Materials and Methods Materials. Phosphofructokinase was obtained as a crys-talline suspension in ammonium sulfate (type III) from Sigma Chemical Co. or purified according to the procedure of Hesterberg and Lee (1980). Comparable results were obtained with both of these enzyme preparations. After PFK was desalted into the appropriate buffer by passage through Sephadex G-25, the absorbance of PFK at 290 nm was used to calculate the concentration of PFK, assuming an extinction coefficient of 0.87 L cm'1 g'1 (Paetkau & Lardy, 1967). Glycerol-3-phosphate dehydrogenase, aldolase, and triose phosphate isomerase used in the coupled assay of PFK were purchased from Calbiochem (La Jolla, CA). All substrates were purchased from Sigma Chemical Co.(St. Louis, MO). Actin was extracted from an acetone powder of rabbit skeletal muscle as described by Pardee and Spudich (1982). The concentration of actin was determined spectrophotometrically, assuming an extinction coefficient at 280 nm of 1.11 L cm'1 g'1 (Houk & Ue, 1974). Tropomyosin was extracted by the method of Smillie (1982), and troponin was extracted by the method of Potter (1982). The protein concentrations of the tropomyosin and the troponin solutions were determined by the microbiuret method (Itzhaki & Gill, 1964). Electron Microscopy. Solutions of F-actin or F-actin plus PFK were prepared in binding assay buffer [50 mM HEPES'-KOH, pH 7.3 at 20 C, 0.2 mM CaCl2, 0.5 mM