ISOLATION AND PROPERTIES OF 3 FORMS OF PHOSPHORYLASE AND PHOSPHORYLASE-KINASE FROM HUMAN SKELETAL-MUSCLE

Three forms of phosphorylase (I, II, and III), two of which (I and II) are active in the presence of AMP and one (III) in its absence, have been produced from human skeletal muscles. The pI values of phosphorylases bI and bII are the same (5.8-5.9). In chromatofocusing, a low molecular-weight protein (20-21 kD) with pl 4.8 is separated from phosphorylase bII. Separation of the protein is accompanied first by an increase in the specific enzyme activity and then by a drop in it. During reconstitution of the complex, the phosphorylase bII activity returns to the original level. In the case of phosphorylation, half as much 32p is incorporated into phosphorylase bII as into rabbit phosphorylase b and human bI. It can be assumed that in the phosphorylase bII molecule isolated from human muscles, one of the two subunits is phosphorylated in vivo. This form of the enzyme was found to be characterized by greater affinity for glycogen and lower sensitivity to allosteric effectors (AMP, glucose-6-phosphate, caffeine) than phosphorylase bI. Thus, form bII is unusual among the forms of phosphorylase obtained, since it is partially phosphorylated by phosphorylase kinase and forms a complex with a low-molecular-weight protein that stabilizes its activity. A partially Purified phosphorylase kinase preparation was isolated from human muscles. Enzyme activity requires Ca2+ ions (c0.5 = 0.63-mu-M). At pH 68 the enzyme is activated by calmodulin (c0.5 = 15-mu-M). The activity ratio at pH 68 and 8.2 is equal to 0. 18.