating microsomes with Triton X-100. The enzymatic dephosphorylation of exogenous dolichyl [ 32P]monophosphate catalyzed by particulate and detergent-solubilized preparations is negligibly affected by equimolar concentrations of ATP and an assortment of phosphomonoesters, including phosphatidic acid and hexadecyl phosphate. A reduction of approximately 40% in dolichyl monophosphatase activity is observed in the presence of equimolar amounts of retinyl monophosphate. Overall, these results represent good evidence for the presence of a neutral polyisoprenyl monophosphatase in central nervous tissue.