ENZYMOLOGICAL CHARACTERISTICS OF PLASMA MEMBRANE PHOSPHATIDATE PHOSPHOHYDROLASE (PAP2) FROM RAT LIVER

Document Type: Regular Paper

Authors

1 Department of Biochemistry, Ilam University of Medical Sciences, Ilam, I. R. of Iran

2 Department of Biochemistry, Esfahan University of Medical Sciences, Esfahan, I. R. of Iran

Abstract

Phosphatidate phosphohydrolase (PAP2b, fraction b) was purified from the plasma membrane of
rat liver cells. The Km 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 S300 in
the presence of Triton X-100. The pH optima obtained for PAP2b were 5.5 and 7 in imidazole and Tris- HCl
buffers, respectively. The membrane homogenate enzyme (PAP2) consumed the lamellar (La) phase of
phosphatidate and was activated (approximately 3-fold) by Lubrol PX, CTAB and Tween 80 and inhibited by
Zn2+ and Mn2+. The inhibition was concentration dependent. These cations affected PAP2b activity through the phase transition of phosphatidate from lamellar (La) to inverted hexagonal (HII) form. Guanidine
hydrochloride and urea increased PAP2 activity (2-fold) up to 20mM concentrations by stabilizing the La
phase. Optimum activity of purified PAP2b was obtained at 3% trehalose and 7% sucrose. The data suggested that the stability of the La form of phosphatidate by detergent micelles may take place through surface dilution processes.

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