A distinction in vitro between rat liver phosphatidate phosphatase and phospholipase C

Hepatocellular membranes (1000 X g) containing membrane-associated, labeled phosphatidic acid were incubated (1-30 min) with 2 mM oleate or 5 mM bromobenzene in the presence or absence of various metals and NaF. Under the appropriate incubation conditions, membranes displayed rapid and significant oleate- and bromobenzene-dependent increases in the dephosphorylation of labeled phosphatidic acid. However, oleate and bromobenzene activated the dephosphorylation of phosphatidate by phosphatidate phosphatase and phospholipase C, respectively. This conclusion is supported by the observation that the phosphatase stimulated by oleate is: (1) Mg2+ -dependent; (2) inhibited by other metals, such as Ca2+; (3) inhibited by NaF; (4) specific for phosphatidic acid; and (5) associated with a rise in liver cell triacylglycerol production. Bromobenzene, however, activated a phospholipase C that is: (1) stimulated by various metals, such as Mg2+, Ca2+ and Ba2+; (2) insensitive to NaF; (3) associated with the degradation of various membrane phospholipids; (4), associated with liver cell injury; and (5) not associated with a rise in liver cell triacylglycerol formation. These results suggest that under appropriate conditions in vitro the dephosphorylation of phosphatidic acid can be used to assess changes in phosphatidate phosphatase and/or phospholipase C activity. The distinction between these enzymes is important, since phosphatidate phosphatase and phospholipase C regulate key steps in phospholipid biosynthesis and degradation, respectively.