Brown H A, Gutowski S, Kahn R A, Sternweis P C
Department of Pharmacology, University of Texas, Southwestern Medical Center, Dallas 75235-9041, USA.
J Biol Chem. 1995 Jun 23;270(25):14935-43. doi: 10.1074/jbc.270.25.14935.
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.
培养细胞的膜中的磷脂酶D（PLD）活性可被鸟苷5'-O-（3-硫代三磷酸）和小的GTP依赖性蛋白Arf激活。虽然这种活性在HL60细胞的膜中很明显，但在各种哺乳动物组织的膜中则低得多或无法观察到。然而，用去污剂提取猪脑膜并随后用SP-琼脂糖凝胶进行色谱分析，发现了一个对Arf敏感的PLD活性大峰。该活性已通过几步色谱法得到富集，并在大小、核苷酸特异性以及对不同Arf和Arf样蛋白的敏感性方面进行了表征。流体动力学分析表明，富集的PLD的沉降系数s20,w为5.1，斯托克斯半径为4.3nm。这些参数表明该酶的表观分子量为95,000Da。GTP以及不可水解的类似物可有效刺激富集的酶。测试的所有Arf亚型都是PLD活性的有效激活剂。源自酵母的Arf可激活哺乳动物PLD，但效力较低。与Arf相关的Arl蛋白无效。高度富集的PLD仍需要磷脂酰肌醇4,5-二磷酸才能有效表达活性。此外，相对于不纯的Arf活性级分，重组或纯化的猪脑Arf刺激PLD活性的能力有所降低。因此，已纯化约5000-10000倍的猪PLD被Arf与随附论文（Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C. (1995) J. Biol. Chem. 270, 14944-14950）中描述的其他胞质成分协同激活。综上所述，这些数据表明，对Arf敏感的PLD的生理调节可能涉及几个相互作用的调节亚基的协同组装。
