Nimmo G A, Cohen P
Eur J Biochem. 1978 Jun 15;87(2):341-51. doi: 10.1111/j.1432-1033.1978.tb12383.x.
Inhibitor-1 is a protein which inhibits phosphorylase phosphatase only when it has been phosphorylated by cyclic-AMP-dependent protein kinase [Huang, F. L. and Glinsmann, W. H. (1976) Eur. J. Biochem. 70, 419--426]. Inhibitor-1 was purified by a heat treatment at 90 degrees C, precipitation with ammonium sulphate, chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and finally rechromatography of the phosphorylated protein on DEAE-cellulose, The protein was purified 4000-fold and 1.5 mg per 1000 g muscle was obtained in seven days corresponding to an overall yield of 15-20%. The purified protein was in a state approaching homogeneity as judged by the criteria of polyacrylamide-gel electrophoresis and ultracentrifugal analysis. The concentration of inhibitor-1 in vivo was calculated to be 1.5 micron, which is at least as high as the concentration of phosphorylase phosphatase. The amino acid composition of inhibitor-1 showed several unusual features. Glutamic acid and proline accounted for nearly one third of the residues, tyrosine, tryptophan and cysteine were absent, and the content of aromatic amino acids was very low. The molecular weight measured by sedimentation equilibrium centrifugation was 19200 and by amino acid analysis was 20800. These values were lower than the mol. wt 26000 determined empirically by gel electrophoresis in the presence of sodium dodecyl sulphate, and much lower than the apparent molecular weight of 60000 estimated by gel filtration on Sephadex G-100. The gel filtration behaviour, stability to heating at 100 degrees C and amino acid composition suggest that inhibitor-1 may possess little ordered structure. The phosphorylated from of inhibitor-1 contained close to one molecule of covalently bound phosphate per mole of protein, which is consistent with the previous finding of a unique decapeptide sequence at the site of phosphorylation, Ile-Arg-Arg-Arg-Arg-Pro-Thr(P)-Pro-Ala-Thr- [Cohen, P., Rylatt, D. B. and Nimmo, G. A. (1977) FEBS Lett. 76, 182-186].the phosphorylated form of inhibitor-1 inhibited phosphorylase phosphatase activity (0.02U) by 50% at a concentration of only 7.0 nM in the standard assay, but the phosphorylated decapeptide was 1000-2000 times less effective as an inhibitor.
抑制因子1是一种蛋白质，只有在被环磷酸腺苷依赖性蛋白激酶磷酸化后，才会抑制磷酸化酶磷酸酶[黄，F. L.和格林斯曼，W. H.（1976年）《欧洲生物化学杂志》70卷，419 - 426页]。通过90℃热处理、硫酸铵沉淀、DEAE - 纤维素柱色谱、Sephadex G - 100凝胶过滤，最后对磷酸化蛋白进行DEAE - 纤维素柱再色谱，对抑制因子1进行了纯化。该蛋白质被纯化了4000倍，7天内每1000克肌肉可获得1.5毫克，总产率为15% - 20%。根据聚丙烯酰胺凝胶电泳和超速离心分析的标准判断，纯化后的蛋白质接近均一状态。体内抑制因子1的浓度经计算为1.5微摩尔，至少与磷酸化酶磷酸酶的浓度一样高。抑制因子1的氨基酸组成显示出几个不寻常的特征。谷氨酸和脯氨酸占残基的近三分之一，酪氨酸、色氨酸和半胱氨酸不存在，芳香族氨基酸的含量非常低。通过沉降平衡离心法测得的分子量为19200，通过氨基酸分析测得的分子量为20800。这些值低于在十二烷基硫酸钠存在下通过凝胶电泳凭经验测定的分子量26000，并且远低于通过Sephadex G - 100凝胶过滤估计的表观分子量60000。凝胶过滤行为、在100℃加热的稳定性和氨基酸组成表明抑制因子1可能几乎没有有序结构。抑制因子1的磷酸化形式每摩尔蛋白质含有接近一分子共价结合的磷酸盐，这与先前在磷酸化位点发现的独特十肽序列Ile - Arg - Arg - Arg - Arg - Pro - Thr(P) - Pro - Ala - Thr - [科恩，P.、赖拉特，D. B.和尼莫，G. A.（1977年）《欧洲生物化学学会联合会快报》76卷，182 - 186页]一致。在标准测定中，抑制因子1的磷酸化形式在浓度仅为7.0纳摩尔时就能抑制50%的磷酸化酶磷酸酶活性（0.02单位），但磷酸化的十肽作为抑制剂的效果要低1000 - 2000倍。
