Abstract for Paper III

The effect of Mg²⁺ on cardiac muscle function; is CaATP the substrate for priming myofibril cross-bridge formation and Ca²⁺ re-uptake by the sarcoplasmic reticulum?

Kinetics are established for the activation of the myofibril from the relaxed state [Smith, Dixon, Kirschenlohr, Grace, Metcalfe and Vandenberg (2000) Biochem J 346, 393−402 (in this series as Paper II)]. These require two troponin-Ca²⁺-binding sites, one for each myosin head, to act as a single unit in initial cross-bridge formation. This defines the first, or activating, ATPase reaction, as distinct from the further activity of the enzyme that continues when a cross-bridge to actin is already established. The pairing of myosin heads to act as one unit suggests a possible alternating mechanism for muscle action. A large positive inotropic (contraction-intensifying) effect of loading the Mg²⁺ chelator citrate, via its acetoxymethyl ester, into the heart has confirmed the competitive inhibition of the Ca²⁺ activation by Mg²⁺, previously seen in vitro. In the absence of a recognized second Ca²⁺-binding site on the myofibril, with appropriate binding properties, the bound ATP is proposed as the second activating Ca²⁺-binding site. As ATP, free or bound to protein, can bind either Mg²⁺ or Ca²⁺, this leads to competitive inhibition by Mg²⁺. Published physico-chemical studies on skeletal muscle have shown that CaATP is potentially a more effective substrate than MgATP for cross-bridge formation. The above considerations allow calculation of the observed variation of fractional activation by Ca²⁺ as a function of [Mg²⁺] and in turn reveal simple Michaelis–Menten kinetics for the activation of the ATPase by sub-millimolar [Mg²⁺]. Furthermore the ability of bound ATP to bind either cation, and the much better promotion of cross-bridge formation by CaATP binding, give rise to the observed variation of the Hill coefficient for Ca²⁺ activation with altered [Mg²⁺]. The inclusion of CaADP within the initiating cross-bridge and replacement by MgADP during the second cycle is consistent with the observed fall in the rate of the myofibril ATPase that occurs after two phosphates are released. The similarity of the kinetics of the cardiac sarcoplasmic reticulum ATPase to that of the myofibril, in particular the positive cooperativity of both Mg²⁺ inhibition and Ca²⁺ activation, leads to the conclusion that this ATPase also has an initiation step that utilizes CaATP. The first-order activation by sub-millimolar [Mg²⁺], similar to that of the myofibril, may be explained by Mg²⁺ involvement in the phosphate release step of the ATPase. The inhibition of both the myofibril and SR Ca²⁺-transporting ATPases by Mg²⁺ offers an explanation for the specific requirement for phosphocreatine (PCr) for full activity of both enzymes in situ and its effect on their apparent affinities for ATP. This explanation is based on the slow diffusion of Mg²⁺ within the myofibril and on the contrast of PCr with both ATP and phosphoenolpyruvate, in that PCr does not bind Mg²⁺ under physiological conditions, whereas both the other two bind it more tightly than the products of their hydrolysis do. The switch to supply of energy by diffusion of MgATP into the myofibril when depletion of PCr raises [ATP]/[PCr] greatly, e.g. during anoxia, results in a local [Mg²⁺] increase, which inhibits the ATPase. It is possible that mechanisms similar to those described above occur in skeletal muscle but the Ca²⁺ cooperativity involved would be masked by the presence of two Ca²⁺ binding sites on each troponin.