Fornés M W, Barbieri M A, Bertini F
Instituto de Histología y Embriología, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo-CONICET, Mendoza, Argentina.
Biocell. 1996 Aug;20(2):111-21.
Sperm from rat cauda epididymis was washed, sonicated and centrifuged to obtain fractions sedimenting at 600 x g for 5 min, 27.000 x g for 5 min, and 100.000 x g for 40 min. All fractions were observed with the electron microscopy and assayed for cytochrome c oxidase activity. The 100.000 x g fraction contained only small membranous vesicles and less than 0.5% of the total enzymatic activity. This fraction was considered to represent sperm plasmalemma and it was extracted with Tris-HCl buffer before treating it with one of the following chemicals: acetate buffer, pH: 4.5; 0.6 M KCl; bicarbonate buffer, pH 11.0; Triton X-100, and Sodium Dodecyl Sulfate (SDS). After centrifuging, the residual sediments were solubilized in hot 2% SDS. The extracts and the solubilized sediments (hot SDS) were analyzed in SDS-PAGE. The extracts obtained with the first three chemicals contained 11,9, and 25% of total proteins respectively. The bicarbonate buffer solubilized 45%, and the detergents 55% and 65% respectively. A total of 30 bands were seen in the extracts and sediments. Acid pH extracted a low number of bands of high mobility and low molecular weight. Instead, the KCl and bicarbonate buffer, extracted a great number of bands over a wide range of molecular weights (23, 38.5, 55, 100, and 140 KD). The detergents had similar effects: both solubilized four new bands. In residual sediments there were no new proteins and the bands corresponded to those extracted with the detergents, but they varied in staining intensity. According to the results obtained with the mild chaotropic agents of 0.6 M KCl and bicarbonate buffer, 50% of the mass of membraneous proteins may be peripheric. Proteins partially extracted with the detergents were also found in the residual sediment, and they may constitute the skeleton of sperm membrane.
从大鼠附睾尾部获取精子，经洗涤、超声处理和离心，分别以600×g离心5分钟、27,000×g离心5分钟、100,000×g离心40分钟，得到不同组分。对所有组分进行电子显微镜观察并测定细胞色素c氧化酶活性。100,000×g组分仅含有小膜泡，且酶活性不到总活性的0.5%。该组分被认为代表精子质膜，在用以下化学物质之一处理前，先用Tris-HCl缓冲液提取：pH 4.5的醋酸盐缓冲液；0.6 M KCl；pH 11.0的碳酸氢盐缓冲液；Triton X-100和十二烷基硫酸钠（SDS）。离心后，将残留沉淀物溶解在热的2% SDS中。提取物和溶解后的沉淀物（热SDS）进行SDS-PAGE分析。用前三种化学物质获得的提取物分别含有总蛋白的11%、9%和25%。碳酸氢盐缓冲液溶解了45%，去污剂分别溶解了55%和65%。提取物和沉淀物中共观察到30条带。酸性pH提取出数量较少、迁移率高且分子量低的条带。相反，KCl和碳酸氢盐缓冲液提取出大量分子量范围广泛的条带（23、38.5、55、100和140 KD）。去污剂有类似效果：两者都溶解出四条新条带。残留沉淀物中没有新蛋白质，条带与去污剂提取的条带相对应，但染色强度不同。根据用0.6 M KCl和碳酸氢盐缓冲液这些温和的离液剂获得的结果，50%的膜蛋白质量可能是外周蛋白。在用去污剂部分提取的蛋白质也存在于残留沉淀物中，它们可能构成精子膜的骨架。
