F. Edmund Hunter

The stability of oxidative and phosphorylative systems in mitochondria under anaerobic conditions

Abstract Anaerobic conditions protect mitochondria from the swelling and loss of DPN-dependent oxidations produced by preincubation at 30° with phosphate, arsenite, Ca ++ , Zn ++ , and a number of other substances. Cyanide inhibition of cytochrome oxidase does not mimic anaerobiosis. Anaerobic addition of oxidizing agents like ferricyanide does mimic aerobic conditions. It is suggested that oxidation of some key group is responsible for swelling and loss of DPN. The phosphorylating mechanism is not rapidly inactivated during preincubation, but rather declines gradually over several hours, independently of the presence or absence of oxygen. Preincubation anaerobically with DNP, a condition which should deplete ATP, does not result in rapid swelling and loss of DPN.

Stability of oxidative phosphorylation and structural changes of mitochondria in ischemic rat liver

Abstract Mitochondria isolated from rat livers ischemic at 24 ° for up to 13 hr exhibited nearly normal capacity to phosphorylate added ADP ( ADP 0 ratios) in the presence of bovine serum albumin (BSA). Respiratory control ratios (rate with ADP/rate without ADP) remained nearly normal for the first 3 hr, then declined somewhat. However, even at 13 hr the ratios were still 2 to 3 compared to normal values of 4 to 6. The decline of respiratory control was due primarily to a decline in the State 3 respiratory rate (i.e., with ADP and substrate). The demonstration of relatively normal oxidative phosphorylation in mitochondria isolated from livers after prolonged periods of ischemia at 24 ° is dependent on the use of BSA in the isolation medium and/or in the test medium. In the complete absence of BSA, or at 37 ° in the presence of BSA, the respiratory control is lost rapidly. These data are compatible with release of fatty acids in the ischemic cell being responsible for the apparent damage to mitochondria. Electron microscopic studies show very slow changes with time in mitochondria in ischemic liver cells, greater changes in the isolated mitochondria. During the isolation procedures and in the test medium, binding by BSA can remove the fatty acids and their uncoupling and inhibitory effects, revealing relatively intact enzymes of oxidative phosphorylation.

Catalytic effect of GSSG on reduction of cytochrome c by GSH-possible model for facilitation of electron transfer and energy conservation by sulfonium ion formation☆

Abstract The reduction of cytochrome c by GSH can occur by two reactions. One is metal catalyzed, prevented by EDTA, and is greatly inhibited by the ionic strength of isotonic solutions. The other is GSSG catalyzed and is completely insensitive to ionic strength. It is suggested that a complex between GSSG and GSH, possibly involving a ring of the three sulfur atoms, facilitates the electron donating activity of GS− and stabilizes the first and second oxidation products, GS· and GS+. The stabilized sulfonium ion might represent a type reaction involved in energy conservation in oxidative phosphorylation.

Is halothane a true uncoupler of oxidative phosphorylation

The ability of isolated rat liver milochondria to accumulate calcium in the presence of various concentrations of halothane was studied. Calcium transport into mitochondria is completely prevented by true uncouplers of oxidative phosphorylation. Mitochondrial calcium accumulation continued in the presence of halothane concentratoins as high as 4 per cent. The rate of calcium accumulation showed as halothane levels increased, but the amount accumulated was the same. The conclusion is that halothane is not a true uncoupler of oxidative phosphorylation, and that any uncoupling with anesthetic levels must be very limited. Malignant hyperpyrexia associated with halothane anesthesia is probably not the result of a direct uncoupling of oxidative phosphorylation in normal mitochondria. Greater susceptibility of mitochondria in individuals prone to hyperpyrexia reactions for genetic reasons is not ruled out.

Phosphorylation coupled to oxidation of thiol groups (GSH) by cytochrome c with disulfide (GSSG) as an essential catalyst II. Demonstration of ATP formation from ADP and HPO42

Abstract The oxidation of GSH by cytochrome c in buffered solution in the presence of EDTA requires GSSG as a catalyst. It appears that an intermediate complex between GSSG and GSH is more active in reducing cytochrome c . During this electron transfer reaction ADP can be generated from AMP and inorganic phosphate. AMP and inorganic pyrophosphate yield ATP. This reaction, which uses common biological materials and occurs in aqueous medium, is a most interesting model for oxidative phosphorylation. It may involve the basic mechanism for coupling of phosphorylation to electron transport in mitochondria.

Chemical sympathectomy by guanidinium adrenergic neuron blocking agents

Abstract Chronic administration of high doses of the adrenergic neuron blocking agent, guanethidine, to neonatal or adult rats produces destruction of the sympathetic nervous system (sympathectomy). Although the mechanism of cytotoxicity is not established, it has been proposed to be due to inhibition of oxidative phosphorylation, an activity of guanethidine which has been demonstrated in vitro in isolated mitochondria. In this study, 40 guanidinium adrenergic neuron blocking agents were examined for cytotoxic effects on sympathetic neurons when administered chronically to neonatal rats. The ability of many of the compounds to concentrate in sympathetic neurons was determined, using, in most cases, an assay based on the development of a phenanthrenequinone derivative. The ability of several of the compounds to inhibit oxidative phosphorylation in isolated rat liver mitochondria was also determined. The results show that only a few compounds, very similar in structure to guanethidine, are cytotoxic in vivo . Only compounds very similar to guanethidine accumulate to high concentrations in the neuronal cell bodies, and this may explain the lack of toxicity of some compounds. However, compounds were found, particularly Pharmacia 881/7, which accumulate in the cell body but are not cytotoxic. In isolated mitochondria, Pharmacia 881/7 was ten times more potent than guanethidine as an inhibitor of oxidative phosphorylation. Some compounds which were cytotoxic in vivo produced little or no inhibition of oxidative phosphorylation. These data indicate that inhibition of oxidative phosphorylation is not the mechanism by which guanethidine destroys sympathetic neurons.

