Arnold Schwartz

Mechanism of cardiac glycoside inhibition of the (Na+-K+)-dependent ATPase from cardiac tissue

Specific digoxin binding to a (Na+−K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from cardiac tissue was determined by use of [3H]digoxin. The binding required ATP and Mg2+, was stimulated by Na+ and depressed by K+. Active cardiac glycosides significantly diluted the [3H]digoxin binding, while inactive ones had no effect. The binding was also observed in the presence of acetyl phosphate substituted for ATP; in this case Na+ had no stimulatory effect while K+ still depressed the binding. Considering the similarity of the increase and decrease of both the digoxin binding and the phosphorylated intermediate of ATPase under various conditions, these results suggest that digoxin binds with the phosphorylated conformation of the enzyme. The mechanism of digoxin inhibition of the ATPase is explained by the stability of the digoxin-intermediate complex.

Purification and properties of a highly active ouabain-sensitive Na+, K+-dependent adenosinetriphosphatase from cardiac tissue

Abstract A highly active and specific Na + , K + -dependent ATPase was obtained from calf-heart muscle by succesive treatments with deoxycholate and NaI. General properties of the enzyme were studied. Simultaneous addition of Na + and K + increased the activity about 20 times above the basic Mg 2+ -dependent level. The activation was completely reversed by 10 −4 M ouabain. The optimal pH was 7.2; the K m for ATP for the Na + ,K + -dependent and Mg 2+ -dependent enzyme activities were 2.4·10 −4 M and 5.0·10 −5 M, respectively. Only CTP could be substituted for ATP, but the activity was 14% of that found with ATP. Azide and histone, which affected the crude ATPase system, had no effect on the purified enzyme. p -Chloromercuribenzoate, N -ethylmaleimide, oligomycin, tributyltin chloride, octylguanidine as well as ouabain were found to inhibit the Na + , K + -dependent component while having little or no effect on the basic Mg 2+ -dependent activity. The K i s were 5·10 −6 M, 6·10 −4 M, 7·10 −6 M, 1.5·10 −5 M, 3·10 −4 M and 10 −6 M, respectively.

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 sodium and potassium-stimulated adenosine triphosphatase from cardiac tissues. I. Preparation and properties.

Abstract In recent years attention has been given to an adenosine triphosphatase (ATPase), which has been implicated in active cation transport across membranes. This enzyme system has been extensively studied in crab nerve (Skou 1957 1960), red blood cell membranes (Post 1959; Dunham and Glynn 1961) and cerebral tissues (Jarnefelt 1961; Aldridge 1962; Schwartz et al 1962; Skou 1962). The present report is concerned with the preparation and some properties of an active sodium and potassium-dependent ATPase from heart muscle.

Kinetic analysis of ouabain-K+ and Na+ interaction on a Na+, K+-dependent adenosinetriphosphatase from cardiac tissue.

Abstract Antagonism by K + of ouabain inhibition of Na + ,K + -dependent adenosinetriphosphatases (Na + ,K + -ATPase), reported earlier by Dunham and Glynn (1961) , Post and Albright (1961) and Auditore (1964) , suggests that ouabain inhibits the enzyme activity by a displacement of K + from a K + -binding site (Skou, 1964). On the other hand, Schatzmann (1965) observed a non-competitive interaction between ouabain and K + or Na + on a red cell ghost ATPase system. The kinetics of the Na + ,K + -ATPase as well as studies of partial reactions of the enzyme system have not been extensively pursued due, in part, to difficulty in purifying the enzyme. The present report is concerned with the kinetics of ouabain inhibition of a highly specific Na + ,K + -ATPase from cardiac tissue ( Matsui and Schwartz, 1966 ). The non-competitive nature of ouabain-K + interaction is described, stressing the dependency of ouabain-induced inhibition upon the Na + K + ratio.

