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[21] Calmodulin as an integral subunit of phosphorylase kinase from rabbit skeletal muscle

1983; Academic Press; Linguagem: Inglês

10.1016/s0076-6879(83)02023-6

1557-7988

Colin Picton, Shirish Shenolikar, Roger J.A. Grand, Philip Cohen,

Cardiomyopathy and Myosin Studies

This chapter discusses the methodology used to study the interaction of phosphorylase kinase with calmodulin and troponin C. It is now established that calmodulin is an integral subunit of phosphorylase kinase that is very tightly bound to the catalytic subunit of the enzyme even in the absence of Ca2+. It, therefore, resembles proteins, such as the troponin complex and calcineurin, in which structurally related Ca2+-binding proteins are tightly complexed with other subunits in the absence of Ca2+. In the skeletal muscles of rabbit, the concentration of calmodulin bound to phosphorylase kinase was estimated by urea-polyacrylamide gel electrophoresis to be 17 mg per 1000 g of muscle. This is in excellent agreement with the value of 19 mg per 1000 g predicted from the known content of phosphorylase kinase in this tissue. In estimating the concentration of calmodulin by gel electrophoresis, the gels are calibrated by running purified calmodulin as an internal standard at a range of concentrations and estimating the amount of calmodulin by densitometry. A slight improvement to this method might be to include a trace of 125I-labeled calmodulin to each sample, prior to electrophoresis, to assess the recovery of calmodulin in each gel track.