Myosin light chain phosphorylation during contraction of turtle heart

The phosphorylation of myosin P-light chain was determined during the contraction cycle of turtle heart beating 5--8 times/min at 5 degrees C. The hearts were freeze-clamped either in systole or diastole, then homogenized and washed in strong acids in order to completely inhibit myosin light chain kinase and phosphatase and isolate the total P-light chain of the heart. The phospho and dephospho forms of P-light chain were separated by two-dimensional gel electrophoresis and were quantitated by densitometry. Alternatively, the hearts were perfused with 32P and the incorporation of [32P]phosphate into the P-light chain was determined. Both methods demonstrated that in hearts frozen in systole more P-light chain was phosphorylated than in hearts frozen in diastole.