Olson S C, Bowman E P, Lambeth J D
Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322.
J Biol Chem. 1991 Sep 15;266(26):17236-42.
Receptor-linked activation of phospholipase D has been demonstrated recently in a variety of intact cell types including granulocytes, but little is known about the enzyme, its cofactor requirements, and regulation. Using [3H]alkyllysophosphatidylcholine to prelable an endogenous phosphatidylcholine substrate pool in conjunction with transphosphatidylation using ethanol to generate labeled phosphatidylethanol, we demonstrated a novel phospholipase D activity in neutrophil subcellular fractions. Guanosine 5'-O-3-(thiotriphosphate) (GTP gamma S) and phorbol 12-myristate 13-acetate (PMA) activated both phosphatidic acid generation and transphosphatidylation. Activity using both activators required the presence of not only plasma membrane but also cytosol, and proteolytic and thermal inactivation demonstrated the requirement for protein factors in both fractions. Using both stimuli, activity increased with increasing cytosol concentration. Product formation was approximately linear for about 10 min with PMA and 30 min with GTP gamma S, and both activators resulted in the total hydrolysis of up to 10% of the labeled phosphatidylcholine. The activity using both activators showed similar broad neutral pH optima, and both required the presence of micromolar levels of calcium, which by itself failed to activate at concentrations up to 1 mM. At low micromolar concentrations of nucleotides, activation was specific for guanine nucleotides and showed a specificity of GTP gamma S greater than guanyl-5'-yl imidodiphosphate greater than GTP, with no effect of GDP and GMP or adenine nucleotides, consistent with the participation of a guanine nucleotide regulatory protein. PMA activation was dependent on the presence of ATP, in particular when dialyzed cytosol was used, and was inhibited by about 50% by staurosporine, supporting a role for protein kinase C. However, purified protein kinase C failed to substitute for cytosol, implicating an additional cytosolic factor(s) in this response. These results indicate that the granulocytic phospholipase D pathway is a complex system that is regulated by at least two activation pathways, each comprised of components in two subcellular compartments.
最近已证实在包括粒细胞在内的多种完整细胞类型中存在受体连接的磷脂酶D激活现象，但对于该酶、其辅因子需求及调控了解甚少。我们使用[3H]烷基溶血磷脂酰胆碱对内源性磷脂酰胆碱底物池进行预标记，并结合使用乙醇进行转磷脂酰基作用以生成标记的磷脂酰乙醇，从而在中性粒细胞亚细胞组分中证实了一种新型磷脂酶D活性。鸟苷5'-O-3-(硫代三磷酸)(GTPγS)和佛波醇12-肉豆蔻酸酯13-乙酸酯(PMA)均可激活磷脂酸生成和转磷脂酰基作用。使用这两种激活剂时的活性不仅需要质膜的存在，还需要胞质溶胶，并且蛋白水解和热失活表明这两个组分中均需要蛋白质因子。使用这两种刺激时，活性随胞质溶胶浓度的增加而增加。PMA作用下产物形成约10分钟呈近似线性，GTPγS作用下则为30分钟，并且两种激活剂均可导致高达10%的标记磷脂酰胆碱完全水解。使用这两种激活剂时的活性表现出相似的宽泛中性pH最佳值，并且两者均需要微摩尔水平的钙存在，而钙本身在高达1 mM的浓度下无法激活。在低微摩尔浓度的核苷酸条件下，激活对鸟嘌呤核苷酸具有特异性，并且显示出GTPγS的特异性大于鸟苷-5'-基亚氨基二磷酸大于GTP，GDP、GMP或腺嘌呤核苷酸无作用，这与鸟嘌呤核苷酸调节蛋白的参与一致。PMA激活依赖于ATP的存在，特别是在使用透析后的胞质溶胶时，并且被星形孢菌素抑制约50%，这支持蛋白激酶C的作用。然而，纯化的蛋白激酶C无法替代胞质溶胶，这表明在此反应中存在其他胞质因子。这些结果表明粒细胞磷脂酶D途径是一个复杂的系统，受至少两条激活途径调控，每条途径由两个亚细胞区室中的组分组成。
