Asano T, Morishita R, Matsuda T, Fukada Y, Yoshizawa T, Kato K
Department of Biochemistry, Aichi Prefectural Colony, Japan.
J Biol Chem. 1993 Sep 25;268(27):20512-9.
To investigate the physiological significance of the diversity of gamma subunits of G proteins, we purified four forms of beta gamma of G proteins from bovine brain (beta gamma-B1, beta gamma-B2, beta gamma-B3), and spleen (beta gamma-S1) by the sequential chromatography on columns of DEAE-Sephacel, Ultrogel AcA 34, heptylamine-Sepharose, phenyl-5PW, and DEAE-5PW. Electrophoretic analyses showed that each beta gamma mainly contained the 36-kDa beta and a distinct but homogeneous gamma. These beta gamma complexes were subjected directly to proteolytic digestion and subsequent amino acid sequence analyses of their fragments. It was revealed that beta gamma-B1, -B2, and -B3 were identical to beta 1 gamma 7 (with a low level of beta 2 gamma 7), beta 1 gamma 2 and beta 1 gamma 3, respectively, while beta gamma-S1 was composed of beta 1 and an unidentified form of gamma. Then we examined the functional differences among these beta gamma complexes and the beta gamma of transducin (beta gamma-T, beta 1 gamma 1). Few differences were observed among all beta gamma complexes to enhance pertussis toxin-catalyzed ADP-ribosylation of the alpha subunits of G(o) and Gt. The four forms of beta gamma complexes purified from brain and spleen showed indistinguishable inhibitory effects on the release of GDP from G(o) alpha, but beta gamma-T was much less effective. Brain and spleen beta gamma complexes were equally effective in inhibiting calmodulin-stimulated adenylyl-cyclase activity, but beta gamma-T had a very weak inhibitory effect. Five forms of beta gamma facilitated metarhodopsin II-catalyzed binding of GTP gamma S to Gt alpha in a concentration-dependent manner with the following rank order of effectiveness: beta gamma-S1 > beta gamma-T > beta gamma-B1 > beta gamma-B2 > beta gamma-B3. Because the beta gamma complexes used in this study mostly contained the same beta subunit, the functional differences must be dependent on the gamma subunits. Thus, it seems likely that the receptor, the alpha subunits, and the effector are able to distinguish between the various gamma subunits.
为了研究G蛋白γ亚基多样性的生理意义，我们通过在DEAE - Sephacel、Ultrogel AcA 34、庚胺 - 琼脂糖、苯基 - 5PW和DEAE - 5PW柱上进行连续层析，从牛脑（βγ - B1、βγ - B2、βγ - B3）和脾脏（βγ - S1）中纯化了四种形式的G蛋白βγ。电泳分析表明，每种βγ主要包含36 kDa的β和一种独特但均一的γ。将这些βγ复合物直接进行蛋白酶消化，随后对其片段进行氨基酸序列分析。结果显示，βγ - B1、 - B2和 - B3分别与β1γ7（含有少量β2γ7）、β1γ2和β1γ3相同，而βγ - S1由β1和一种未鉴定形式的γ组成。然后我们研究了这些βγ复合物与转导素的βγ（βγ - T，β1γ1）之间的功能差异。在增强百日咳毒素催化的G(o)和Gt的α亚基的ADP - 核糖基化方面，所有βγ复合物之间几乎没有观察到差异。从脑和脾脏中纯化的四种形式的βγ复合物对G(o)α释放GDP的抑制作用难以区分，但βγ - T的效果要差得多。脑和脾脏的βγ复合物在抑制钙调蛋白刺激的腺苷酸环化酶活性方面同样有效，但βγ - T的抑制作用非常弱。五种形式的βγ以浓度依赖的方式促进视紫红质II催化的GTPγS与Gtα的结合，其有效性顺序如下：βγ - S1 > βγ - T > βγ - B1 > βγ - B2 > βγ - B3。由于本研究中使用的βγ复合物大多含有相同的β亚基，功能差异必定取决于γ亚基。因此，受体、α亚基和效应器似乎能够区分各种γ亚基。
