The indirect flight muscles (IFM) of insects are nature’s versatile engines highly specialized to produce power for flight. Myosin, the motor... Show moreThe indirect flight muscles (IFM) of insects are nature’s versatile engines highly specialized to produce power for flight. Myosin, the motor protein, plays an important role in this process, supporting the rapid wingbeat frequency and converting chemical energy into mechanical force. A novel efficient protocol for preparing functional myosin from Manduca sexta flight muscle is described in this study. We tested the biochemical properties of myosin from the dorsal subunit and ventral subunit of the dominant flight muscle. High salt solubilization and low salt precipitation was used to extract myosin from the homogenate of moth split thorax muscles since low ionic strength allows filament formation and precipitation of myosin while high ionic strength helps myosin solubility. Coomassie Blue stained 10% SDS PAGE analysis showed the purity was 50% and the result was confirmed by western blot with anti-myosin antibody MAC 147. This revealed the final pellet of purification was myosin and was of moderate purity and homogeneity, showing that this method was feasible for isolating myosin from this species. The focus of this study was to compare the catalytic efficiency of extracted ventral DLM1 myosin and dorsal DLM1 myosin in vitro, as myosin was the dominant catalytic protein which hydrolyze ATP to utilize energy in muscle contraction. The rate of an identical ATP hydrolysis reaction was measured catalyzed by myosin extracted from DLM1D and DLM1V, and the activation energy (Ea) was calculated using Arrhenius equation. The reaction catalyzed by dorsal myosin exhibited higher Ea than that catalyzed by ventral myosin, which meant a lower catalytic efficiency of DLM1D myosin. This indicated catalytic heterogeneity of DLM1D and DLM1V myosin in a single muscle. Additionally, a significant within muscle Ea difference of ATP hydrolysis could be partially responsible for the known temperature gradient, within the DLM1 of Manduca demonstrated previously(Nicole T George & Daniel, 2011), considering that the hydrolysis of ATP is responsible for the heat generated during muscle contraction as well as muscle force and lengthening. M.S. in Biology, May 2016 Show less