Koide A, Titani K, Ericsson L H, Kumar S, Neurath H, Walsh K A
Biochemistry. 1978 Dec 26;17(26):5657-72. doi: 10.1021/bi00619a012.
The sequence of the amino-terminal 349 residues of rabbit muscle glycogen phosphorylase (EC 2.4.1.1) has been determined. Limited proteolysis of native phosphorylase b (841 residues, subunit molecular weight 97 412) by subtilisin BPN', Streptomyces alkaline protease, or elastase yielded two large segments (light and heavy). The light segment isolated from the subtilisin digest was cleaved at methionyl bonds with cyanogen bromide to yield eight major fragments and two minor overlapping fragments. The alignment of the major fragments was obtained by analysis of the two minor fragments, of five tryptic peptides containing methionine and of one large fragment generated by cleavage of an aspartylproline bond. Analysis of two cyanogen bromide fragments (CB14 and CB17) isolated from the intact molecule identified the sites susceptible to limited proteolysis and the overlap between the light and the heavy segments. Serine-14 and tyrosine-155 were identified as the residues involved in the covalent and allosteric controls of the enzyme, respectively. Residues 108 and 142 were identified as the cysteine residues reported to be involved in the aggregation of subunits.
已确定兔肌肉糖原磷酸化酶（EC 2.4.1.1）氨基末端349个残基的序列。用嗜热栖热放线菌蛋白酶BPN'、链霉菌碱性蛋白酶或弹性蛋白酶对天然磷酸化酶b（841个残基，亚基分子量97412）进行有限的蛋白水解，产生了两个大片段（轻链和重链）。从嗜热栖热放线菌蛋白酶消化物中分离出的轻链片段用溴化氰在甲硫氨酰键处切割，产生八个主要片段和两个较小的重叠片段。通过分析两个较小片段、五个含甲硫氨酸的胰蛋白酶肽段以及一个由天冬氨酰脯氨酸键切割产生的大片段，获得了主要片段的排列顺序。对从完整分子中分离出的两个溴化氰片段（CB14和CB17）进行分析，确定了易受有限蛋白水解作用的位点以及轻链和重链片段之间的重叠部分。丝氨酸-14和酪氨酸-155分别被确定为参与该酶共价控制和别构控制的残基。残基108和142被确定为据报道参与亚基聚集的半胱氨酸残基。
