Quantitation of myosin light chain phosphorylation in small tissue samples.
A method to quantitate the extent of phosphorylation of the 20,000-dalton phosphorylatable myosin light chain (P-light chain) in cardiac, smooth, or skeletal muscle samples which have limited amounts of tissue and myosin is presented. Native myosin is isolated from other cellular proteins in crude homogenates prepared from a few milligrams of muscle by pyrophosphate-polyacrylamide gel electrophoresis. The extent of P-light chain phosphorylation is maintained throughout this procedure by the inclusion of myosin light chain kinase and phosphatase inhibitors. Myosin obtained by pyrophosphate-gel electrophoresis is subjected to isoelectric focusing on polyacrylamide gels to separate the phosphorylated from the nonphosphorylated forms of the P-light chain. Following staining with ammonial-silver phosphorylation is quantitated on a mole of phosphate incorporated per mol of P-light chain basis by densitometric scanning of the isoelectric focusing gels. Direct comparison of this methodology with another method used for quantitating phosphorylation revealed no difference between the techniques in measuring the extent of P-light chain phosphorylation in muscle biopsy samples. This methodology provides the means for examination of the possible regulatory roles of P-light chain phosphorylation in contraction of cardiac, smooth, skeletal muscle.
D000818:Animals; D001011:Aorta / Q000032:analysis; D007525:Isoelectric Focusing / Q000379:methods; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000032:analysis; D009132:Muscles / Q000032:analysis*; D009206:Myocardium / Q000032:analysis*; D009218:Myosins / Q000302:isolation & purification*; D010766:Phosphorylation; D051381:Rats
D009218:Myosins