Uncoupled ATPase Activity and Heat Production by the Sarcoplasmic Reticulum Ca2+-ATPase

Abstract

Sarcoplasmic reticulum vesicles of rabbit skeletal muscle accumulate Ca2؉ at the expense of ATP hydrolysis. The heat released during the hydrolysis of each ATP molecule varies depending on whether or not a Ca 2؉ gradient is formed across the vesicle membrane. After Ca 2؉ accumulation, a part of the Ca 2؉-ATPase activity is not coupled with Ca 2؉ transport (Yu, X., and Inesi, G. (1995) J. Biol. Chem. 270, 4361-4367). I now show that both the heat produced during substrate hydrolysis and the uncoupled ATPase activity vary depending on the ADP/ATP ratio in the medium. With a low ratio, the Ca 2؉ transport is exothermic, and the formation of the gradient increases the amount of heat produced during the hydrolysis of each ATP molecule cleaved. With a high ADP/ATP ratio, the Ca 2؉ transport is endothermic, and formation of a gradient increased the amount of heat absorbed from the medium. Heat is absorbed from the medium when the Ca 2؉ efflux is coupled with the synthesis of ATP (5.7 kcal/mol of ATP). When there is no ATP synthesis, the Ca 2؉ efflux is exothermic (14 -16 kcal/Ca 2؉ mol). It is concluded that in the presence of a low ADP concentration the uncoupled ATPase activity is the dominant route of heat production. With a high ADP/ATP ratio, the uncoupled ATPase activity is abolished, and the Ca 2؉ transport is endothermic. The possible correlation of these findings with thermogenesis and anoxia is discussed.This work deals with two interconnected subjects: (i) the mechanism of energy interconversion by enzymes and (ii) heat generation, a process that plays a key role in the metabolic activity and energy balance of the cell. The biological preparation used was vesicles derived from the sarcoplasmic reticulum of rabbit white skeletal muscle. These vesicles retain a membrane-bound Ca 2ϩ -ATPase, which is able to interconvert different forms of energy. During Ca 2ϩ transport, the chemical energy derived from ATP hydrolysis is used by the ATPase to pump Ca 2ϩ across the vesicle membrane, leading to the formation of a transmembrane Ca 2ϩ gradient (see reactions 1-6 forward in Figs. 1 and 2). In this process, chemical energy derived from ATP hydrolysis is converted into osmotic energy. After Ca 2ϩ accumulation, the catalytic cycle of the enzyme can be reversed, and the accumulated Ca 2ϩ leaves the vesicles through the Ca 2ϩ -ATPase synthesizing ATP from ADP and P i (read reactions 6 to 1 backward in Figs. 1 and 2). During synthesis, osmotic energy is converted back into chemical energy (1-6). In the steady state, the Ca 2ϩ concentrations inside the vesicles and in the assay medium remain constant, but the ATPase operates simultaneously forward (ATP hydrolysis and Ca 2ϩ uptake) and backwards (Ca 2ϩ efflux and ATP synthesis), and chemical and osmotic energy are continuously interconverted by the ATPase.The catalytic cycle of the ATPase varies depending on the Ca 2ϩ concentration in the vesicle lumen. When the free Ca 2ϩ concentration inside the vesicles is kept in the micromolar range, the reaction cycle flows as shown i...

The Slippage of the Ca2+ Pump and Its Control by Anions and Curcumin in Skeletal and Cardiac Sarcoplasmic Reticulum

“…Experimental evidence obtained in our own (3) and other laboratories (4,5) indicates that slippage of the pump occurs when the concentration of luminal Ca 2ϩ is high, thereby raising the steady state level phosphoenzyme intermediates undergoing unproductive cleavage (Scheme 1). We attempt here to distinguish various phosphoenzyme species that may undergo hydrolytic cleavage through an unproductive cycle, rather than completing productive cycles in the forward or reverse direction of the pump.…”

“…Considering that mM concentrations of ATP, ADP, and P i are normally present in muscle fibers, it is apparent that slippage occurs to a significant extent in situ, concomitant with Ca 2ϩ signaling waves, and may play an important physiological role for thermogenesis (5). The extent of slippage is dependent on how effectively cytosolic Ca 2ϩ is lowered by the action of the pump, and ATP reformed from ADP following its utilization for cellular functions.…”

“…3, A and B). In this case, the reduction of net Ca 2ϩ uptake is due, at least in part, to Ca 2ϩ efflux by reversal of the pump rather than slippage (5 …”

“…On the other hand, it was later demonstrated that the catalytic and transport cycle can undergo significant slippage through branched reactions, resulting in lower coupling ratios (3)(4)(5). Slippage is influenced by the lipid composition of the membrane (6) and can be reduced by curcumin (7).…”

Heat of PPi Hydrolysis Varies Depending on the Enzyme Used

“…In this case, a part of the chemical energy derived from ATP cleavage is used for Ca 2ϩ transport, and a part is converted into heat. The second route is a shortcut of the transport cycle where the cleavage of ATP is completed in a step that precedes the translocation of Ca 2ϩ through the membrane, and all the energy derived from ATP hydrolysis is converted into heat (4,5,8,10,(13)(14)(15). Thus, during the course of the reaction, the amount of heat released varies depending on how much ATP is cleaved in each of these two routes.…”