TISSUE-SPECIFIC DISTRIBUTION AND SUBCELLULAR-DISTRIBUTION OF PHOSPHOLIPASE-D IN RAT - EVIDENCE FOR DISTINCT RHOA-RIBOSYLATION AND ADP-RIBOSYLATION FACTOR (ARF)-REGULATED ISOENZYMES

Phospholipase D (PLD) is regulated by many factors including the small G-proteins, RhoA and ADP-ribosylation factor (ARF). The present study examined the distribution of RhoA- and ARF-responsive PLD in membrane s, microsomes and cytosol of rat tissues and in rat liver subcellular fractions. PLD was present in all tissue fractions examined and was st imulated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]), with the h ighest specific activities being in lung, kidney and spleen. When myri stoylated recombinant ARF (mARF) was added with GTP[S], the PLD activi ty was stimulated further, but the addition of RhoA was without effect . However, in extracts from crude membranes both mARF and RhoA enhance d the stimulation by GTP[S], with high specific activities of PLD bein g observed in all tissues except muscle. The response to mARF was usua lly greater than to RhoA, and the responses were additive, except for liver, which showed synergism. When the PLD activity of subcellular fr actions of liver was examined, GTP[S] caused increases in all fraction s except microsomes and mitochondria, which exhibited low activity. Al l fractions except mitochondria showed responses to RhoA and mARF, wit h the response to RhoA being greater in plasma membranes and that to m ARF being greater in Golgi and nuclei. Western blotting showed that Rh oA was located mainly in the cytosol and plasma membranes, whereas ARF was principally in the cytosol. These findings demonstrate the widesp read occurrence of significant activity of both Rho- and ARF-responsiv e forms of PLD in membranes from all tissues except muscle, and the pr esence of both forms in liver subcellular fractions except mitochondri a. The large variations in the relative responses of PLD to Rho and AR F observed in different tissues and fractions support the existence of different isoforms of the enzyme.