Assessment of Ca2+-Calmodulin Formation in Intact Vertebrate Skeletal Muscle

D. R. Manning, J. T. Stull

Research output: Contribution to journalArticle

Abstract

The extent of intracellular myosin light-chain kinase activation, determined by the extent of kinase complexation with Ca2+-calmodulin, is manifest in the rate of P-light chain phosphorylation. Although the apparent rate of P-light chain phosphorylation is actually determined by concomitant kinase and phosphatase activities, the influence of the latter can easily be assessed. Myosin phosphatase activity is probably not subject to intracellular regulation and is probably a simple function of substrate concentration.4,5 Moreover, the intracellular concentration of light-chain phosphatase and its intrinsic catalytic rate are low relative to light-chain kinase, so that in instances when light-chain kinase is appreciably activated phosphatase activity can be disregarded. The rate and extent of P-light chain phosphorylation in relation to muscle contractile activity have been previously studied by the methods described.4,8 Tetanic stimulation of rat EDL muscle for 1 sec at 200 Hz resulted in a time-dependent increase in P-light chain phosphate content (Fig. 3). Phosphate content attained a maximum value of 0.7 mol of phosphate per mole of P-light chain within 20 sec and thereafter declined in a slow, monoexponential manner. Presumably, sarcoplasmic calcium influx and consequent Ca2+-calmodulin formation during tetany resulted in light-chain kinase activation. The maximum rates of intracellular P-light chain phosphorylation and dephosphorylation, 2 and 0.035 μmol/min per gram of tissue, respectively, were comparable to those measured in muscle homogenates under optimal conditions for expression of light-chain kinase and phosphatase activities. P-light chain phosphate content in intact muscle was also found to be dependent upon muscle stimulation frequency and duration.3 Measurements of myosin P-light chain phosphorylation in skeletal muscle provide a simple index of light-chain kinase activation, and consequently of Ca2+-calmodulin formation. Correlations established among other intracellular events and P-light chain phosphorylation may thus be used as a means of corroborating biochemical evidence that these events are indeed Ca2+-calmodulin-dependent. Comparisons between P-light chain phosphorylation and other putatively Ca2+-dependent processes, moreover, may serve to elucidate more fully the means whereby Ca2+ regulates various intracellular events. Such comparisons, for instance, have been made among P-light chain phosphorylation, muscle tension development and phosphorylase a formation.8 The latter two processes are ostensibly dependent upon Ca2+ binding to troponin and to Ca2+ activation of phosphorylase kinase via free or tightly bound calmodulin. A positive correlation has also been established between P-light chain phosphorylation and posttetanic potentiation of peak twitch tension (Fig. 3).4,8 Thus, measurement of the latter phenomenon may also provide a simple, though less substantiated, index of Ca2+-calmodulin formation.

Original languageEnglish (US)
Pages (from-to)74-82
Number of pages9
JournalMethods in Enzymology
Volume102
Issue numberC
DOIs
StatePublished - Jan 1 1983

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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