Singh I, Goll D E, Robson R M, Stromer M H
Biochim Biophys Acta. 1977 Mar 28;491(1):29-45. doi: 10.1016/0005-2795(77)90038-1.
Highly purified bovine cardiac alpha-actinin is obtained by successive chromatography on DEAE-cellulose and hydroxyapatite of a crude fraction obtained by salting out low ionic strength extracts of bovine cardiac muscle between 0 and 30% ammonium sulfate saturation. Hydroxyapatite chromatography removes a 43 000-dalton polypeptide chain that is difficult to remove by successive DEAE-cellulose columns. Removal of all 43 000-dalton material by hydroxyapatite chromatography is accompanied by disappearance of a very small 9 to 10 S boundary in analytical ultracentrifuge diagrams of DEAE-cellulose-purified 6.2S alpha-actinin. Approximately 95% of the protein in DEAE-cellulose and hydroxyapatite-purified alpha-actinin is the 100 000-dalton alpha-actinin polypeptide as estimated by SDS-polyacrylamide gel electrophoresis. Purified bovine cardiac, porcine skeletal, chicken gizzard, and chicken breast alpha-actinins all contain leucine as the C-terminal amino acid of both polypeptide chains in the alpha-actinin molecule. Bovine cardiac and porcine skeletal alpha-actinins contain arginine as the amino acid penultimate to C-terminal leucine. None of the four different alpha-actinins studied had a N-terminal amino group available for reaction with dansyl chloride, but all four alpha-actinins contained 1.6 to 1.8 acetate residues per molecule (200 000 daltons) of alpha-actinin. It seems likely that the N-terminal amino groups of both polypeptide chains in these four alpha-actinins are acetylated. A peptide having the composition N-Ac-Asp2-Glu4 was isolated from a proteolytic digest of bovine cardiac alpha-actinin. alpha-Actinin seems to be a conserved protein molecule found in many different motile systems.
通过对牛心肌低离子强度提取物在0至30%硫酸铵饱和度下盐析得到的粗级分先后进行DEAE - 纤维素柱色谱和羟基磷灰石柱色谱，可获得高度纯化的牛心肌α - 辅肌动蛋白。羟基磷灰石柱色谱可去除一条43000道尔顿的多肽链，而连续的DEAE - 纤维素柱很难将其去除。通过羟基磷灰石柱色谱去除所有43000道尔顿的物质后，DEAE - 纤维素纯化的6.2Sα - 辅肌动蛋白在分析超速离心图谱中的一个非常小的9至10S边界消失。通过SDS - 聚丙烯酰胺凝胶电泳估计，DEAE - 纤维素和羟基磷灰石纯化的α - 辅肌动蛋白中约95%的蛋白质是100000道尔顿的α - 辅肌动蛋白多肽。纯化的牛心肌、猪骨骼肌、鸡砂囊和鸡胸α - 辅肌动蛋白在α - 辅肌动蛋白分子的两条多肽链中，C末端氨基酸均为亮氨酸。牛心肌和猪骨骼肌α - 辅肌动蛋白在C末端亮氨酸的前一个氨基酸为精氨酸。所研究的四种不同的α - 辅肌动蛋白均没有可与丹磺酰氯反应的N末端氨基，但所有四种α - 辅肌动蛋白每分子（200000道尔顿）α - 辅肌动蛋白含有1.6至1.8个乙酸盐残基。这四种α - 辅肌动蛋白的两条多肽链的N末端氨基似乎都被乙酰化了。从牛心肌α - 辅肌动蛋白的蛋白水解消化物中分离出一种组成为N - Ac - Asp2 - Glu4的肽。α - 辅肌动蛋白似乎是一种在许多不同运动系统中都存在的保守蛋白质分子。
