Abstract
It had been reported previously that U.V irradiation of myosin A led to the formation of dimers and that it also caused alterations in enzymatic activity of the protein(1). To obtain further information about the changes induced by U.V., irradiated protein was subjected to trypsin digestion and the products of this treatment were studied by ultracentrifugation, diffusion, electrophoresis and ATP-ase measurements.
Materials and methods. Myosin A was prepared as described by Mommaerts(2), The experimental techniques of ultracentrifugation, diffusion and electrophoresis, as well as ATPase determination have been described(1). U.V. irradiation was performed in a cold room (2-4 C) on a 3.2 mm thick (10 mg/ml) myosin A layer under constant slow mechanical stirring. Myosin A was dissolved in a solution containing 0.5 M KC1 and 0.1 M tris buffer at pH 7.5. The output of the G15R8 General Electric germicidal lamp was measured by a dose-rate meter(3) and was found to be 8.54 × 1014 quanta cm2 second. All samples were spun after irradiation in a Spinco model L ultracentrifuge at 20,000 RPM for 45 minutes. Trypsin digestion was performed at room temperature (23±1°C) for 60 minutes under constant mechanical stirring. The concentration of the myosin A was 7.5 mg/ml and that of trypsin, 0.02 mg/ml. The digestion was stopped by adding 0.03 mg/ml soybean trypsin inhibitor to the mixture. The solubility of the proteins at various ammonium sulphate concentrations was measured as published by Szent-Gyorgyi(4) and the amount of the 5% TCA soluble fraction as described by Mihalyi(5).
Results. After 60 minutes of trypsin digestion of native myosin, the well-known peaks of the heavy and light meromyosins were demonstrated in the analytical ultracentrifuge (5,6,7,8). If, however, the 60-minute irradiated-myosin A was digested by trypsin under identical conditions, a different ultra-centrifugal pattern was obtained.
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