Uncoupling effect of fatty acids on heart muscle mitochondria and submitochondrial particles.

The effect of ATP/ADP-antiporter inhibitors on palmitate-induced uncoupling was studied in heart muscle mitochondria and inside-out submitochondrial particles. In both systems palmitate is found to decrease the respiration-generated membrane potential. In mitochondria, this effect is specifically abolished by carboxyatractylate (CAtr) a non-penetrating inhibitor of antiporter. In submitochondrial particles, CAtr does not abolish the palmitate-induced potential decrease. At the same time, bongkrekic acid, a penetrating inhibitor of the antiporter, suppresses the palmitate effect on the potential both in mitochondria and particles. Palmitoyl-CoA which is known to inhibit the antiporter in mitochondria as well as in particles decreases the palmitate uncoupling efficiency in both these systems. These data are in agreement with the hypothesis that the ATP/ADP-antiporter is involved in the action of free fatty acids as natural uncouplers of oxidative phosphorylation.     

The effect of inorganic phosphate on calcium influx into rat heart mitochondria

The effect of inorganic phosphate on the accumulation of Ca²⁺ by heart mitochondria has been reinvestigated. Inorganic phosphate has no effect on the initial rate of Ca²⁺ uptake supported by respiration on either ascorbate $\text{plus}$ tetramethylenephenylene diamine, pyruvate $\text{plus}$ malate, or glutamate $\text{plus}$ malate, although it does increase the final amount of Ca²⁺ accumulated; evidence suggests that the latter phenomenon requires phosphate influx via the phosphate carrier. It is concluded that the earlier reports that phosphate augments the initial rate of Ca²⁺ influx reflects an effect of phosphate on succinate oxidation, which was employed in the previous studies, rather than an Ca²⁺ transport itself.     

The effect of lead on the calcium-handling capacity of rat heart mitochondria.

1. Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate-NADP+ oxidoreductase, EC 1.1.1.49) from baker's yeast (Saccharomyces cerevisiae) was immobilized on CNBr-activated Sepharose 4B with retention of about 3% of enzyme activity. This uncharged preparation was stable for up to 4 months when stored in borate buffer, pH7.6, at 4 degrees C. 2. Stable enzyme preparations with negative or positive overall charge were made by adding valine or ethylenediamine to the CNBr-activated Sepharose 4B 30min after addition of the enzyme. 3. These three immobilized enzyme preparations retained 40-60% of their activity after 15 min at 50 degrees C. The soluble enzyme is inactivated by these conditions. 4. The soluble enzyme lost 45 and 100% of its activity on incubation for 3h at pH6 and 10 respectively. The three immobilized-enzyme preparations were completely stable over this entire pH range. 5. The pH optimum of the positively and negatively charged immobilized-enzyme preparations were about 8 and 9 respectively. The soluble enzyme and the uncharged immobilized enzyme had an optimum pH at about 8.5 6. Glucose 6-phosphate dehydrogenase immobilized on CNBr-activated Sephadex G-25 was unstable, as was enzyme attached to CNBr-activated Sepharose 4B to which glycine, asparitic acid, valine or ethylenediamine was added at the same time as the enzyme.     

The effect of inorganic phosphate on creatine kinase in respiring rat heart mitochondria

The ability of creatine to stimulate the respiration of rat heart mitochondria in vitro is reversibly affected by the concentration of inorganic phosphate. The rate of oxygen consumption due to post-ADP state-4 respiration in the presence of 20 mm creatine is reduced significantly when the potassium phosphate concentration is raised from 5 to 20 mm. State-3 respiration is reduced only by potassium phosphate concentrations higher than 20 mm. The rate of synthesis of creatine phosphate is also affected by phosphate concentration, and the apparent K_m of the coupled reactions for ADP is significantly higher at 25 mm phosphate as compared to that at 5 mm phosphate. These observations are consistent with the hypothesis that inorganic phosphate acts as an effector molecule, regulating creatine phosphate synthesis by favoring the dissociation of mitochondrial creatine kinase from the mitochondrial membrane. Such regulation may be important in the case of cells undergoing partial or severe ischemia, where changes in phosphate concentration within this range have been reported.     

The formation of intracristal structures induced in skeletal muscle mitochondria by high pressure

Following the application of high pressure to skeletal muscle for extended periods, intracristal structures are found in the mitochondria. In addition, electron dense granules of 70–170 nm diameter are found in the matrix of these mitochondria. In contrast, pressure-treated liver mitochondria show only large (300–400 nm) matrix granules but not the intracristal structures. Both the inner and outer mitochondrial membranes appear intact after pressure-treatment. Short periods of pressuretreatment have little effect on either the morphology of mitochondria or the pH of the tissues. It is suggested that the formation of the intracristal structures may be due to the effects of pH rather than pressure alone. This finding raises the possibility that intracristal structures may occur as a preparative artefact particularly where the tissue has undergone considerable manipulation.     

