Nucleotide binding by myosin in glycerol-extracted rabbit skeletal muscle fibres at temperatures between -35 degrees C and 10 degrees C.
Glycerol-extracted rabbit psoas fibres were incubated at temperatures between -35 degrees C and +10 degrees C in a low-ionic-strength relaxing solution containing 50% ethyleneglycol, 100 microM [3H]MgATP, 1 mM [14C]mannitol and less than 0.01 microM Ca2+. The fibres were then rinsed in a solution containing 1 mM ATP and the bound nucleotide eluted in trichloroacetic acid; all these operations were carried out at the cold temperature. Residual bound nucleotide was eluted with trichloroacetic acid at room temperature. The fibres were found to bind approximately 180 microM nucleotide, which is consistent with binding to the enzymatic site of myosin. The eluate, obtained in the cold, was analysed on poly(ethyleneimine)-cellulose for its ATP and ADP content. At temperatures down to -22 degrees C most of the bound nucleotide was ADP and there was little variation of this fraction with temperature. As the temperature was lowered below -22 degrees C the ATP fraction rose sharply; by -35 degrees C it predominated. These results are similar in type to those found by Biosca et al. [(1984) Biochemistry 23, 1947-1953] on isolated subfragment 1, but are displaced to a much lower temperature range. Thus in a muscle fibre only a low thermal energy is needed for myosin to hold its nucleotide in a constant balance between ATP and ADP.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004734:Energy Metabolism; D005990:Glycerol; D066298:In Vitro Techniques; D009132:Muscles / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D013696:Temperature
D009711:Nucleotides; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009218:Myosins; D005990:Glycerol