Long term objective of this project is to elucidate molecular sources of observed variations in energetic and mechanical properties of striated muscle contraction. Properties to be investigated include: rate of energy liberation during an isometric tetanus and rate of mechanical relaxation. Experiments will be performed on fibers isolated with intact membranes from frog skeletal muscles. Individual fibers will be studied in order to take advantage of naturally occurring variation among fibers in muscles. Hypotheses and specific aims are: I. Isometric energy liberation is a function of composition of the myosin molecule and concentration of parvalbumin (PV). Energy liberation due to cross-bridge cycling at 0 degrees C will be related to heavy and light chain composition of myosin molecule. Correlations will be sought between concentration of PV and magnitude of labile energy liberation. Myosin and PV isoforms will be determined by polyacrylamide gel electrophoresis. II. Parvalbumin is a soluble relaxing factor. Time course of progressive slowing of relaxation and subsequent recovery will be determined as a function of tetanus duration and temperature from 0 to 20 degrees C. Correlations will be sought between concentration of PV and extent of slowing of relaxation at 0 degrees C. Using purified frog PV and stopped-flow techniques at 0 to 20 degrees C, rate of dissociation from PV of: a) Mg2+ will be compared to time course of slowing of relaxation (and evolution of labile energy) and b) Ca2+ will be compared to subsequent recovery of relaxation speed. Ratio of affinities of Ca2+ and Mg2+ for PV will be related to amount of labile energy. Rate of Ca2+ uptake by isolated sarcoplasmic reticulum from 0 to 20 degrees C will be compared to rate of relaxation in prolonged tetani in fibers. PV probably exists in all fast contracting skeletal muscles. Concentration is high in frog, making it useful for study. Proposed experiments provide stringent tests for the putative role of PV in muscle function.