Properties of phosphatidate phosphohydrolase in rat adipose tissue.
Previously we have identified the presence of two different phosphatidate phosphohydrolase (PPH) activities in rat adipose tissue, based on Mg(2+)-dependency. In the present investigation, we have further characterized these isoenzymes, using both aqueous dispersed and membrane-bound phosphatidate as substrates and differentiated these activities on the basis of both Mg(2+)-dependency and N-ethylmaleimide (NEM)-sensitivity. These two distinguishing criteria gave identical estimates of PPH activities present in the different subcellular fractions. The microsomal and cytosol fractions contained mainly the Mg(2+)-dependent (NEM-sensitive) form, which was inhibited by various thiol reagents, was inactivated by heating at 55 degrees C for 20 min, and was decreased significantly within 2 h after intraperitoneal administration of cystamine (200 mg/kg). Such treatments had no effects on the Mg(2+)-independent (NEM-insensitive) form of PPH, which was mainly located in the plasma membranes, mitochondrial and microsomal fractions. Addition of Lipid A and guanosine 5'-[gamma-thio]triphosphate to the assay mixture had no effect on the PPH activities. The Mg(2+)-independent PPH form, which was thermostable in the intact subcellular fractions, became thermolabile when these fractions were disrupted in the presence of Triton X-100. The present studies demonstrate that: (1) the thermostability is not a satisfactory index to differentiate these isoenzymes; (2) the thiol/disulphide exchange may be involved in the regulation of Mg(2+)-dependent PPH activity; and (3) the PPH isoenzymes do not seem to be under G-protein control in adipose tissue, as reported previously in the mesangial cell line.
Jamdar
S C
SC
Department of Anesthesiology, College of Physicians & Surgeons, Columbia University, New York, NY 10032.
Cao
W F
WF
D000273:Adipose Tissue / Q000201:enzymology* / Q000648:ultrastructure; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D005033:Ethylmaleimide / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006358:Hot Temperature; D007527:Isoenzymes / Q000378:metabolism*; D008050:Lipid A / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008861:Microsomes / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D010711:Phosphatidate Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D007527:Isoenzymes; D008050:Lipid A; D013439:Sulfhydryl Reagents; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010711:Phosphatidate Phosphatase; D008274:Magnesium; D005033:Ethylmaleimide