Dependence of adenosine triphosphatase activity of rabbit psoas muscle fibres and myofibrils on substrate concentration.
The rate of hydrolysis of adenosine triphosphate (ATP) by chemically skinned rabbit muscle fibres was measured as a function of Mg ATP concentration in the range 5 microM to 5 mM. Pyruvate kinase and lactate dehydrogenase were used to link adenosine diphosphate formation to oxidation of nicotinamide adenine dinucleotide which was followed by the change in absorption at 340 nm. The ATPase rate of a fully activated fibre (pCa = 4.5) increased monotonically with Mg ATP concentration in a manner that could be readily fitted by a hyperbola. At 15 degrees C, pH 7 and an ionic strength of 0.2 M the rate at saturating Mg ATP (Vm) was 1.78 +/- 0.2 s-1 per myosin head (mean +/- S.D.; n = 6) and the Mg ATP concentration needed for half the maximal rate (Km) was 16.6 +/- 2 microM. The ATPase of fibres that had been stabilized by cross-linking with 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC) was also investigated. Cross-linking did not significantly affect the Vm or Km and these fibres proved useful for investigating the adequacy of the pyruvate kinase activity for regenerating hydrolysed ATP. Myofibrils were cross-linked with EDC or glutaraldehyde to prevent shortening. Their ATPase properties were investigated: the values of Vm were 0.85 +/- 0.18 (mean +/- S.D.; n = 14) and 0.82 +/- 0.05 s-1 (n = 6) and of Km were 18.0 +/- 2.8 and 12.4 +/- 2.4 microM respectively. The values of Vm and Km for EDC cross-linked myofibrils were fairly insensitive to ionic strength, the Km decreasing 40% and the Vm increasing 50% for a change from 0.2 to 0.3 M. This slight dependence on ionic strength is considered in relation to the ionic strength dependence of the elementary rate constants of the actomyosin subfragment-1 ATPase cycle.
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D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D006868:Hydrolysis; D066298:In Vitro Techniques; D009119:Muscle Contraction; D009132:Muscles / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism*; D011770:Pyruvate Kinase / Q000378:metabolism; D011817:Rabbits
D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase; D000251:Adenosine Triphosphatases; D005022:Ethyldimethylaminopropyl Carbodiimide