A sodium-and potassium-stimulated adenosine triphosphatase from cardiac tissues—II: The effects of ouabain and other agents that modify enzyme activity

Abstract An ATP-hydrolyzing enzyme system (ATPase) has been found in the ‘microsomal’ fraction of heart muscle homogenates, which appears to consist of at least two components, one, Mg2+-dependent and the other, Mg2+ + Na+ + K+-dependent. Various inhibitory agents were employed in order to differentiate these two components or sites of activity. Amytal and ethyl alcohol are representative of compounds that inhibit the Mg2+ + Na+ + K+-dependent activity to a greater extent than the Mg2+-dependent activity. Ouabain and p-hydroxymercuribenzoate. (POMB) specifically inhibit the Mg2+ + Na+ + K+-ATPase activity while having little or no effect on the basic Mg2+-activity. The POMB-sensitive portion of the enzyme system appears to be sensitive to ouabain. Sodium azide, guanidine, and trinitrobenzenesulfonic acid represent compounds which preferentially inhibit the basic Mg2+ activity of the heart muscle, while being almost unreactive on a similar enzyme system from brain. These substances markedly increase the sensitivity of the enzyme system to both ouabain and to sulfhydryl group inhibitors. The possibility that certain charged groups are involved in the effect of ouabain on the membrane ATPase system from heart muscle is discussed.

Active Transport of Potassium Ion in Heart Mitochondria

Several factors affecting K+ transport by rabbit heart mitochondria were examined, using a K+-sensitive electrode. The histone fractions f2a and β-7 produced an energy-dependent efflux of K+. Inorganic phosphate was required for optimal activity; Km for phosphate was 60 μM. Both rate and extent of K+ efflux decreased as K+ concentration in the reaction medium was increased. The direction of valinomycin-induced K+ movements was shown to depend on the net resultant of an active transport mechanism and increased membrane permeability. The detergent triton X-100 produced a nonspecific increase in membrane permeability that led to a rapid efflux of K+. Evidence is presented for competition between ion transport and ATP formation for some common energy intermediate. Possible mechanisms of action of histones and other agents affecting heart mitochondrial K+ transport are considered.

A sodium and potassium-stimulated adenosine triphosphatase from cardiac tissues: IV. Localization and further studies of a basic protein inhibitory factor

Abstract A crude factor has been separated from heart muscle and other tissues which preferentially inhibits one site of a microsomal ATPase system. The evidence suggests that the factor is a histone or a histone-like compound which may be present in the nucleus of the cell. Differences in sensitivity of microsomal ATPases from brain and from heart to the factor are described and discussed in terms of isolation procedure and function. A number of highly purified histone fractions derived chiefly from calf thymus closely mimic the inhibitory effects of the crude factors. Both the factors as well as the purified histones modify the action of ouabain on the microsomal ATPases.

A sodium and potassium-stimulated adenosine triphosphatase from cardiac tissues—III: The properties of an endogenous inhibitory factor

Abstract An endogenous factor has been solubilized from cardiac muscle preparations, which exerts a preferential inhibitory action on the Mg 2+ -dependent site of a heart microsomal ATPase system. Similar factors have been isolated from brain and parotid gland. The effects of this material resemble those induced by certain nitrogenous compounds and those observed after ‘aging’ of the enzyme system. Possible relationships to basic polypeptides are discussed.

Isotopic labelling of heart mitochondria as a function of respiratory state

Abstract Studies in this laboratory have demonstrated marked effects of specific histone fractions on mitochondrial activity (1,2). It was suggested that the mechanism may involve an interaction of the basic protein with acidic polyelectrolytes present in mitochondria (3). Two possible candidates are RNA and DNA, both of which are now known to occur in mammalian mitochondria (4–9). The latter polynucleotide is usually associated with histones in the nucleus. Studies involving the separation and characterization of DNA and RNA from mitochondria were undertaken and the effects of histones on incorporation of precursors into mitochondrial RNA, were studied. Several aspects of this investigation suggested that the functional state of the mitochondria may play a role in the incorporation process. Some of these results are reported in this communication.