Subunit structure of rabbit skeletal muscle phosphorylase kinase

Abstract

Rabbit skeletal muscle phosphorylase kinase was treated with sodium dodecyl sulfate and fractionated by gel filtration chromatography into two peaks. Peak I was shown to consist of previously identified subunits A and B having molecular weights of 118,000 and 108,000, respectively. Peak II was shown to consist of subunit C having a molecular weight of 41,000. Peak I showed slight heterogeneity on sedimentation equilibrium. The average molecular weight for this fraction was found to be 103,000 in 0.1 % sodium dodecyl sulfate and 113,000 in buffer at pH 10.3. Peak II showed a molecular weight of 43,000 in 0.1% sodium dodecyl sulfate. The amino acid composition of peak I was shown to differ T A he preceding paper (Hayakawa et al., 1973) described a purification procedure for rabbit skeletal muscle phosphorylase kinase (ATP-phosphorylase phosphotransferase, EC 2.7.138) which yielded an essentially homogeneous enzyme. Physicochemical characterization of the kinase showed it to have a molecular weight of 1.33 X 106 and to be composed of three types of subunits with molecular weights of 118,000, 108,000. and 41,000 as revealed by disc gel electrophoresis in the presence of sodium dodecyl sulfate. These subunits, referred to as A, B, and C, respectively, were found to be present in amounts suggesting that the quaternary structure of the intact enzyme can be expressed by the formula, A4B4CV In the present paper an effort has been made to characterize more fully the subunits of phosphorylase kinase. Enzyme dissociated by sodium dodecyl sulfate was divided into a heavy (mixture of subunits A and B) and a light fraction (subunit C) by gel filtration chromatography. Each of these fractions was characterized by sedimentation equilibrium and amino acid analysis. Activation of the kinase by enzymatic phosphorylation was studied as a function of phosphate uptake in the individual subunits. Preliminary observations were made as to the effect of tryptic activation on the subunit structure of phosphorylase kinase. Experimental SectionMaterials. The purification of nonactivated rabbit skeletal muscle phosphorylase kinase was carried out as described t From the

Self-association of the α Subunit of Phosphorylase Kinase as Determined by Two-hybrid Screening

“…Studies have shown that activation of PhK by multiple effectors occurs concomitantly with common conformational changes in the ␣ and ␤ subunits (4 -6); moreover, the activity of free ␥ subunit is inhibited by the ␣ and ␤ subunits (7,8). It has been proposed that the ␣ and ␤ subunits of PhK impose steric constraint upon ␥ that, when removed, leads to activation of the kinase (7)(8)(9)(10).…”

Calcium and calmodulin activation of muscle phosphorylase kinase

“…Limited tryptic proteolysis of phosphorylase kinase is known to cause extensive structural changes, both in terms of cleaving preferentially the A and B subunits [3,4,7,8] as well as generating enzyme molecules oflower molecular weight [7]. However, basic catalytic activity can be preserved following this treatment.…”

“…The muscle enzyme was shown to be composed of three different subunit types, A, B, and C, arranged in a probable stoichiometry of (ABC)4 [3,4]; recent studies [5] have also demonstrated the presence of a fourth component, mol. wt 17 000, that appears to be identical to the Ca2+dependent regulator protein or calmodulin.…”

“…Calmodulin is thought to be present at a stoichiometry of close to 1: 1 with respect to the other subunit types [6]. Phosphorylase kinase as purified is relatively inactive at pH 6.8 compared with pH 8.2 [3,4]. A well-known means of activating the enzyme, which is most evident at pH 6.8, involves the proteolytic degradation of the A and B subunits such as by limited trypsin action [3,4,7,8];during proteolytic activation, the proportions of the C subunit and calmodulin remain essentially unchanged [3,4,8].…”

“…A well-known means of activating the enzyme, which is most evident at pH 6.8, involves the proteolytic degradation of the A and B subunits such as by limited trypsin action [3,4,7,8];during proteolytic activation, the proportions of the C subunit and calmodulin remain essentially unchanged [3,4,8]. Another important property of phosphorylase kinase is its reversible activation by Ca" [3,4]. The additional presence of calmodulin leads to a further activation of the kinase.…”