Guanine-nucleotide- and adenine-nucleotide-dependent regulation of phospholipase D in electropermeabilized HL-60 granulocytes.
Guanine-nucleotide- and adenine-nucleotide-dependent regulation of phospholipase D in electropermeabilized HL-60 granulocytes.
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电透化 HL-60 粒细胞中磷脂酶 D 的鸟嘌呤核苷酸和腺嘌呤核苷酸依赖性调节。
DOI：
10.1042/bj2780081
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Dubyak,GR
中科院分区：
文献类型：
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作者：
Xie,MS;Dubyak,GR
我们已经表征了磷脂酶D （PLD）在电渗透HL-60粒细胞中的调节，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′-[β - γ -酰亚胺]三磷酸处理这些通透化细胞，诱导磷脂酸（PA）持续（近线性累积长达60分钟）积累。在乙醇存在下，还观察到持续生产磷脂酰乙醇（PEt）。随着乙醇浓度的增加，PEt的形成增加，PA的形成减少；这表明一种pld型效应酶参与其中。GTP[S]刺激PLD活性的能力依赖于Mg(2+)，并被GDP及其不可水解的β -硫代类似物抑制。Ca2+浓度小于或等于nM时，对GTP[S]依赖性PLD活性没有影响。然而，较高浓度的Ca2+产生了这种活性的显著增强。包括MgATP（大于或等于0.1 mM），但不包括其他核苷三磷酸，也诱导了GTP[S]依赖性PLD激活的大幅增强。在缺乏鸟嘌呤核苷酸的情况下，MgATP没有引起PLD的显著激活。值得注意的是，ATP的这种作用不会被腺苷5 ' -[β - γ -亚甲基]三磷酸（一种不可水解的ATP类似物）所模仿。相反，这种类似物抑制了基础和atp增强的GTP[S]依赖性PLD活性。这表明ATP增强GTP[S]依赖性PLD活性的能力涉及磷酸转移酶作用，而不是腺嘌呤核苷酸结合诱导的简单变构效应。经不同时间的预孵育后，经MgATP增强的GTP[S]依赖性PLD活性的绝对强度降低了90%。当通透化的细胞在GTP的存在下预孵育时，可以防止这种时间依赖性的mgatp诱导的增强丧失[S]。这些结果表明，电渗透HL-60粒细胞可以用来区分gtp结合蛋白(s)和atp依赖过程（激酶？）在磷脂酶D活性调节中的协同作用。
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.