Ryu S H, Cho K S, Lee K Y, Suh P G, Rhee S G
J Biol Chem. 1987 Sep 15;262(26):12511-8.
We previously reported (Ryu, S. H., Cho, K. S., Lee, K. Y., Suh, P. G., and Rhee, S. G. (1986) Biochem. Biophys. Res. Commun. 141, 137-144) that cytosolic fractions of bovine brain contain two phosphoinositide-specific phospholipase C (PLC), PLC-I and PLC-II. In this paper purification procedures and properties of these two forms of enzyme are presented. The two enzymes exhibit similar substrate specificity. Both PLC-I and PLC-II catalyze the hydrolysis of phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2). Yet, they respond differently to activators such as Ca2+ and nucleotides and to inhibitory divalent metal ions such as Hg2+ and Cd2+. In addition, they are immunologically distinct as evidenced by the fact that monoclonal antibodies directed against either enzyme do not cross-react with the other. Their activities are Ca2+ concentration-dependent. PIP and PIP2 are better substrates than PI for both PLC-I and PLC-II when the concentration of Ca2+ is in the micromolar range. Study of the effect of nucleotides, such as GTP, guanosine 5'-(3-O-thio)triphosphate, guanyl-5'-yl imidodiphosphate, and ATP, on the activities of both isozymes with PIP2 as substrate revealed that (i) in the absence of Ca2+, PLC-I activity is enhanced by 400% by either GTP or ATP. In the presence of Ca2+ (a condition in which PLC-I exhibits much higher activity), the activation factor by nucleotides is diminished to approximately 140%. (ii) without Ca2+, PLC-II activity is too low to measure with or without added nucleotides. The effect of nucleotides on PLC-II activity is trivial in the presence of Ca2+. In addition, studies on the effect of metal ions on PI hydrolysis showed that the activities of both PLC-I and PLC-II are not affected by 50 microM of Mg2+, Mn2+, Ca2+, or Ni2+. However, Hg2+, Zn2+, and Cu2+ inhibited both PLC-I and PLC-II, with PLC-II exhibiting much higher sensitivity to these metal ions than PLC-I. For example, the value of I0.5 for Hg2+ inhibition is 0.2 microM for PLC-II and 1 microM for PLC-I. Cd2+ selectively inhibits PLC-II with a I0.5 value of 5 microM. Most of these metal ions' inhibition can be overcome by either dithiothreitol or EDTA.
我们之前报道过（柳，S. H.，赵，K. S.，李，K. Y.，徐，P. G.，和李，S. G.（1986年）《生物化学与生物物理研究通讯》141卷，第137 - 144页），牛脑的胞质部分含有两种磷酸肌醇特异性磷脂酶C（PLC），即PLC - I和PLC - II。本文介绍了这两种酶的纯化过程及性质。这两种酶表现出相似的底物特异性。PLC - I和PLC - II都催化磷脂酰肌醇（PI）、磷脂酰肌醇 - 4 - 磷酸（PIP）和磷脂酰肌醇 - 4,5 - 二磷酸（PIP2）的水解。然而，它们对诸如Ca2 +和核苷酸等激活剂以及诸如Hg2 +和Cd2 +等抑制性二价金属离子的反应不同。此外，它们在免疫学上是不同的，这一点可由针对其中一种酶的单克隆抗体不与另一种酶发生交叉反应这一事实得到证明。它们的活性依赖于Ca2 +浓度。当Ca2 +浓度处于微摩尔范围内时，PIP和PIP2对PLC - I和PLC - II来说都是比PI更好的底物。以PIP2为底物研究核苷酸（如GTP（鸟苷 - 5'-三磷酸）、鸟苷5' -（3 - O - 硫代）三磷酸、鸟苷 - 5'-亚氨基二磷酸和ATP）对两种同工酶活性的影响，结果表明：（i）在没有Ca2 +的情况下，GTP或ATP可使PLC - I的活性提高400%。在有Ca2 +存在时（在这种情况下PLC - I表现出高得多的活性），核苷酸的激活因子降至约140%。（ii）在没有Ca2 +时，PLC - II的活性太低，无论是否添加核苷酸都无法测量。在有Ca2 +存在时，核苷酸对PLC - II活性的影响微不足道。此外，关于金属离子对PI水解作用的研究表明，50微摩尔的Mg2 +、Mn2 +、Ca2 +或Ni2 +对PLC - I和PLC - II的活性均无影响。然而，Hg2 +、Zn2 +和Cu2 +抑制PLC - I和PLC - II，其中PLC - II对这些金属离子的敏感性比PLC - I高得多。例如，Hg2 +抑制的I0.5值对于PLC - II为0.2微摩尔，对于PLC - I为1微摩尔。Cd2 +选择性抑制PLC - II，I0.5值为5微摩尔。这些金属离子的大多数抑制作用可被二硫苏糖醇或EDTA克服。
