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ISOLATION AND KINETIC COMPARISON OF INDIRECT FLIGHT MUSCLE MYOSIN IN MANDUCA SEXTA
Title
ISOLATION AND KINETIC COMPARISON OF INDIRECT FLIGHT MUSCLE MYOSIN IN MANDUCA SEXTA
Creator
Liu, Yang
Date
2016, 2016-05
Description
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
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THE LENGTH-TENSION CURVE OF AND LATTICE SPACING CHANGES IN SKINNED FLIGHT MUSCLE IN MANDUCA SEXTA
Title
THE LENGTH-TENSION CURVE OF AND LATTICE SPACING CHANGES IN SKINNED FLIGHT MUSCLE IN MANDUCA SEXTA
Creator
Shaoshuai, Chen
Date
2016, 2016-05
Description
The synchronous indirect flight muscle (DLM1) of the Hawk moth, Manduca Sexta, has similarities to cardiac striated muscle in its...
Show moreThe synchronous indirect flight muscle (DLM1) of the Hawk moth, Manduca Sexta, has similarities to cardiac striated muscle in its physiological properties. In particular, both operate in vivo on the so-called ascending limb of the length-tension curve. The length-tension curve is a classical experiment to study the physiological properties of muscle. The length tension curve of DLM1 of Manduca sexta has previously been reported with a descending limb that is steeper than what would be expected given likely dimensions for the thick and thin filaments. Excessive rundown of the muscle preparations is a likely cause of these observations. Factors caused muscle rundown are many, such as the time allowed for the muscle to relax, the time for muscle spends contracting or the time spent on sarcomere length adjusting. Insights into the factors mentioned above were obtained by conducting a series of experiments designed to systematically explore the causes of rundown. These included Constant Sarcomere Length Experiments, Reciprocating Stretch Experiments and Back to SL 3.25 μm Experiment. Then a modified protocol for carrying out the length-tension experiment was developed based on these findings. A new length-tension curve was plotted and shows a shape closer to what might be expected theoretically. The force went to zero at about SL 5.7μm. This result is constant with other measurements of the length of the myofilaments. Finally, the lattice spacing experiment was carried out to figure out how the interfilament lattice spacing changes across the SL growing. Result shows that the lattice spacing did not change much over the plateau region of the length-tension curve (SL 2.9- 3.1) but increased substantially as SL decreases further in the ascending limb of the length tension curve.
M.S. in Biology, May 2016
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