George E. Lindenmayer

Reevaluation of Oxidative Phosphorylation in Cardiac Mitochondria from Normal Animals and Animals in Heart Failure

For an adequate evaluation of mitochondria from diseased hearts, basic characteristics of isolation, storage, media, ultrastructure and type of assay were first determined using mitochondria from normal animals. A proteinase procedure yielded mitochondria from small laboratory animals, with low respiratory control and marked permeability changes. The isolation medium yielding the most stable mitochondria with the highest respiratory control contained 0.18M KCl, 10mM EDTA, and 0.5% to 1% bovine serum albumin at pH 7.2. Heart failure in guinea pigs and rabbits was produced by varying degrees of stenosis of the ascending aorta. An aberration in respiratory control was found in mitochondria from hearts in severe failure. The quantitative differences between normal and experimental respiratory control values were greatest when the highest possible normal respiratory control levels were obtained. The difference between mitochondria prepared by a proteinase method from control and failing hearts was minimal. No changes in oxidative phosphorylation were noted in mitochondria from hearts arrested by nitrogen, suggesting that acute hypoxia does not irreversibly damage energy-liberating reactions. It is concluded that severe heart failure is characterized by defects in mitochondrial oxidative phosphorylation, and that techniques of isolation or assay or both are probably not causing the abnormalities.

A possible molecular mechanism of the action of digitalis: ouabain action on calcium binding to sites associated with a purified sodium-potassium-activated adenosine triphosphatase from kidney.

SUMMARY Calcium binding at 0°C to a purified sheep kidney Na+,K+-ATPase was described by linear Scatchard plots. Binding at saturating free calcium was 65–80 nmol/mg of protein, or 30–40 mol of calcium/mol of enzyme. Aqueous emulsions of lipids extracted from Na+,K+-ATPase yielded dissociation constants and maximum calcium-binding values that were similar to those for native Na+,K+-ATPase. Phospholipase A treatment markedly reduced calcium binding. Pretreatment of native Na+,K+-ATPase with ouabain increased the dissociation constant for calcium binding from 131 ± 7 to 192 ± 7 &mgr;M without altering maximum calcium binding. Ouabain pretreatment did not affect calcium binding to extracted phospholipids, ouabain-insensitive ATPases, or heat-denatured Na+,K+-ATPase. Na+ and K+ (5–20 mM) in-creased the dissociation constants for calcium, which suggests competition between the monovalent cations and calcium for the binding sites. At higher concentrations of monovalent cations, ouabain increased the apparent affinity of binding sites for calcium. Extrapolation to physiological cation concentrations revealed that the ouabain-induced increase in apparent affinity for calcium may be as much as 2- to 3-fold. These results suggest: (1) calcium binds to phospholipids associated with Na+ ,K-ATPase; (2) ouabain interaction with Na+,K+-ATPase induces a perturbation that is transmitted to adjacent phospholipids, altering their affinity for calcium; and (3) at physiological concentrations of Na+ or K+, or both, ouabain interaction with Na+,K+-ATPase may lead to an increased pool of membrane-bound calcium.

A kinetic description for sodium and potassium effects on (Na++K+)-adenosine triphosphatase: a model for a two-nonequivalent site potassium activation and an analysis of multiequivalent site models for sodium activation

1. Dissociation constants for sodium and potassium of a site that modulates the rate of ouabain‐(Na++K+)‐ATPase interaction were applied to models for potassium activation of (Na++K+)‐ATPase. The constants for potassium (0·213 m M) and for sodium (13·7 m M) were defined, respectively, as activation constant, Ka and inhibitory constant, Ki.

EFFECTS OF SODIUM AND POTASSIUM ON THE PARTIAL REACTIONS OF A HIGHLY PURIFIED (NA++ K+)‐ATPASE: MODULATION OF THE RATE OF OUABAIN INTERACTION*

Sodium and potassium interactions with (Na+ + K+)-ATPase are thought t o require specific sites on the enzyme. The sodium-activation sites are located o n the internal surface of the membrane and have high affinity for sodium and low affinity for potassium. The potassium-activation sites are located on the external surface of the membrane and have high affinity for potassium and low affinity for sodium. Potassium interaction with the sodium-activation sites or sodium with the potassium-activation sites inhibits (Na+ t K+)-ATPase activity. The stoichiometry of the sodium pump is believed t o be three sodium ions pumped out per two potassium ions pumped in per molecule of ATP hydrolyzed. This implies that there must be three sodium-activation sites and two potassium-activation sites per enzyme molecule. The multiple sites for sodium or potassium may be equivalent or nonequivalent and interrelated in a cooperative manner or indeKinetic analysis of sodium and potassium activation of (Na+ + K+)ATPase in broken membrane preparations has proven difficult because of the above complexities, which are further aggravated by the fact that it is not possible t o selectively study cation effects a t only one surface. Recently, however, we were able t o show that the rate of ouabain interaction with a beef-brain (Na+ t K+)-ATPase preparation was modulated by sodium and potassium competition for a common site.4 Sodium stimulated and potassium retarded the rate of binding.