Effect of Alloxan-Diabetes and Subsequent Treatment with Insulin on Kinetic Properties of Succinate Oxidase Activity from Rat Liver Mitochondria

We evaluated early and late effects of alloxan-diabetes and subsequent insulin treatment on the kinetic properties of succinate oxidase (SO) in rat liver mitochondria. Diabetic state lowered the SO activity; insulin treatment was effective in restoring the activity only in oneweek diabetic rats. The energies of activation in low and high temperature ranges (EH and EL) decreased significantly in diabetic animals; once again insulin treatment was partially effective only in the one-week diabetic group. The total phospholipids (TPL) and cholesterol (CHL) contents did not change in one-week groups. In one-month diabetic animals TPL decreased while CHL increased; insulin treatment induced further changes without restoring normality. The lysophospholipid (Lyso), sphingomyelin (SPM), phosphatidylinositol (PI) and phosphatidylserine (PS) content increased in the diabetic state while phosphatidylcholine (PC) and phosphatidylethanolamine (PE) decreased. Insulin treatment had a partial restorative effect. The changes in EH correlated negatively with SPM. The phase transition temperature, Tt, decreased in diabetic and insulin-treated groups. These changes correlated positively with the ratios of TPL/PI and TPL/PS. The membrane fluidity decreased in the diabetic state; insulin had a restorative effect only in the one-week group.

Functional changes in rat liver mitochondria on administration of 2-methyl-4-dimethylaminoazobenzene

Administration of 2-methyl-4-dimethylaminobenzene in the diet (0.1%, w/w) for 85-90 days doubled the content of mitochondria in the livers of rats. The azodye was covalently bound to liver proteins, and about 15% of the amount found in liver was associated with the mitochondrial fraction. Mitochondria isolated from the livers of azodye-fed animals showed drastically lowered ability to oxidize NAD+-linked substrates. The inhibited electron-transfer step was the reduction of ubiquinone. The organelles showed a large increase in succinate oxidase activity. The activity of cytochrome oxidase and the content of cytochrome aa3 were substantially higher in these organelles. Azodye-fed animals showed depressed serum cholesterol concentrations. The content of ubiquinone in liver also registered a small increase.

Nitroxide destruction and flavin-photosensitized damage in inner mitochondrial membranes

Nitroxide free radicals lost their paramagnetic absorption spectrum when they were illuminated with visible light in aged but not freshly isolated inner mitochondrial membranes (SMP's). The action spectrum of spin loss rates coincided with a flavin absorption spectrum. Spin loss consisted of one-electron reduction and "destruction", the latter being defined as spin loss that cannot be reversed by ferricyanide oxidation. By placing SMP's in gas permeable tubing and illuminating alternately under nitrogen and air, it was possible to discriminate qualitatively between spin reduction and destruction. Aerobic spin loss consisted entirely of destruction. When aged SMP's were centrifuged, spin loss was observed in supernatants but not pellets. Flavin fluorescence was observed in the supernatants, suggesting that free flavins catalyzed spin loss. However, addition of exogenous flavins to fresh SMP's did not cause spin loss; hence some other factor was required to cause nitroxide destruction. This factor accumulates during either aerobic or anaerobic aging of SMP's and may be present in mitochondria. Supernatants of aged SMP's, when added to freshly isolated SMP's, induced rapid nitroxide destruction, slightly accelerated photodamage to succinate oxidase, and considerably increased photo-induced lipid peroxidation.