Regulation of muscle phosphorylase b kinase activity by inorganic phosphate and calcium ions

[摘要]

The regulation of the activity of blowfly flight-muscle phosphorylase b kinase by P_i and Ca²⁺ was studied, and the actions of these effectors on the kinases from insect flight and rabbit leg muscles were compared. Preincubation of blowfly kinase with P_i increased activity severalfold. The effect was concentration-dependent, with an apparent K_m of about 20mm, and time-dependent, requiring at least 10min for maximal activation. Neither ATP nor cyclic AMP was needed, suggesting that a protein kinase may not be involved. Maximal activation of the insect kinase required Mg²⁺ in addition to P_i. The apparent K_m for Mg²⁺ was 3mm. Rabbit leg-muscle phosphorylase b kinase was slightly inhibited, rather than stimulated, by P_i, and was strongly inhibited by K⁺, Na⁺ and Li⁺. At physiological concentrations, Ca²⁺ activated the phosphorylase b kinases from both blowfly flight and rabbit leg muscles. However, the responses to Ca²⁺ of the enzymes from the two tissues were different. The mammalian kinase had virtually no activity in the absence of Ca²⁺, and showed a large increase in activity over a narrow range of Ca²⁺ concentrations. Flight-muscle kinase had appreciable activity in the absence of Ca²⁺, and had a smaller increase over a wide range of Ca²⁺ concentration. The concentrations of Ca²⁺ required for half-activation were 0.1 and 1μm for the blowfly and rabbit enzymes respectively. The pH–activity profiles of the non-activated, phosphate- and Ca²⁺-activated kinase revealed considerable enhancement of activity with little, if any, increase in the ratio of activities at pH6.8 to those at 8.2. These results are discussed in relation to the mechanism coupling contraction to glycogenolysis and to the biochemical distinction between asynchronous and synchronous types of muscle.