Phosphorylase b kinase was purifed from fresh rabbit skeletal muscle by a procedure involving mild acid precipitation (pH 6.1), differential centrifugation, and free electrophoresis. The extent of purzcation achieved was 100-to 150-fold. In the procedure phosphorylase b was separated from the kinase by sedimentation of a glycogen-phosphorylase b complex at 80,000 x g for 1 hour. The kinase itself was sedimented when centrifuged at 100,000 x g for 3 hours, and there was evidence that the activity was associated with a component having a sedimentation coefficient in the range of 22-24 S. The form of phosphorylase b kinase purified in this preparation was referred to as nonactivated phosphorylase b kinase to distinguish it from activated kinase obtained by incubation of the enzyme with ATP or with trypsin. Nonactivated phosphorylase b kinase has a very high K,,, (Michaelis constant) for phosphorylase b which was decreased in the presence of glycogen. The K, for activated phosphorylase b kinase was much lower than that of nonactivated kinase. Activation of phosphorylase b kinase by incubation with ATP occurred more rapidly in the presence of adenosine-3',5'-phosphate but did require this nucleotide. Glycogen and heparin also increased the rate of activation of phosphorylase b kinase by ATP. No gross changes in the sedimentation pattern of purified phosphorylase b kinase were caused by activation.Muscle phosphorylase b kinase catalyzes the conversion of phosphorylase b to phosphorylase a according to the following equation (Krebs et al., 1958):The terminal phosphate of ATP is transferred to a specific seryl residue (Fischer et al., 1959) in the phosphorylase subunit, two of which are present in phosphorylase b and four of which are found in phosphorylase a. The latter has twice the molecular weight of phosphorylase b (Keller, 1955). Phosphorylase a formation can be followed conveniently by measuring phosphorylase activity in absence of AMP or by determining the extent of incorporation of 3*P into the protein Krebs et al., 1958). It is also possible to follow the reaction by coupling it with the pyruvic kinase-lactic dehydrogenase system, so that the ADP produced can be measured spectrophotometrically (Gonzales, 1962). No reversal of the kinase reaction has been demonstrated by any of several means employed .Purification of muscle phosphorylase b kinase was undertaken to facilitate further studies on the mechanism of phosphorylase b to a reaction and as a part of a general program to resolve the numerous components involved in the complex system regulating glycogenolysis. It is known that glycogenolysis is accelerated in muscle under circumstances in which phosphorylase a formation is increased. Thus, when resting muscle containing phosphorylase predominantly in the b form (Krebs and Fischer, 1955) is stimulated electrically, phosphorylase a is formed and glycogenolysis ensues (Cori, 1956). Muscle glycogenolysis produced by epinephrine administration is also associated with phosphorylase a formation (Sutherland, 1951; ...

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Older coronary patients successfully improve peak aerobic capacity after 3 and 12 months of supervised aerobic conditioning compared with control subjects. The mechanism of the increase in peak aerobic capacity is associated almost exclusively with peripheral skeletal muscle adaptations, with no discernible improvements in cardiac output or calf blood flow.

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