Partial purification and characterization of Arf-sensitive phospholipase D from porcine brain

H. A. Brown, S. Gutowski, R. A. Kahn, P. C. Sternweis

Research output: Contribution to journalArticle

126 Citations (Scopus)

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 s(20,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 Ar1 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.

Original languageEnglish (US)
Pages (from-to)14935-14943
Number of pages9
JournalJournal of Biological Chemistry
Volume270
Issue number25
DOIs
StatePublished - 1995

Fingerprint

Phospholipase D
Purification
Brain
Swine
Membranes
Chromatography
Guanosine Triphosphate
Guanosine 5'-O-(3-Thiotriphosphate)
HL-60 Cells
Molecular mass
Enzymes
Hydrodynamics
Phosphatidylinositols
Detergents
Yeast
Sepharose
Cultured Cells
Proteins
Nucleotides
Yeasts

ASJC Scopus subject areas

  • Biochemistry

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Partial purification and characterization of Arf-sensitive phospholipase D from porcine brain. / Brown, H. A.; Gutowski, S.; Kahn, R. A.; Sternweis, P. C.

In: Journal of Biological Chemistry, Vol. 270, No. 25, 1995, p. 14935-14943.

Research output: Contribution to journalArticle

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