Brain dolichyl pyrophosphate phosphatase. Solubilization, characterization, and differentiation from dolichyl monophosphate phosphatase activity.
Dolichyl [beta-32P]pyrophosphate ([beta-32P]Dol-P-P) has been prepared chemically to study Dol-P-P phosphatase in calf brain. Calf brain microsomes catalyze the enzymatic release of 32Pi from exogenous [beta-32P]Dol-P-P by a bacitracin-sensitive reaction. [32P]Pyrophosphate was not detected with the water-soluble product even when 1 mM sodium pyrophosphate was added to impede pyrophosphatase activity. A substantial fraction of the Dol-P-P phosphatase activity can be solubilized by treating brain microsomes with 3% Triton X-100. The detergent extracts catalyze the enzymatic release of 32Pi from [beta-32P]Dol-P-P and the conversion of [14C]undecaprenyl pyrophosphate to [14C]undecaprenyl monophosphate. The solubilized Dol-P-P phosphatase activity: 1) is optimal at neutral pH; 2) is inhibited by Mn2+ and stimulated by EDTA; 3) exhibits an apparent Km = 20 microM for Dol-P-P; 4) is competitively inhibited by undecaprenyl pyrophosphate, and 5) is blocked by bacitracin. Solubilized Dol-P-P phosphatase activity differs from Dol-P phosphatase activity present in the same detergent extracts with respect to: 1) thermolability at 50 degrees C, 2) effect of 20 mM EDTA, and 3) sensitivity to phosphate and fluoride ions. These studies describe the chemical synthesis of [beta-32P]Dol-P-P for use in a convenient assay of Dol-P-P phosphatase activity. A procedure for the solubilization of Dol-P-P phosphatase activity from microsomes is presented, and an enzymological comparison indicates that Dol-P-P and Dol-P phosphatase are separate enzymes in calf brain.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002250:Carbon Radioisotopes; D002417:Cattle; D004288:Dolichol Phosphates; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D011755:Pyrophosphatases
D002250:Carbon Radioisotopes; D004288:Dolichol Phosphates; C026406:dolichol pyrophosphate; D010744:Phosphoric Monoester Hydrolases; C020088:dolichyl-phosphatase; D011755:Pyrophosphatases; C023082:dolichyldiphosphatase