Characterization of dolichol and dolichyl phosphate phosphatase from soya beans (Glycine max).
A series of polyprenols, ranging in length from 15 to 22 isoprene units, has been isolated from soya beans (Glycine max) and purified by high-pressure liquid chromatography. N.m.r., i.r. and mass spectra of the compounds indicated that they are alpha-saturated polyprenols of the dolichol type. The amount present in dry seeds was about 9 mg/100 g, whereas dolichyl phosphate (Dol-P) was present only in trace amounts. Dol-P phosphatase activity was detected in the microsomal fraction of 5-day-old germinating soya-bean cotyledons. The Dol-P phosphatase activity was linear with respect to time and protein concentration and exhibited a broad pH optimum (pH 7-9). Triton X-100 was necessary for significant enzyme activity. Enzyme activity was slightly enhanced by EDTA, whereas dithiothreitol was without effect. An apparent Km of 5 microM was determined for Dol-P. Bivalent metal ions were not required for enzyme activity. A number of phosphorylated compounds tested as enzyme substrates (including a number of nucleoside phosphates, glucose 6-phosphate, sodium beta-glycerophosphate and Na4P2O7) did not compete with [1-3H]Dol-P as substrate. A number of phospholipids were also tested for their ability to act as Dol-P phosphatase substrates. At 1 mM concentration, phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid and lysophosphatidic acid each inhibited enzymic activity. However, at 0.1 mM concentration, phosphatidylcholine and phosphatidylethanolamine were slightly stimulatory, whereas phosphatidic acid and lysophosphatidic acid were still inhibitory. Phosphatidic acid showed competitive inhibition.
D002851:Chromatography, High Pressure Liquid; D004224:Diterpenes / Q000378:metabolism*; D004286:Dolichols / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D017830:Octoxynol; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D011092:Polyethylene Glycols / Q000494:pharmacology; D013025:Glycine max / Q000201:enzymology; D013055:Spectrophotometry, Infrared; D013379:Substrate Specificity
D004224:Diterpenes; D004286:Dolichols; D011092:Polyethylene Glycols; D017830:Octoxynol; D010744:Phosphoric Monoester Hydrolases; C020088:dolichyl-phosphatase