Purification and properties of ATPase inhibitor from rat liver mitochondria

(1) The ATPase inhibitor protein has been isolated from rat liver mitochondria in purified form. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis is approximately 9500, and the isoelectric point is 8.9.

(2) The protein inhibits both the soluble ATPase and the particle-bound ATPase from rat liver mitochondria. It also inhibits ATPase activities of soluble F₁, and inhibitor-depleted submitochondrial particles derived from bovine heart mitochondria.

(3) On particle-bound ATPase the inhibitor has its maximal effect if incubated in the presence of Mg²⁺. ATP at slightly acidic pH.

(4) The inhibitor has a minimal effect on P_i-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimulates P_i-ATP exchange activity in whole mitochondria while the low ATPase activity of the mitochondria is not affected. The possible mechanism of action of the inhibitor on intact mitochondria is offered.     

The interaction of glucagon treatment and uncoupler concentration on ATPase activity of rat liver mitochondria

Mitochondria isolated from livers of rats treated briefly with glucagon show an increased ATPase activity in the presence of appropriate concentrations of protonophoric uncouplers (Yamazaki, R. K., Sax, R.D., and Hauser, M.A. (1977) FEBS Lett. 75, 295-299). With the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) the effect of glucagon treatment was most evident at concentrations of uncoupler higher than required for maximal stimulation of ATPase in control mitochondria. In this range of FCCP concentrations that produced the greatest contrast in ATPase activity of control and hormone-stimulated mitochondria, there were no significant differences in delta pH, delta psi, or delta p between the two groups. The presence of added succinate in the ATPase assay system mimicked the effect of glucagon treatment, permitting greater activity at high concentrations of uncoupler without significantly affecting delta p. No significant effect of glucagon treatment or uncoupler concentrations on mitochondrial volumes was observed. Following treatment with glucagon, the mitochondria retained a greater content of Mg+ and K+ throughout the range of FCCP concentrations tested. In the upper range of FCCP concentrations there was appreciable loss of K+ from the mitochondria which was greater in control mitochondria than in mitochondria from glucagon-treated rats or in mitochondria assayed in the presence of succinate. The activity of the uncoupler-dependent ATPase was greatly stimulated by increased concentrations of potassium chloride in the assay medium without significantly diminishing the hormone effect. It is proposed that the intrinsic peptide inhibitor of ATPase is dissociated from the enzyme to an increased degree following glucagon treatment and that high levels of uncoupler inhibit by causing an increased association of the enzyme and its inhibitor.     

Effect of alpha-tocopherol and its derivatives on ATPase activity and oxidative phosphorylation in rat liver mitochondria

The effects of alpha-tocopherol and its derivatives on the energy metabolism in mitochondria were studied. It was shown that alpha-tocopherol derivatives with short hydrocarbon chain produce uncoupling effects, while alpha-tocopherol itself as well as its long-chain derivatives (tocopheryl acetate and tocopheryl stearate) exert no effect. All the derivatives studied produced an inhibitory effect on DNP-stimulated mitochondrial ATPase. As far as these compounds did not influence the activity of mitochondrial F1 factor, it was concluded that the effects of the compounds were due to membrane modification.     

Calcium-activated proteolytic activity in rat liver mitochondria

Soluble extracts from sonicated rat liver mitochondria and rat liver cytosol were each chromatographed on DEAE-cellulose columns, and the fractions assayed for Ca²⁺-activated proteolytic activity using ¹⁴C-casein as a substrate. The mitochondrial preparations were shown to be free of cytosolic and microsomal contamination by the lack of alcohol dehydrogenase activity, a cytosolic marker enzyme, and by a lack of cytochrome P-450 activity, a microsomal marker enzyme. Two peaks of Ca²⁺-activated neutral endoprotease activity were resolved from the mitochondrial fractions. One protease was half-maximally activated with 25 μM Ca²⁺, and the other by 750 μM Ca²⁺. Rat liver cytosol contained only a high Ca²⁺-requiring protease peak. This is the first demonstration of Ca²⁺-activated proteases in mitochondria.     

Insulin-status-dependent modulation of FoF1 ATPase activity in rat kidney mitochondria

The early and late effects of alloxan-diabetes and insulin treatment on kinetic properties of mitochondrial FoF1 ATPase were examined. Diabetic state resulted in significant decrease in the activity while insulin treatment caused hyper-stimulation. In control animals the enzyme activity resolved in three kinetic components. In diabetic condition only component I and II were present. With insulin treatment component III was restored but component II was abolished. Diabetic state and insulin treatment had varied effects on Km values of the three components, whereas the Vmax values were generally on the higher side. Evaluation of the AppKcat/Km values revealed that diabetic state resulted in increased catalytic efficiency; insulin treatment brought back these values to normality. Temperature kinetics studies indicated that the phase transition temperature decreased significantly in the diabetic and insulin-treated diabetic animals. The energy of activation in low temperature range increased in the diabetic animals. Insulin treatment corrected the Arrhenius pattern at early stage of diabetes; at late stage the pattern was reversed. The results are suggestive of subtle insulin-status-dependent alterations in membrane structure - function relationships.     

Purification and properties of ATPase inhibitor from rat liver mitochondria.

(1) The ATPase inhibitior protein has been isolated from rat liver mitochondria in purified form. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis is approximately 9500, and the isoelectric point is 8.9. (2) The protein inhibits both the soluble ATPase and the particle-bound ATPase from rat liver mitochondria. It also inhibits ATPase activities of soluble F1, and inhibitor-depleted submitochondrial particles derived from bovine heart mitochondria. (3) On particle-bound ATPase the inhibitor has its maximal effect if incubated in the presence of Mg2+. ATP at slightly acidic pH. (4) The inhibitor has a minimal effect on Pi-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimules Pi-ATP exchange activity in whole mitochondria while the low ATPase activity of the mitochondria is not affected. The possible mechanism of action of the inhibitor on intact mitochondria is offered.     

Detection of an ATPase activity in rat liver peroxisomes

An ATPase co-sedimenting with rat liver peroxisomes has been detected after subcellular fractionation. The activity is Mg2+ dependent, with pH optimum of 7.5 and is inhibited by NEM and DCCD but not by oligomycin. Partial inhibition of the mitochondrial ATPase allows to detect the peroxisomal activity in the gradients. Protease inactivation and solubilization data suggests that the activity resides in a protein of the peroxisomal membrane, exposed to the cytosol.     

Cytochrome c degrading activity in rat liver mitochondria

Benzophenone can be used as an extrinsic triplet state probe, as its phosphorescence, a broad band centered at 445 nm, is readily observable in aqueous solution at room temperature. When bound covalently as an acyl enzyme at the active site of chymotrypsin, the benzophenone probe produces phosphorescence which is unusually resistant to quenching by O₂, $\text{trans}$-cinnamic acid, and H₃O⁺. Sodium 2-naphthalenesulfonate quenches the phosphorescence, probably indirectly. The quenching data indicate that the local protein structure at the enzyme active site provides a rigid and protective substrate environment, which is not penetrated by even the smallest triplet quenchers.     

Cyclic succinic dehydrogenase activity in rat liver mitochondria

Succinic dehydrogenase activity was assayed in isolated rat liver mitochondria. Rats had been exposed for at least two weeks to a 24 hour feeding-fasting schedule, 5 hours feeding, 19 hours fasting. Enzyme activity was determined at nine specific hours over a 24 hour period. Lowest enzyme activity occured six hours after the feeding cages had been closed. The highest activity, which was 158 percent greater than the lowest activity, occurred during the next feeding period. It is concluded that time of sacrifice is an important consideration when determining succinic dehydrogenase activity.