Hines D S, Wee S, Grogan W M
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond 23298-0614, USA.
Lipids. 1996 Dec;31(12):1233-43. doi: 10.1007/BF02587907.
A 28 kDa inhibitory protein was purified from rat testis cytosol by sequential 40-65% ammonium sulfate precipitation, cation exchange chromatography, anion exchange chromatography, and preparative SDS-polyacrylamide gel electrophoresis. The heat-stable, trypsin-labile protein exhibited nonenzymatic, concentration-dependent inhibition of testicular and pancreatic cholesteryl ester hydrolases at all stages of purification. Copurifying at each stage was a 26.5 kDa protein which comprised 25% of the mass of the two proteins. Polyclonal antibodies raised to either or both 28 kDa and 26.5 kDa proteins by direct injection of excised electrophoretic bands cross-reacted with both proteins on western blots, immunoprecipitated both proteins, and neutralized inhibitory activity. Amino acid compositions of the individual proteins electroeluted from SDS-polyacrylamide gels were different from those of other surface-active proteins of similar molecular weights. Both proteins exhibited identical pl of 4.8 on chromatofocusing columns and two-dimensional gel electrophoresis. Although the subcellular distribution of the 28 kDa protein is unknown, its testicular cytosolic concentration, calculated from the purified protein mass, was 8 X 10(-9) mols/L, which probably underestimates the actual concentration by an order of magnitude. This is greater than the minimum concentration required for in vitro inhibition (10(-9) mols/L), consistent with a physiological role for this protein.
通过40%-65%硫酸铵分步沉淀、阳离子交换色谱、阴离子交换色谱和制备性SDS-聚丙烯酰胺凝胶电泳，从大鼠睾丸细胞质中纯化出一种28 kDa的抑制蛋白。这种热稳定、对胰蛋白酶敏感的蛋白在纯化的各个阶段均表现出对睾丸和胰腺胆固醇酯水解酶的非酶促、浓度依赖性抑制作用。在每个阶段共纯化出一种26.5 kDa的蛋白，其占这两种蛋白总质量的25%。通过直接注射切除的电泳条带产生的针对28 kDa和26.5 kDa蛋白中任意一种或两种的多克隆抗体，在蛋白质印迹法中与这两种蛋白发生交叉反应，免疫沉淀这两种蛋白，并中和抑制活性。从SDS-聚丙烯酰胺凝胶中电洗脱的单个蛋白的氨基酸组成与其他分子量相似的表面活性蛋白不同。在聚焦色谱柱和二维凝胶电泳上，这两种蛋白均表现出相同的4.8的等电点。尽管28 kDa蛋白的亚细胞分布尚不清楚，但根据纯化蛋白质量计算，其在睾丸细胞质中的浓度为8×10⁻⁹ mol/L，这可能将实际浓度低估了一个数量级。这一浓度高于体外抑制所需的最低浓度（10⁻⁹ mol/L），这与该蛋白的生理作用相符。
