THE 87-KDA PROTEIN, A MAJOR SPECIFIC SUBSTRATE FOR PROTEIN-KINASE-C - PURIFICATION FROM BOVINE BRAIN AND CHARACTERIZATION
THE 87-KDA PROTEIN, A MAJOR SPECIFIC SUBSTRATE FOR PROTEIN-KINASE-C - PURIFICATION FROM BOVINE BRAIN AND CHARACTERIZATION
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DOI：
10.1073/pnas.84.20.7046
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发表时间：
1987-10-01
影响因子：
11.1
通讯作者：
GREENGARD, P
中科院分区：
文献类型：
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作者：
ALBERT, KA;NAIRN, AC;GREENGARD, P
87-kDa的蛋白质，蛋白激酶C的主要特异性底物，已被纯化500倍，从牛前脑上清液的表观同质性。纯化过程包括DE-52（DEAE-纤维素）分批吸附、（NH 4）2SO 4沉淀、DEAE-Sephacel层析、Bio-Gel HTP（羟基磷灰石）层析、Sephacryl S-400层析和快速蛋白液相色谱ProRPC。氨基酸组成是值得注意的，其高比例的丙氨酸（28.6摩尔%）和其富集谷氨酸/谷氨酰胺（18.1摩尔%），甘氨酸（12.6摩尔%），和脯氨酸（11.3摩尔%）。偏比容为0.702毫升/克，斯托克斯半径和沉降系数为85埃。和2.11S。尽管在NaDodSO 4/8%PAGE上蛋白质的相对分子量为87-90 kDa，但由上述值确定的分子量为68 kDa。摩擦比为3.2，轴比为60，表明87-kDa蛋白质是一个非常细长的单体。通过纯化的蛋白激酶C将纯化的87-kDa蛋白磷酸化至每摩尔87-kDa蛋白2.2摩尔32 P的化学计量（使用68 kDa的分子量值计算）。磷酸化仅在丝氨酸残基上。
The 87-kDa protein , a major specific substrate for protein kinase C, has been purified 500-fold to apparent homogeneity from bovine forebrain supernatant. The purification procedure included batch adsorption to DE-52 (DEAE-cellulose), (NH4)2SO4 precipitation, and chromatography on DEAE-Sephacel, Bio-Gel HTP (hydroxylapatite), Sephacryl S-400, and fast protein liquid chromatography ProRPC. The amino acid composition was notable for its high proportion of alanine (28.6 mol%) and its enrichment in glutamate/glutamine (18.1 mol%), glycine (12.6 mol%), and proline (11.3 mol%). The partial specific volume was 0.702 ml/g; the Stokes radius and sedimentation coefficient were 85 .ANG. and 2.11 S, respectively. Although the relative molecular mass of the protein on NaDodSO4/8% PAGE was 87-90 kDa, the molecular mass as determined from the above values was 68 kDa. The frictional ratio was 3.2, and the axial ratio was 60, indicating that the 87-kDa protein is an extremely elongated monomer. The purified 87-kDa protein was phosphorylated by purified protein kinase C to a stoichiometry of 2.2 mol of 32P per mol of 87-kDa protein (calculated using a value of 68 kDa for the molecular mass). Phosphorylation was exclusively on serine residues.