PROPERTIES OF PHOSPHATIDATE PHOSPHOHYDROLASE IN RAT ADIPOSE-TISSUE

Previously we have identified the presence of two different phosphatid ate phosphohydrolase (PPH) activities in rat adipose tissue, based on Mg2+-dependency. In the present investigation, we have further charact erized these isoenzymes, using both aqueous dispersed and membrane-bou nd phosphatidate as substrates and differentiated these activities on the basis of both Mg2+-dependency and N-ethylmaleimide (NEM)-sensitivi ty. These two distinguishing criteria gave identical estimates of PPH activities present in the different subcellular fractions. The microso mal and cytosol fractions contained mainly the Mg2+-dependent (NEM-sen sitive) form, which was inhibited by various thiol reagents, was inact ivated by heating at 55 degrees C for 20 min, and was decreased signif icantly within 2 h after intraperitoneal administration of cystamine ( 200 mg/kg). Such treatments had no effects on the Mg2+-independent (NE M-insensitive) form of PPH, which was mainly located in the plasma mem branes, mitochondrial and microsomal fractions. Addition of Lipid A an d guanosine 5'-[gamma-thio]triphosphate to the assay mixture had no ef fect on the PPH activities. The Mg2+-independent PPH form, which was t hermostable in the intact subcellular fractions, became thermolabile w hen these fractions were disrupted in the presence of Triton X-100. Th e present studies demonstrate that: (1) the thermostability is not a s atisfactory index to differentiate these isoenzymes; (2) the thiol/dis ulphide exchange may be involved in the regulation of Mg2+-dependent P PH activity; and (3) the PPH isoenzymes do not seem to be under G-prot ein control in adipose tissue, as reported previously in the mesangial cell line.