Purification and properties of phosphatidic acid phosphatase from porcine thymus membranes.
We purified phosphatidic acid phosphatase (EC 3.1.3.4) 2300-fold from porcine thymus membranes. The enzyme was solubilized with beta-octyl glucoside and Triton X-100 and fractionated with ammonium sulfate. The purification was then achieved by chromatography in the presence of Triton X-100 with Sephacryl S-300, hydroxylapatite, heparin-Sepharose, and Affi-Gel Blue. The final enzyme preparation gave a single band of M(r) = 83,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The native enzyme, on the other hand, was eluted at M(r) = 218,000 in gel filtration chromatography with Superose 12 in the presence of Triton X-100. The enzyme was judged to be specific to phosphatidic acid, since excess amounts of dicetylphosphate or lysophosphatidic acid did not inhibit the enzyme activity. In this respect, the enzyme was inhibited by 1,2-diacylglycerol but not by 1- or 2-monoacylglycerol and triacylglycerol. The enzyme required Triton X-100 or deoxycholate for its activity. Although the enzyme appeared to be an integral membrane protein, we could not detect its phospholipid dependencies. The activity was independent of Mg2+, and other cations were strongly inhibitory. The specific enzyme activity was 15 mumol/min/mg of protein when assayed using phosphatidic acid as Triton X-100 mixed micelles. The Km for the surface concentration of phosphatidic acid was 0.30 mol%. The enzyme was inhibited by sphingosine and chloropromazine, and less potently, by propranolol and NaF. The enzyme was insensitive to thio-reactive reagents like N-ethylmaleimide.
Kanoh
H
H
Department of Biochemistry, Sapporo Medical College, Japan.
Imai
S
S
Yamada
K
K
Sakane
F
F
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003902:Detergents / Q000494:pharmacology; D017886:Durapatite; D005989:Glycerides / Q000494:pharmacology; D006882:Hydroxyapatites; D007700:Kinetics; D008970:Molecular Weight; D010711:Phosphatidate Phosphatase / Q000302:isolation & purification* / Q000378:metabolism*; D010743:Phospholipids / Q000494:pharmacology; D013552:Swine; D013950:Thymus Gland / Q000201:enzymology*
D003902:Detergents; D005989:Glycerides; D006882:Hydroxyapatites; D010743:Phospholipids; D017886:Durapatite; D010711:Phosphatidate Phosphatase