ENZYMOLOGICAL CHARACTERISTICS OF PLASMA MEMBRANE PHOSPHATIDATE PHOSPHOHYDROLASE (PAP_2) FROM RAT LIVER

摘要

Phosphatidate phosphohydrolase (PAP_(2b), fraction b) was purified from the plasma membrane of rat liver cells. The K_m for the surface concentration of phosphatidic acid was 0.43 mol%. The subunit of the enzyme had an M.W. of 33.8 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native enzyme shows a molecular weight of 182 kDa in a gel filtration column packed with Sephacryl S_(300) in the presence of Triton X-100. The pH optima obtained for PAP_(2b) were 5.5 and 7 in imidazole and Tris- HC1 buffers, respectively. The membrane homogenate enzyme (PAP_2) consumed the lamellar (L_a) phase of phosphatidate and was activated (approximately 3-fold) by Lubrol PX, CTAB and Tween 80 and inhibited by Zn~(2+) and Mn~(2+). The inhibition was concentration dependent. These cations affected PAP_(2b) activity through the phase transition of phosphatidate from lamellar (L_a) to inverted hexagonal (H_Ⅱ) form. Guanidine hydrochloride and urea increased PAP_2 activity (2-fold) up to 20mM concentrations by stabilizing the L_a phase. Optimum activity of purified PAP_(2b) was obtained at 3% trehalose and 7% sucrose. The data suggested that the stability of the L_a form of phosphatidate by detergent micelles may take place through surface dilution processes.