Three myosin heavy chain isoforms with different actin-activated Mg(2+)-ATPase activities were found in the fast skeletal muscle from carp (Cyprinus carpio) acclimated to 10 and 30 degrees C. The composition of three types of myosin heavy chain was dependent on acclimation temperature, demonstrating the presence of temperature-specific myosin isoforms in carp. Subsequently, the temperature-dependence of the sliding velocity of fluorescent F-actin in myosins isolated from 10 degrees C- and 30 degrees C-acclimated carp was measured. At 8 degrees C, the filament velocity was three times higher for myosin from 10 degrees C- than from 30 degrees C-acclimated fish. Activation energies (E(a)) for the sliding velocity of F-actin were 63 and 111 kJ mol(-1) for myosins from 10 degrees C- and 30 degrees C-acclimated fish, respectively. Activation energy for actin-activated Mg(2+)-ATPase activity was 0.46 kJ mol(-1) in myosin from 10 degrees C-acclimated fish and 0.54 kJ mol(-1) in myosin from 30 degrees C-acclimated fish. The inactivation rate constant (K(D)) of Ca(2+)-ATPase was 7.5x10(-4)s(-1) at 30 degrees C for myosin from 10 degrees C-acclimated fish, which was approximately twice that for myosin from 30 degrees C-acclimated fish. It is suggested that these differences in thermostability reflect a more flexible structure of the myosin molecule in cold-acclimated carp, which results in a reduced activation enthalpy for contraction and, hence, a higher sliding velocity at low temperatures. Structural analysis of cDNAs encoding the carp myosin heavy chain demonstrated striking differences in two surface loops of myosin subfragment-1 (S1), loops 1 and 2, between the 10 degrees C and 30 degrees C types, which were predominantly expressed in carp acclimated to 10 degrees C and 30 degrees C, respectively. Chimeric myosins composed of Dictyostelium discoideum myosin backbones with loop sequences of carp S1 heavy chain isoforms demonstrated that the diversity of the loop 2 sequence of carp S1 affected the V(max) of actin-activated Mg(2+)-ATPase activity.