Stozharov A N
Biokhimiia. 1985 Feb;50(2):337-46.
Affinity adsorption on immobilized DNAase I and the measurements of the protein mobility upon SDS-PAGE electrophoresis were used for the identification of the actin-like protein as well as for the study of its biosynthesis is liver mitochondria of hepatectomized rats. The kinetics of biosynthesis showed a maximum on the 10th min after intraperitoneal injection of the label. Fractionation of mitochondria demonstrated that more than 50% of the whole amount of the "de novo" synthesized protein is localized in the intermembrane space, approximately 30%--in the mitochondrial matrix. The purity of the fractions was controlled by analyzing the polypeptide content of the samples and by measuring the marker enzyme activity. Besides, additional identification of the actin-like protein was carried out directly in the mitochondrial matrix and intermembrane space by two-dimensional electrophoresis in polyacrylamide gel performed by the O'Farrell method. The subsequent staining of the gels with silver revealed the presence of two basic isoforms of non-muscle action (beta- and gamma-actins). The presence of the actin-like protein in the inner mitochondrial compartments characterized by a high rate of metabolism may be regarded as compelling evidence of its mitochondrial localization.
利用固定化脱氧核糖核酸酶I上的亲和吸附以及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）中蛋白质迁移率的测量，来鉴定肌动蛋白样蛋白，并研究其在肝切除大鼠肝线粒体中的生物合成。生物合成动力学显示，在腹腔注射标记物后第10分钟达到最大值。线粒体分级分离表明，“从头”合成的蛋白质总量中，超过50%位于膜间隙，约30%位于线粒体基质。通过分析样品的多肽含量和测量标记酶活性来控制各组分的纯度。此外，通过奥法雷尔（O'Farrell）方法在聚丙烯酰胺凝胶中进行二维电泳，直接在线粒体基质和膜间隙对肌动蛋白样蛋白进行了额外鉴定。随后用银对凝胶进行染色，发现存在两种非肌肉肌动蛋白的基本同工型（β-肌动蛋白和γ-肌动蛋白）。在内线粒体区室中存在肌动蛋白样蛋白，且这些区室具有高代谢率，这可被视为其定位于线粒体的有力证据。
