Glycogen phosphorylase kinases in soluble fractions of various rat tissues were examined for the pH 6.8/8.5 activity ratio, Ca2+-dependency, activation by cyclic AMP-dependent protein kinase (protein kinase A), and reactivity with anti-skeletal muscle phosphorylase kinase serum. The enzymes could be divided into at least two major groups; muscle and liver types. The muscle type, that has a low value of pH 6.8/8.5 activity ratio, is highly dependent on Ca2+, markedly activated by protein kinase A, and strongly inhibited by the antiserum. Inversely, the liver type, that has a high value of pH 6.8/8.5 activity ratio, is poorly dependent on Ca2+, not activated by protein kinase A, and weakly inhibited by the antiserum. The enzymes from heart and skeletal muscle were similar and belonged to the former entity. Whereas, the enzymes from liver, kidney, spleen, lung, and testis appeared to belong to the latter entity. The enzyme from brain apparently differs from these entities, and seems to be an intermediate type or a hybrid of the two.

Guanyl-5'-yl imidodiphosphate (Gpp(NH)p), a nucleotide phosphohydrolase-resistant analog of GTP, caused inhibitory and stimulatory effects on the basal adenylate cyclase activity of rat synaptosomal fractions when manganese was present in the assay mixture, whereas the nucleotide caused only a stimulatory effect when magnesium was employed. In the presence of manganese, the inhibitory and stimulatory effects of Gpp(NH)p could be seen at around concentrations of 10(-7) M and 10(-4) M Gpp(NH)p, respectively. The inhibitory and stimulatory effects of Gpp(NH)p were both antagonized competitively by GTP; these effects of the analog were the opposite of those observed with GTP, which was stimulatory and inhibitory for fat call adenylate cyclase at 10(-7) M and 10(-4) M, respectively (Yamamura, H., Lad, P.M., and Rodbell, M. (1977) J. Biol. Chem. 252, 7964--7966). The degree of inhibition by Gpp(NH)p did not depend on the concentration of manganese nor on the addition of ethylene glycol bis(beta-aminoethyl ether)-N, N, N', N'-tetraacetic acid.

Although muscle glycogen phosphorylase kinase reacts preferentially with an inactive form of phosphorylase, the enzyme is able to phosphorylate in vitro multiple species of unidentified endogenous proteins in mammalian tissues such as liver. The reactions absolutely require Ca2+. Phosphate acceptor proteins are most abundant in the soluble and microsomal fractions. Sodium lauryl sulfate-slab gel electrophoresis analysis has revealed that the spectrum of phosphate acceptor proteins entirely differs from that for cyclic AMP-dependent protein kinase, although the biological significance of these reactions is unclear. Nevertheless, it is suggested that the enzyme is potentially multifunctional and plays roles in controlling some of the Ca2+-dependent processes. In contrast, myosin light chain kinase which is another species of calmodulin-dependent protein kinase seems to be strictly specific for this particular protein, and does not utilize any other endogenous protein so far tested.

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