Smrcka A V, Sternweis P C
Department of Pharmacology, Southwestern Graduate School of Biomedical Science, University of Texas Southwestern Medical Center, Dallas 75235-9041.
J Biol Chem. 1993 May 5;268(13):9667-74.
Specific antisera were produced to peptides representing the carboxyl termini of three subtypes of phosphatidylinositol-specific phospholipase C (PIPLC) beta which have been identified by isolation of cDNAs (Kriz, R., Lin, L., Sultzman, L., Ellis, C., Heldin, C., Pawson, T., and Knopf, J. (1990) Ciba Found. Symp. 150, 112-127). Screening with the antisera indicates that PIPLC beta 3 is present in a variety of cell lines and rat tissues, whereas the distribution of PIPLC beta 1 and beta 2 is more restricted. A combination of conventional and immunoaffinity chromatographic techniques was used to purify PIPLC beta 1 and beta 3 from rat brain membranes. PIPLC beta 2 was purified from cytosol of HL60 cells. All three subtypes were activated by purified G protein alpha q/11 subunits with the following relative efficacies: PIPLC beta 3 > or = PIPLC beta 1 >> PIPLC beta 2. All three PIPLC subtypes were also activated by G protein beta gamma subunits with varying efficacies. The presence of beta gamma subunits depressed the ability of alpha q/11 to activate PIPLC beta 1 and beta 3 at low Mg2+ concentrations (1 mM). At higher concentrations of Mg2+ (2 mM or greater), activation of PIPLC beta 3, but not PIPLC beta 1, by beta gamma and alpha q/11 became additive. PIPLC beta 3 was activated by alpha q/11 even in the presence of a saturating concentration of beta gamma subunits. This indicates that there are separate sites for interaction of PIPLCs with G protein subunits and that this interaction differs depending on the enzyme subtype and the concentration of Mg2+.
针对代表磷脂酰肌醇特异性磷脂酶C（PIPLC）β三种亚型羧基末端的肽段制备了特异性抗血清，这些亚型是通过cDNA分离鉴定出来的（克里兹，R.，林，L.，苏尔茨曼，L.，埃利斯，C.，赫尔丁，C.，帕森，T.，和克诺夫，J.（1990年）《Ciba Found. Symp.》150，112 - 127）。用这些抗血清进行筛选表明，PIPLCβ3存在于多种细胞系和大鼠组织中，而PIPLCβ1和β2的分布则更为局限。采用常规色谱技术和免疫亲和色谱技术相结合的方法从大鼠脑膜中纯化PIPLCβ1和β3。从HL60细胞的胞质溶胶中纯化出PIPLCβ2。所有三种亚型都被纯化的G蛋白αq/11亚基激活，其相对活性如下：PIPLCβ3≥PIPLCβ1>>PIPLCβ2。所有三种PIPLC亚型也被G蛋白βγ亚基以不同的活性激活。βγ亚基的存在降低了αq/11在低镁离子浓度（1 mM）下激活PIPLCβ1和β3的能力。在较高的镁离子浓度（2 mM或更高）下，βγ和αq/11对PIPLCβ3的激活作用相加，但对PIPLCβ1则不然。即使在存在饱和浓度的βγ亚基的情况下，PIPLCβ3仍能被αq/11激活。这表明PIPLC与G蛋白亚基相互作用的位点是分开的，并且这种相互作用因酶亚型和镁离子浓度的不同而有所差异。