Regulatory effect of pig heart phospholipids on heart muscle AMP-deaminase

AMP-deaminase purified from pig heart has been found to be activated by liposomes prepared from phospholipids extracted from pig heart mitochondria, microsomes and cytoplasm, as well as by intact microsomes. The activation by phospholipids occurred only in the presence of ATP and after the enzyme had been preincubated with liposomes for 30 min. Liposomes prepared from cardiolipin displayed an inhibitory effect on pig heart AMP-deaminase.     

The effect of acetate oxidation on the endogenous adenine nucleotides of rat heart mitochondria

When citrate cycle substrates or glutamate are oxidized by rat heart mitochondria, AMP comprises only a few percent of the endogenous pool of adenine nucleotides. However, when acetate is oxidized, greater than half or about 30% of the total mitochondrial pool of adenine nucleotides is converted to AMP in the ADP-stimulated or resting state, respectively. Supporting substrates which form GTP as a result of their oxidation partially, but not completely, reverse the accumulation of AMP which results from acetate metabolism.     

The effect of malonyl-coa on fatty acid oxidation in rat muscle and liver mitochondria

The effect of malonyl-CoA on palmitate oxidation was compared for skeletal muscle and liver mitochondria from fed rats and rats starved for 18 and 42 h. The nutritional state did not influence the palmitate oxidation rate by both types of mitochondria. Muscle mitochondria are more sensitive to malonyl-CoA inhibition of palmitate oxidation than are liver mitochondria. Their response is not influenced by the nutritional state, in contrast to that of liver mitochondria. The extent of inhibition was not dependent on the carnitine concentration (above 0.5 mM), but higher at lower palmitate:albumin ratio or palmitate concentration.     

The effect of anthracyclines on heart mitochondria respiration during the action of creatine phosphokinase

It was shown that during glutamate+malate oxidation in the presence of creatine, antitumour anthracycline antibiotics strongly inhibit the rate of oxygen uptake by rat heart mitochondria; ADP excess activated the respiration up to the initial level, i.e., that observed after the first addition of ADP. Carboxyatractyloside addition to a system containing creatine (or hexokinase+glucose) results in the stimulation of rubomycin-induced mitochondrial respiration. Substitution of carboxyatractyloside by oligomycin gives very similar results. It is supposed that anthracycline antibiotics exert a manyfold effect on heart mitochondrial membranes which results in impaired compartmentation of enzymatic systems providing for oxidative phosphorylation.     

Immobilized enzymes at the surface of rat heart muscle mitochondria

Of the three pairs of complementary replicas mentioned in the previous paper (1985, J. Ultrastruct. Res. 91, 38-50) one pair consisted of fracture faces exposing the cytoplasmic surface of the outer surface membrane while the complementary face exposed the cytosol at the membrane surface. The latter face was particulate with randomly distributed particles in the size range of 100 to 200 A. These particles could be shown to be located in the cytosol at the membrane surface. They qualify as particles that are loosely bound to this surface, and it is proposed that at least part of these particles consist of glycolytic enzymes.     

High‐fat diet affects skeletal muscle mitochondria comparable to pressure overload‐induced heart failure

In heart failure, high‐fat diet (HFD) may exert beneficial effects on cardiac mitochondria and contractility. Skeletal muscle mitochondrial dysfunction in heart failure is associated with myopathy. However, it is not clear if HFD affects skeletal muscle mitochondria in heart failure as well. To induce heart failure, we used pressure overload (PO) in rats fed normal chow or HFD. Interfibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM) from gastrocnemius were isolated and functionally characterized. With PO heart failure, maximal respiratory capacity was impaired in IFM but increased in SSM of gastrocnemius. Unexpectedly, HFD affected mitochondria comparably to PO. In combination, PO and HFD showed additive effects on mitochondrial subpopulations which were reflected by isolated complex activities. While PO impaired diastolic as well as systolic cardiac function and increased glucose tolerance, HFD did not affect cardiac function but decreased glucose tolerance. We conclude that HFD and PO heart failure have comparable effects leading to more severe impairment of IFM. Glucose tolerance seems not causally related to skeletal muscle mitochondrial dysfunction. The additive effects of HFD and PO may suggest accelerated skeletal muscle mitochondrial dysfunction when heart failure is accompanied with a diet containing high fat.