Phosphorylation coupled to oxidation of thiol groups (GSH) by cytochrome c with disulfide (GSSG) as an essential catalyst IV. Stability of intermediates and possible mechanism of reaction

Abstract The primary intermediates responsible for energy conservation during GSSG catalyzed electron transfer from GSH to cytochrome c under anaerobic conditions are labile but have a life time of a few minutes. The presence of phosphate results in relatively stable intermediates, presumably by forming a more stable charge transfer complex or phosphorylated form. The formation of 1 ∼ P per cytochrome c reduced requires postulation of a one electron transfer mechanism or a 2 electron transfer mechanism with an unrecognized acceptor for the second electron.

The effect of chlorpromazine, promethazine, and diphenhydramine on swelling of isolated liver mitochondria

Abstract Diphenhydramine, chlorpromazine, and promethazine do not inhibit the initial adjustment of isolated liver mitochondria to hypotonie medium. Chlorpromazine and promethazine in optimum concentrations do inhibit glutathione- or ascorbate-induced swelling-lysis, late isotonic swelling, and a late or second phase of hypotonie swelling. In isotonic medium they do not inhibit electron transport-dependent swelling except in very much greater concentrations, and then only partially. Diphenhydramine does not inhibit glutathione- or ascorbate-induced swelling-lysis, late isotonic swelling, or electron transport-dependent swelling in isotonic medium. In most cases it does inhibit late swelling of mitochondria in hypotonie media. Late swelling of fresh mitochondria in very hypotonie medium may have some relation to electron transport, since it is inhibited by 2, 4-dinitrophenol and most electron transport inhibitors, but late swelling under other circumstances is not. The late swelling of fresh mitochondria in very hypotonic medium, like the late swelling in isotonic medium and most other circumstances, is inhibited by the antioxidant butylated hydroxyanisole, so that lipid peroxidation may be responsible for the changes. With DPHA there is clear indication of a membrane-stabilizing effect independent of electron transport inhibition, uncoupling, or antioxidant activity. With the phenothiazines, the powerful antioxidant action makes it more difficult to determine whether the stabilizing action is direct like that of DPHA or is the result of preventing peroxidation, but they do inhibit swelling in some circumstances when BHA is inactive. The aging of isolated mitochondria results in some membrane change which greatly slows their initial adjustment to hypotonic medium.

Glutathione peroxidase II of guinea pig liver cytosol: relationship to glutathione S-transferases.

Abstract GSH peroxidase II activity is not associated with all GSH- S -transferase (EC 2.5.1.18) proteins. In guinea pig liver GSH peroxidase II (nonseleno and specific for organic hydroperoxides) is associated almost entirely with GSH- S -transferase peak aa and a smaller peak designated aa′. Transferase a shows a slight peroxidase activity, transferase b is absent, and transferase c has no peroxidase activity. GSH peroxidase II of guinea pig liver has an isoelectric point of 8.9 and a molecular weight of 45,000. It consists of two subunits of similar size (26,000). The GSH peroxidase II and the GSH- S -transferase activities of transferase aa have not been resolved into separate proteins and presumably reside in the same protein. In rat liver GSH peroxidase II activity is present with the highest specific activity in GSH- S -transferase AA. There is no AA′. Transferase B also shows peroxidase activity. Transferases A and C show low but measurable peroxidase activity. Transferase peak E shows peroxidase activity, but it is contaminated by large amounts of GSH peroxidase I (EC 1.11.1.9), recognized by its activity on H 2 O 2 .

Extension of the blood half-life of glyceryl trinitrate: Inhibition of glutathione organic nitrate ester reductase activity in the rat and guinea pig

Abstract Cephalothin, penicillin G and probenecid inhibited GSH organic nitrate ester reductase (ONER) and several other enzymatic activities of GSH- S -transferases (EC 2.5.1.18) from rat and guinea pig liver. Erythrityl tetranitrate, a substrate for ONER, inhibited the aryl and alkyl transferase activities of two guinea pig liver GSH- S -transferases. These findings support the concept that ONER is one of the several activities possessed by the GSH- S -transferases. In an examination of possible in vivo action, parenteral administration of these inhibitors 2–30 min prior to i.v. administration of [ 14 C]glyceryl trinitrate resulted in a 50–100 per cent increase in the half-time of the metabolism phase of [ 14 C]glyceryl trinitrate clearance from the blood and postponed the appearance of metabolites. This presumably occurs through the in vivo inhibition of GSH-ONER activity of the GSH- S -transferases and suggests a possible means of prolonging the pharmacologie action of nitrate esters.