Malencik D A, Zhao Z, Anderson S R
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331-7305.
Mol Cell Biochem. 1993 Nov;127-128:31-43. doi: 10.1007/BF01076755.
Limited proteolysis of rabbit muscle phosphorylase kinase catalyzed by chymotrypsin generates a 33 kD product whose kinase activity is independent of both calcium and pH over the range of 6.8 to 8.3 (Malencik, D.A. & Fischer, E.H. Calcium and Cell Function III: 161-188, 1982). This active preparation consists of three related species containing residues 1-290, 1-296, and 1-298 of the 44.7 kD gamma-subunit of phosphorylase kinase (Harris, W.R., Malencik, D.A., Johnson, C.M., Carr, S.A., Roberts, G.D., Byles, C.E., Anderson, S.R., Heilmeyer, L.M.G., Fischer, E.H. & Crabb, J.W.J. Biol. Chem. 265:11740-11745, 1991). Good recoveries of catalytic activity--with varying degrees of calcium dependence--result upon the digestion of phosphorylase kinase with assorted proteases. However, especially high yields of the chymotryptic fragment are obtainable, with purification on an Ultrogel-34 column and a DEAE Sepharose CL-6B column giving 23% of the maximum possible protein. Physical characterization shows that the 33 kD chymotryptic fragment is globular, with S20,w = 2.9S, and that it has an isoelectric point of 5.3. Our continuous catalytic assay, based on differences in the binding of the fluorescent dye 1-anilinonaphthalene-8-sulfonate by phosphorylase a and b, shows that, on a molar basis, the activity of the fragment is 2.8 fold greater than that of phosphorylase kinase (Malencik, D.A., Zhao, Z. and Anderson, S.R. Biochem. Biophys. Res. Comm. 174: 344-350, 1991). The active fragment also undergoes autophosphorylation. Incubation with Mg[gamma-P32] ATP results in the reaction of 0.7 mol 32P/mol fragment. When the catalytic subunit of the cAMP-dependent protein kinase is also present, the amount of 32P incorporated increases to 1.1 mol/mol. In the former case, phosphorylation occurs primarily at Ser30 while in the latter an additional reaction takes place at Ser81. The phosphopeptides correspond to sequences occurring in the gamma-subunit of phosphorylase kinase.
胰凝乳蛋白酶催化的兔肌肉磷酸化酶激酶有限蛋白水解产生一种33kD的产物，其激酶活性在6.8至8.3的范围内与钙和pH均无关（马伦西克，D.A.和费舍尔，E.H.《钙与细胞功能III》：161 - 188，1982）。这种活性制剂由三种相关的物种组成，它们包含磷酸化酶激酶44.7kDγ亚基的1 - 290、1 - 296和1 - 298位残基（哈里斯，W.R.，马伦西克，D.A.，约翰逊，C.M.，卡尔，S.A.，罗伯茨，G.D.，拜尔斯，C.E.，安德森，S.R.，海尔迈尔，L.M.G.，费舍尔，E.H.和克拉布，J.W.《生物化学杂志》265:11740 - 11745，1991）。用各种蛋白酶消化磷酸化酶激酶后，可获得具有不同程度钙依赖性的催化活性的良好回收率。然而，用胰凝乳蛋白酶消化可获得特别高产量的片段，在Ultrogel - 34柱和DEAE琼脂糖CL - 6B柱上进行纯化，得到的蛋白质占最大可能量的23%。物理特性表明，33kD的胰凝乳蛋白酶片段呈球状，S20,w = 2.9S，其等电点为5.3。我们基于荧光染料1 - 苯胺基萘 - 8 - 磺酸盐与磷酸化酶a和b结合差异的连续催化测定表明，以摩尔为基础，该片段的活性比磷酸化酶激酶高2.8倍（马伦西克，D.A.，赵，Z.和安德森，S.R.《生物化学与生物物理研究通讯》174: 344 - 350，1991）。该活性片段也会进行自身磷酸化。与Mg[γ - P32]ATP一起温育会导致每摩尔片段有0.7摩尔32P发生反应。当也存在cAMP依赖性蛋白激酶的催化亚基时，掺入的32P量增加到1.1摩尔/摩尔。在前一种情况下，磷酸化主要发生在Ser30，而在后一种情况下，在Ser81会发生额外的反应。磷酸肽对应于磷酸化酶激酶γ亚基中出现的序列。
