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1. Two forms of phosphorylase kinase having mol. wt of 1,260,000 (form I) and 205,000 (form II) have been identified by gel filtration chromatography of rabbit liver crude extracts. 2. Form I was the majority when the homogenization buffer was supplemented with a mixture of proteinase inhibitors. This form has been purified through a protocol including ultracentrifugation, gel filtration and affinity chromatography on Sepharose-heparin. 3. Form II was purified by a combination of chromatographic procedures including ion exchange, gel filtration and affinity chromatography on Sepharose-Blue Dextran and Sepharose-histone. 4. Upon electrophoresis in the presence of sodium dodecyl sulfate two subunits of 69,000 and 44,000 were identified for this low molecular weight enzyme. Thus, a tetrameric structure comprising two subunits of each kind can be proposed. 5. Treatment of form I with either trypsin or chymotrypsin gave an active fragment having a molecular weight similar to that of form II. On the contrary, other dissociating treatments with salts, thiols and detergents failed in producing forms of lower molecular weight. 6. The similarities between proteolyzed forms I and II were stressed by their behavior in front of antibodies raised against the muscle isoenzyme of phosphorylase kinase. 7. The study of the effect of magnesium and fluoride ions on the activity of both forms showed an inhibitory effect of magnesium when its concentration exceeded that of ATP. 8. The inhibition could nevertheless be reverted by including 50 mM NaF in the reaction mixture. 9. Form I and form II could be distinguished by their pH dependence in the presence of an excess of magnesium ions over ATP, whereas the affinity for both substrates was not significantly different.

1. Various mitogens such as concanavalin A, phytohaemagglutinin, the pokeweed mitogen and trypsin were found to produce a rapid and transient activation of glycogen phosphorylase activity of lymphocytes incubated in a Krebs-Ringer-bicarbonate-glucose buffer. 2. Activation of the enzyme by these mitogens was always accompanied by an increase in the intracellular cyclic AMP concentration. 3. The presence of calcium ions in the incubation buffer was essential for obtaining the mitogen effects. Addition of ionophore A-23187 also produced an activation of glycogen phosphorylase, similar to that found in mitogen activation but without increase in intracellular cyclic AMP concentration. Dibutyril cyclic AMP also produced lymphocyte phosphorylase activation, even in the absence of extracellular calcium ions. 4. It is proposed that phosphorylase activation by mitogens occurs through a mechanism that involves the participation of both calcium ions and cyclic AMP.

1. Purified native rabbit liver phosphorylase kinase becomes activated during the assay of its activity while low molecular weight forms of the same enzyme do not. 2. The activation requires ATP and magnesium ions, suggesting the phosphorylation of the enzyme by a protein kinase as the mechanism involved. 3. The activation of the enzyme can be reverted by the action of a type I protein phosphatase isolated from the same tissue. 4. The activation can also be catalyzed by the catalytic subunit of cAMP-dependent protein kinase in a process that requires a much lower ATP concentration to proceed. 5. The activation is believed to be due to an autocatalytic phosphorylation of phosphorylase kinase itself. In support of this hypothesis are the regulation of the process through calcium ions, the low levels of endogenous protein kinase detected in the purified preparation, the high ATP concentrations required in the absence of cAMP dependent protein kinase and the fact that the process cannot be blocked by an excess of the heat stable inhibitor specific for the later enzyme. 6. The low molecular weight forms of the enzyme on their side are not affected by the action of neither protein phosphatase 1 nor cyclic AMP dependent protein kinase. 7. Both activated and nonactivated phosphorylase kinase are partially dependent on calcium ions, the affinity of the former being higher than that of the latter. The low molecular forms do not require calcium ions to express their activity.

Two phosphorylase phosphatase activities (I and III) have been purified from rabbit liver, with respective molecular weights of 117,000 and 230,000. Phosphatase III contained three different subunits of molecular weights 35,000, 67,000 and 80,000. Phosphatase I although majoritary in the preparation, was not homogeneous. Both phosphatases were dissociated by 2-mercaptoethanol treatment, releasing a catalytic subunit with a molecular weight of about 35,000. Phosphatases I and III activities responded very differently to incubation with trypsin and to ethanol precipitation. Phosphatase III was much more sensitive to inactivation by several ions and ATP than phosphatase I. On the basis of the obtained data, phosphatase I can be classified as a type-1 phosphatase and phosphatase III as a type-1 phosphatase.