PARTIAL-PURIFICATION AND CHARACTERIZATION OF ARF-SENSITIVE PHOSPHOLIPASE-D FROM PORCINE BRAIN

Phospholipase D (PLD) activity from membranes of cultured cells can be activated by guanosine 5'-O-(3-thiotriphosphate) and the small GTP-de pendent protein, Arf. While this activity was readily apparent in memb ranes from HL60 cells, it was much lower or not observable in membrane s from various mammalian tissues. However, extraction of porcine brain membranes with detergent and subsequent chromatography with SP-Sephar ose revealed a large peak of Arf-sensitive PLD activity. This activity has been enriched through several steps of chromatography and charact erized with respect to size, nucleotide specificity, and sensitivity t o different Arf and Arf-like proteins. Hydrodynamic analysis indicated that the enriched PLD had an s(20,w) of 5.1 and a Stokes radius of 4. 3 nm. These parameters indicate that the enzyme has an apparent molecu lar mass of 95,000 Da. Effective stimulation of the enriched enzyme wa s achieved with GTP as well as nonhydrolyzable analogs. All of the Arf subtypes tested were effective activators of PLD activity. Arf derive d from yeast could activate mammalian PLD but with lower potency. The Arf-related Arl proteins were ineffective. PLD that has been highly en riched retained a requirement for phosphatidylinositol 4,5-bisphosphat e for efficient expression of activity. Additionally, the ability of r ecombinant or purified porcine brain Arf to stimulate PLD activity was reduced relative to impure fractions of Arf activity. Thus, porcine P LD that has been purified about 5,000-10,000-fold is synergistically a ctivated by Arf in combination with other cytosolic components that ar e described in the accompanying paper (Singer, W. D., Brown, H. A., Bo koch, G. M., and Sternweis, P. C. (1995) J. Biol. Chem. 270, 14944-149 50). Taken together, these data suggest that physiological regulation of Arf-sensitive PLD may involve the coordinate assembly of several in teracting regulatory subunits.