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Abstract
Abstract Methods for preparing troponin and tropomyosin from ethanol-extracted muscle are presented. Examination of these proteins by disc electrophoresis indicated that the troponin preparation is homogeneous. If precautions were taken to assure that the —SH groups of tropomyosin were not permitted to oxidize during preparation or electrophoresis, tropomyosin appeared to be nearly homogeneous. Traces of troponin, however, appeared in all samples. Neither troponin nor tropomyosin was found to make actomyosin superprecipitation sensitive to the removal of Ca++. Combined, they were effective. A moderately wide range of troponin to tropomyosin ratios (between 1:1 and 3:1) were found to provide optimal sensitization of actomyosin. At a troponin to tropomyosin ratio of 1.3:1, 35 µg of this protein mixture inhibited by 85% 200 µg of actomyosin. The ability of troponin to collaborate with tropomyosin in the sensitization of actomyosin was lost to varying degrees if the troponin —SH groups were allowed to react with p-chloromercuribenzoate or N-ethylmaleimide or if the —SH groups were not protected during preparation. In contrast, the function of tropomyosin was found not to correlate with the state of its —SH groups. Troponin was found to be a potent calcium-binding substance, possessing 24 µmoles of high affinity calcium-binding sites per g. The high affinity site had an apparent affinity constant of approximately 2.4 x 106 m-1. It is hypothesized that the binding of calcium to this site inactivates troponin and that this is the mechanism whereby calcium activates myofibrillar contraction. It is concluded that tropomyosin does not possess a similar class of calcium-binding sites. The affinity of troponin for calcium was unaltered by reaction of its —SH groups. The number of high affinity calcium-binding sites was, however, doubled to approximately 40 µmoles of sites per g by reaction of troponin with —SH reagents.
Highlights
Methods for preparing troponin and tropomyosin from ethanol-extracted muscle are presented
The apparent presence of troponin in this preparation probably explains the ability of the Mueller type of tropomyosin to sensitize actomyosin preparations
Comparison of the disc electrophoretic patterns of tropomyosin prepared as described by Bailey and Mueller indicated that one of the bands in the Mueller preparation is present only to a minor degree in the Bailey preparation
Summary
Desensitized actomyosin was prepared from myosin B as described by Perry, Davies, and Hayter [21]. The washed actomyosin was suspended in a volume of 50y0 (v/v) glycerol adjusted to give a protein concentration of 4 mg per ml, and stored at -20”. The calcium sensitivity of desensitized actomyosin in the presence and absence of preparations of tropomyosin or troponin was collected by centrifugation and redissolved in 1 M KC1 (adjusted to pH 7.0). The tropomyosin was subjected to a series of isoelectric precipitations as described by Mueller [10]. The protein was dissolved in water (pH 7.0), and ammonium sulfate was added to 40% saturation (pH 7.0). The precipitate was discarded, and ammonium sulfate was added to 70y0 saturation. The precipitate (or both) was assayed by observing the effects of EGTA on superprecipitation
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