Phospholipase D activation in a cell-free system from human neutrophils by phorbol 12-myristate 13-acetate and guanosine 5'-O-(3-thiotriphosphate). Activation is calcium dependent and requires protein factors in both the plasma membrane and cytosol.
Phospholipase D activation in a cell-free system from human neutrophils by phorbol 12-myristate 13-acetate and guanosine 5'-O-(3-thiotriphosphate). Activation is calcium dependent and requires protein factors in both the plasma membrane and cytosol.
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DOI：
10.1016/s0021-9258(19)47364-x
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发表时间：
1991-09
期刊：
影响因子：
--
通讯作者：
S. Olson;E. Bowman;J. Lambeth
中科院分区：
文献类型：
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作者：
S. Olson;E. Bowman;J. Lambeth
磷脂酶D的受体连接激活最近已被证明在各种完整的细胞类型，包括粒细胞，但鲜为人知的酶，其辅因子的要求，和调节。使用[3 H]烷基溶血磷脂酰胆碱prelable一个内源性磷脂酰胆碱底物池结合转磷脂使用乙醇产生标记的磷脂酰乙醇，我们证明了一种新的磷脂酶D活性在中性粒细胞亚细胞组分。鸟苷5 '-O-3-（硫代三磷酸）（GTP γ S）和佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）激活磷脂酸生成和转磷脂酰化。使用这两种激活剂的活性不仅需要质膜的存在下，但也胞质溶胶，和蛋白水解和热灭活证明在这两个馏分中的蛋白质因子的要求。使用这两种刺激，活动随着细胞质浓度的增加而增加。PMA和GTP γ S的产物形成约为10分钟和30分钟近似线性，两种活化剂导致高达10%的标记磷脂酰胆碱的总水解。使用两种活化剂的活性显示出相似的宽中性pH最适值，并且两者都需要微摩尔水平的钙的存在，其本身在高达1 mM的浓度下不能活化。在低微摩尔浓度的核苷酸下，活化对鸟嘌呤核苷酸是特异性的，并且显示出GTP γ S的特异性大于鸟苷基-5 '-基亚氨基二磷酸的特异性大于GTP，而GDP和GMP或腺嘌呤核苷酸没有影响，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，特别是当使用透析的胞质溶胶时，并且被星形孢菌素抑制约50%，支持蛋白激酶C的作用。然而，纯化的蛋白激酶C未能取代胞质溶胶，暗示在这种反应中有额外的胞质因子。这些结果表明，粒细胞磷脂酶D途径是一个复杂的系统，至少有两个激活途径，每个组成的组件在两个亚细胞区室。
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.