Singer W D, Brown H A, Bokoch G M, Sternweis P C
Department of Pharmacology, University of Texas, Southwestern Medical Center, Dallas 75235-9041, USA.
J Biol Chem. 1995 Jun 23;270(25):14944-50. doi: 10.1074/jbc.270.25.14944.
Phospholipase D, which has been extracted from porcine brain membranes and chromatographically enriched 100-fold, was activated better by impure preparations of Arf than by purified or recombinant Arf. Examination of brain cytosol with this enriched preparation of PLD activity revealed at least three stimulatory components. One of these is Arf or the first cytoplasmic factor. A second peak of PLD-stimulating activity (cytoplasmic factor II, CFII) was resolved from Arf by anion exchange and gel filtration. This CFII can be further separated into multiple activities by chromatography with heparin-agarose. The activities were differentiated by their stimulatory properties as measured in the absence or presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) alone and in the presence of added Arf and GTP gamma S. While all of the CFII pools stimulated PLD activity to some degree and showed synergistic activation when administered in conjunction with Arf, they could be classified into two groups with distinct behavior. When used together, pools from the two respective groups showed synergistic activation of PLD. The first set of pools contained the RhoA monomeric G protein. Recombinant RhoA was used to show that it could indeed activate this enriched PLD activity and act synergistically with Arf proteins. A related monomeric G protein, Cdc42, was also effective. The second set of CFII pools were devoid of RhoA and, in contrast to the first group, demonstrated significant stimulating activity in the absence of guanine nucleotides. These data indicate that the PLD activity from brain can be modulated by several cytosolic factors and that Arf-sensitive PLD may represent a complex activity that can be regulated in an interactive fashion by a variety of cellular signaling events.
磷脂酶D已从猪脑膜中提取并通过色谱法富集了100倍，与纯化的或重组的Arf相比，它被不纯的Arf制剂激活得更好。用这种富集的磷脂酶D活性制剂检测脑细胞质溶胶发现至少有三种刺激成分。其中一种是Arf或第一个细胞质因子。通过阴离子交换和凝胶过滤从Arf中分离出磷脂酶D刺激活性的第二个峰（细胞质因子II，CFII）。这种CFII可以通过肝素-琼脂糖色谱进一步分离成多种活性。通过在单独存在或不存在鸟苷5'-O-(3-硫代三磷酸)(GTPγS)以及添加Arf和GTPγS的情况下测量其刺激特性来区分这些活性。虽然所有的CFII组分都在一定程度上刺激了磷脂酶D活性，并且在与Arf联合使用时表现出协同激活作用，但它们可以分为两组，行为不同。当将两组各自的组分一起使用时，它们显示出对磷脂酶D的协同激活作用。第一组组分包含RhoA单体G蛋白。重组RhoA被用于证明它确实可以激活这种富集的磷脂酶D活性并与Arf蛋白协同作用。一种相关的单体G蛋白Cdc42也有效。第二组CFII组分不含RhoA，与第一组相反，在没有鸟嘌呤核苷酸的情况下表现出显著的刺激活性。这些数据表明，脑中的磷脂酶D活性可以被几种细胞质因子调节，并且Arf敏感的磷脂酶D可能代表一种复杂的活性，它可以通过多种细胞信号事件以相互作用的方式被调节。
