Novotny J, Kvapil P, Bokoch G M, Ransnäs L A
Wallenberg Laboratory for Cardiovascular Research, Gothenburg University, Sweden.
Arch Physiol Biochem. 1995 May;103(2):202-10. doi: 10.3109/13813459508996134.
A novel competitive ELISA has been developed for the determination of levels of the beta subunit of guanine-nucleotide-binding protein (G-protein) using antipeptide antibodies directed against the amino terminus of the beta subunit. Because beta subunits form highly hydrophobic.heterodimeric complexes with gamma subunits of G-proteins, specific assay conditions were required. Optimal concentrations of antibodies, detergents, Mg2+ as well as ionic strength were determined. In addition, we found that an effective binding of the used antibodies to the beta subunit was ensured only after denaturation of the beta gamma complexes. Subsequently, this ELISA was used for quantitation of the beta subunit in subcellular fractions of S49 lymphoma cells during isoproterenol-mediated desensitization of beta-adrenergic controlled transmembrane signalling system. A 10 min as well as 60 min treatment of the cells with isoproterenol (1 nmol/ml) resulted in a significant shift of G-protein beta subunits (presumably as beta gamma complexes) from the plasma membrane fractions to low-density microsomal fractions. No significant change was detected after the hormone action in the distribution of plasma membrane constitutive enzymes. In conclusion, the developed ELISA helped us to reveal that beta-adrenergic stimulation can induce redistribution of the beta gamma dimer from plasma membranes to low-density microsomes.
已开发出一种新型竞争性酶联免疫吸附测定法（ELISA），用于使用针对鸟嘌呤核苷酸结合蛋白（G蛋白）β亚基氨基末端的抗肽抗体来测定其水平。由于β亚基与G蛋白的γ亚基形成高度疏水的异二聚体复合物，因此需要特定的测定条件。确定了抗体、去污剂、Mg2+以及离子强度的最佳浓度。此外，我们发现只有在βγ复合物变性后，所用抗体才能有效地与β亚基结合。随后，该ELISA用于定量异丙肾上腺素介导的β-肾上腺素能控制跨膜信号系统脱敏过程中S49淋巴瘤细胞亚细胞组分中的β亚基。用异丙肾上腺素（1 nmol/ml）处理细胞10分钟和60分钟，导致G蛋白β亚基（可能以βγ复合物形式）从质膜组分显著转移到低密度微粒体组分。激素作用后，质膜组成酶的分布未检测到显著变化。总之，所开发的ELISA帮助我们揭示了β-肾上腺素能刺激可诱导βγ二聚体从质膜重新分布到低密度微粒体。
