Xie M S, Dubyak G R
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106.
Biochem J. 1991 Aug 15;278 ( Pt 1)(Pt 1):81-9. doi: 10.1042/bj2780081.
We have characterized the regulation of phospholipase D (PLD) in electropermeabilized HL-60 granulocytes in which endogenous phospholipids were pre-labelled with [3H]oleic acid. Treatment of these permeabilized cells with the non-hydrolysable GTP analogues guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta gamma-imido]triphosphate induced a sustained (near-linear for up to 60 min) accumulation of phosphatidic acid (PA). In the presence of ethanol a sustained production of phosphatidylethanol (PEt) was also observed. With increasing concentrations of ethanol, PEt formation increased, whereas PA formation declined; this indicated involvement of a PLD-type effector enzyme. The ability of GTP[S] to stimulate this PLD activity was Mg(2+)-dependent and was inhibited by GDP and its non-hydrolysable beta-thio analogue. Ca2+, at concentrations less than or equal to nM, had no effect on the GTP[S]-dependent PLD activity. However, higher concentrations of Ca2+ produced a significant potentiation of this activity. Inclusion of MgATP (greater than or equal to 0.1 mM), but not other nucleoside triphosphates, also induced a large potentiation of GTP[S]-dependent PLD activation. In the absence of guanine nucleotides, MgATP elicited no significant activation of PLD. Significantly, this effect of ATP was not mimicked by adenosine 5'-[beta gamma-methylene]triphosphate, a non-hydrolysable ATP analogue. Rather, this analogue inhibited both basal and ATP-potentiated GTP[S]-dependent PLD activity. This suggests that the ability of ATP to potentiate GTP[S]-dependent PLD activity involves phosphotransferase action rather than simple allosteric effects induced by adenine nucleotide binding. The absolute magnitude of the GTP[S]-dependent PLD activity which could be potentiated by MgATP was decreased by 90% when the permeabilized cells were preincubated for various times before addition of these stimulatory agents. This time-dependent loss of MgATP-induced potentiation was prevented when the permeabilized cells were preincubated in the presence of GTP[S]. These results demonstrate that electropermeabilized HL-60 granulocytes can be used to discriminate synergistic roles for a GTP-binding protein(s) and an ATP-dependent process (kinase?) in the regulation of phospholipase D activity.
我们已对电通透HL - 60粒细胞中磷脂酶D（PLD）的调节进行了表征，其中内源性磷脂预先用[3H]油酸标记。用不可水解的GTP类似物鸟苷5'-[γ-硫代]三磷酸（GTP[S]）和鸟苷5'-[βγ-亚氨基]三磷酸处理这些通透细胞，会诱导磷脂酸（PA）持续积累（长达60分钟近乎呈线性）。在乙醇存在下，还观察到磷脂酰乙醇（PEt）持续生成。随着乙醇浓度增加，PEt形成增加，而PA形成减少；这表明涉及一种PLD型效应酶。GTP[S]刺激这种PLD活性的能力依赖于Mg(2+)，并受到GDP及其不可水解的β-硫代类似物的抑制。Ca2+浓度小于或等于nM时，对GTP[S]依赖的PLD活性无影响。然而，较高浓度的Ca2+会显著增强这种活性。加入MgATP（大于或等于0.1 mM），而非其他核苷三磷酸，也会显著增强GTP[S]依赖的PLD激活。在不存在鸟嘌呤核苷酸的情况下，MgATP不会引起PLD的显著激活。重要的是，5'-[βγ-亚甲基]三磷酸腺苷（一种不可水解的ATP类似物）不会模拟ATP的这种作用。相反，这种类似物会抑制基础和ATP增强的GTP[S]依赖的PLD活性。这表明ATP增强GTP[S]依赖的PLD活性的能力涉及磷酸转移酶作用，而非由腺嘌呤核苷酸结合诱导的简单变构效应。当通透细胞在添加这些刺激剂之前预先孵育不同时间时，MgATP可增强的GTP[S]依赖的PLD活性的绝对幅度降低了90%。当通透细胞在GTP[S]存在下预先孵育时，可防止这种MgATP诱导增强作用的时间依赖性丧失。这些结果表明，电通透的HL - 60粒细胞可用于区分GTP结合蛋白和ATP依赖过程（激酶？）在磷脂酶D活性调节中的协同作用。
