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...

Glutamate uptake into synaptic vesicles is driven by an electrochemical proton gradient formed across the membrane by a vacuolar H ؉ -ATPase. Chloride has a biphasic effect on glutamate transport, which it activates at low concentrations (2-8 mM) and inhibits at high concentrations (>20 mM). Stimulation with 4 mM chloride was due to an increase in the V max of transport, whereas inhibition by high chloride concentrations was related to an increase in K m to glutamate. Both stimulation and inhibition by Cl ؊ were observed in the presence of A23187 or (NH 4 ) 2 SO 4 , two substances that dissipate the proton gradient (⌬pH). With the use of these agents, we show that the transmembrane potential regulates the apparent affinity for glutamate, whereas the ⌬pH antagonizes the effect of high chloride concentrations and is important for retaining glutamate inside the vesicles. Selective dissipation of ⌬pH in the presence of chloride led to a significant glutamate efflux from the vesicles and promoted a decrease in the velocity of glutamate uptake. The H ؉ -ATPase activity was stimulated when the ⌬pH component was dissipated. Glutamate efflux induced by chloride was saturable, and half-maximal effect was attained in the presence of 30 mM Cl ؊ . The results indicate that: (i) both transmembrane potential and ⌬pH modulate the glutamate uptake at different levels and (ii) chloride affects glutamate transport by two different mechanisms. One is related to a change of the proportions between the transmembrane potential and the ⌬pH components of the electrochemical proton gradient, and the other involves a direct interaction of the anion with the glutamate transporter.

Brain hexokinase is associated with the outer membrane of mitochondria, and its activity has been implicated in the regulation of ATP synthesis and apoptosis. Reactive oxygen species (ROS) are by-products of the electron transport chain in mitochondria. Here we show that the ADP produced by hexokinase activity in rat brain mitochondria (mt-hexokinase) controls both membrane potential (⌬⌿ m ) and ROS generation. Exposing control mitochondria to glucose increased the rate of oxygen consumption and reduced the rate of hydrogen peroxide generation. Mitochondrial associated hexokinase activity also regulated ⌬⌿ m , because glucose stabilized low ⌬⌿ m values in state 3. Interestingly, the addition of glucose 6-phosphate significantly reduced the time of state 3 persistence, leading to an increase in the ⌬⌿ m and in H 2 O 2 generation. The glucose analogue 2-deoxyglucose completely impaired H 2 O 2 formation in state 3-state 4 transition. In sharp contrast, the mt-hexokinase-depleted mitochondria were, in all the above mentioned experiments, insensitive to glucose addition, indicating that the mt-hexokinase activity is pivotal in the homeostasis of the physiological functions of mitochondria. When mt-hexokinase-depleted mitochondria were incubated with exogenous yeast hexokinase, which is not able to bind to mitochondria, the rate of H 2 O 2 generation reached levels similar to those exhibited by control mitochondria only when an excess of 10-fold more enzyme activity was supplemented. Hyperglycemia induced in embryonic rat brain cortical neurons increased ROS production due to a rise in the intracellular glucose 6-phosphate levels, which were decreased by the inclusion of 2-deoxyglucose, N-acetyl cysteine, or carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Taken together, the results presented here indicate for the first time that mt-hexokinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.

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