Purification and characterization of N-ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP2) from rat liver.
N-Ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP; EC 3.1.3.4) was purified 5900-fold from rat liver. The enzyme was solubilized from membranes with octylglucoside, fractionated with (NH4)2SO4, and purified in the presence of Triton X-100 by chromatography on Sephacryl S300, hydroxyapatite, heparin-Sepharose and Affi-Gel Blue. Silver-stained SDS/PAGE indicated that the enzyme was an 83 kDa polypeptide. Sephacryl S-300 gel filtration also produced a second peak of enzyme activity, which was eluted from all of the chromatography columns at a different position from the purified enzyme. SDS/PAGE indicated that it contained three polypeptides (83 kDa, 54 kDa and 34 kDa), and gel filtration suggested that it was not an aggregate of the purified enzyme. Both forms were sensitive to inhibition by amphiphilic amines, Mn2+ and Zn2+, but not by N-ethylmaleimide. Purified PAP required detergent for activity, but was not activated by Mg2+, fatty acids or phospholipids. The enzyme was able to dephosphorylate lysophosphatidic acid or phosphatidic acid, and was inhibited by diacylglycerol and monoacylglycerol. No evidence was obtained for regulation of PAP by reversible phosphorylation.
Fleming
I N
IN
Department of Biochemistry and Genetics, Medical School, University of Newcastle-upon-Tyne, UK.
Yeaman
S J
SJ
D000588:Amines / Q000494:pharmacology; D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004075:Diglycerides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology*; D005227:Fatty Acids / Q000737:chemistry; D005989:Glycerides / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D008246:Lysophospholipids / Q000378:metabolism / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D010711:Phosphatidate Phosphatase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D014280:Triglycerides / Q000494:pharmacology
D000588:Amines; D004075:Diglycerides; D005227:Fatty Acids; D005989:Glycerides; D008246:Lysophospholipids; D008670:Metals; D014280:Triglycerides; D010711:Phosphatidate Phosphatase; D005033:Ethylmaleimide