Abstract
Phospholipase D (PLD) activity from membranes of cultured cells can be activated by guanosine 5'-O-(3-thiotriphosphate) and the small GTP-dependent protein, Arf. While this activity was readily apparent in membranes from HL60 cells, it was much lower or not observable in membranes from various mammalian tissues. However, extraction of porcine brain membranes with detergent and subsequent chromatography with SP-Sepharose revealed a large peak of Arf-sensitive PLD activity. This activity has been enriched through several steps of chromatography and characterized with respect to size, nucleotide specificity, and sensitivity to different Arf and Arf-like proteins. Hydrodynamic analysis indicated that the enriched PLD had an s20,w of 5.1 and a Stokes radius of 4.3 nm. These parameters indicate that the enzyme has an apparent molecular mass of 95,000 Da. Effective stimulation of the enriched enzyme was achieved with GTP as well as nonhydrolyzable analogs. All of the Arf subtypes tested were effective activators of PLD activity. Arf derived from yeast could activate mammalian PLD but with lower potency. The Arf-related Arl proteins were ineffective. PLD that has been highly enriched retained a requirement for phosphatidylinositol 4,5-bisphosphate for efficient expression of activity. Additionally, the ability of recombinant or purified porcine brain Arf to stimulate PLD activity was reduced relative to impure fractions of Arf activity. Thus, porcine PLD that has been purified about 5,000-10,000-fold is synergistically activated by Arf in combination with other cytosolic components that are described in the accompanying paper (Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C. (1995) J. Biol. Chem. 270, 14944-14950). Taken together, these data suggest that physiological regulation of Arf-sensitive PLD may involve the coordinate assembly of several interacting regulatory subunits.
Highlights
PLD that has been highly enriched retained a requirement for phosphatidylinositol 4,5-bisphosphate for efficient expression of activity
The low activities observed in tissues and some cell lines are probably due to factors that interfere with the assay of PLD activity
This was suggested in part by the observation that the Arf in cytosol from brain was not effective in stimulating PLD activity in HL60 membranes until it was partially resolved by a step of chromatography [9]
Summary
Vol 270, No 25, Issue of June 23, pp. 14935-14943, 1995 Printed in U.S.A. Partial Purification and Characterization of Arf-sensitive Phospholipase D from Porcine Brain*. This enzymatic activity hydrolyzes phospholipids into phosphatidic acid (PA) and their respective polar head groups and is distinct from the better characterized PLD activity that hydrolyzes phosphatidylinositol-glycans [6] Signaling via this reaction is accomplished through the production of PA, which may act directly as a second messenger or serve as a precursor to diacylglycerol or lysophosphatidic acid. Stimulation of PLD activity via hormones that act through either G protein-dependent or tyrosine kinase-linked mechanisms has been reported (for review, see Ref. 5). We report the partial purification of Arf-dependent PLD from porcine brain and its initial characterization with respect to substrate specificity, sensitivity to Arf proteins, and additional cytosolic factors that will be described in more detail in the accompanying manuscript [20]
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