The Thermogenic Function of the Ca2 ATPase Uncoupled Ca2 Efflux and Uncoupled ATP Hydrolysis

The catalytic cycle of the ATPase varies depending on the Ca2+ concentration in the vesicles' lumen. When the free Ca2+ concentration inside the vesicles is kept in the micromolar range, the reaction cycle flows as shown in Figure 1. The main feature of this cycle is that the hydrolysis of each ATP molecule is coupled with the translocation of two Ca2+ ions across the membrane (4-7). This stoichiometry is only measured when the Ca2+ concentration in the vesicles' lumen is kept below 0.2 mM [68-71]. Under physiological conditions, however, the Ca2+ concentration inside the reticulum rises to the millimolar range and in this condition the stoichiometry falls to a value varying between 0.3 and 0.6. Evidence obtained in different laboratories indicates that the enzyme cycles through two more sets of intermediary reactions when the Ca2+ concentration inside the vesicles rises to the millimolar range. These are ramifications of the catalytic cycle and are denoted as dashed lines in Figure 2. In one of them, a part of the Ca2+ accumulated by the vesicles leaks through the enzyme without catalyzing the synthesis of ATP.

This is referred to as uncoupled Ca2+ efflux and is represented by reactions 7, 8, and 9 in Figure 2 [72-76]. In another ramification of the catalytic cycle, the progressive rise in the lumenal Ca2+ concentrations promotes ATP hydrolysis without Ca2+ translocation [71,77]. The uncoupled ATP hydrolysis is derived from the cleavage of the phosphoenzyme form 2Ca: E1 ~ P (reaction 10 in Figure 2).

Until recently, it was assumed that the amount of heat produced during the hydrolysis of an ATP molecule is always the same, as if the energy released during ATP cleavage were divided into two non-interchangeable parts, one for Ca2+ transport and the other converted into heat. In my laboratory [14-17, 78-85] we found that during Ca2+ transport, a fraction of both chemical energy derived from ATP cleavage and osmotic energy derived from the Ca2+ gradient is converted by the ATPase into heat. Thus, depending on the conditions used, the amount of heat released during the hydrolysis of ATP may vary between 7 and 32kcal/mol. We found that this variability is derived from the uncoupled Ca2+ efflux (reactions 7, 8, and 9 in Figure 2) and from the uncoupled ATPase (reaction 10). When

the Ca2+ concentrations on the two sides of the membrane are kept below 0.1 mM, the amount of heat released during the hydrolysis of each mol of ATP varies between 9 and 12 kcal/mol. This was measured using leaky vesicles that are not able to accumulate Ca2+. In this condition, there is no gradient, and no ramification of the catalytic cycle, and the cleavage of ATP is completed after the hydrolysis of the "low-energy" phosphoenzyme E2-P. The yield of heat increases to the range of 2032 kcal/mol when intact vesicles are used and the Ca2+ concentration in the vesicles' lumen rises to the millimolar range; a part of the Ca2+ accumulated leaks through the ATPase leading to the conversion of the osmotic energy into heat [77,82]. The major source of heat, however, is derived from the uncoupled ATPase (reaction 10). The high intravesicular Ca2+ concentration forces the reversal of reactions 5 and 4 leading to accumulation of the "high-energy" phosphoenzyme 2Ca:E1 ~ P, which is then hydrolyzed. During reactions 4 and 5 in Figure 2, a part of the chemical energy derived from ATP cleavage is converted into osmotic energy. If the hydrolysis of ATP is completed before Ca2+ translocation through the membrane (reaction 10 in Figure 2), then there is no conversion of chemical into osmotic energy, and during catalysis more chemical energy is left available to be converted into heat [77,84,85]. The rates of uncoupled Ca2+ efflux and uncoupled ATPase activity are modulated by different drugs, temperature and water activity of the assay medium [78,80,81,83-85]. When taken together, these findings indicate that the Ca2+ -ATPase is able to handle the energy derived from ATP hydrolysis in such a way as to determine how much is used for Ca2+ transport and how much is dissipated as heat. In this view, the total amount of energy released during ATP hydrolysis is always the same, but the fraction of the total energy that is converted into work or heat seems to be modulated by the Ca2+ -ATPase. At present my work is concentrated on the possible thermogenic function of the Ca2+ -ATPase. The aim is to see if there is a correlation between heat production by the Ca2+ -ATPase and control of heat production and thermogenesis in animals. The general interest in this subject has increased during the past decade due to its implications in health and disease. Heat generation plays a key role in the regulation of the energy balance of the cell, and alterations of thermogenesis are noted in several diseases, such as adiposity and thyroid-hormone alterations.

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