Reinstatement of the ATP high energy paradigm.

The paradigm that the hydrolysis of ATP releases high Gibbs energy able to perform work has increasingly been questioned over the last two decades. Results from theoretical and experimental studies have been interpreted to indicate that the synthesis of ATP from ADP and P(i) does not require energy supply and that binding of ATP per se can transmit utilizable energy to an enzyme. As has recently been concluded, all this has led to a change of the ATP high energy paradigm in bioenergetics. Starting from this challenge, the present review singles out the striking sources of the apparent dichotomy in bioenergetics, and endeavours to eliminate the apparent contradictions by the application of the prior knowledge on both the participation of the enzyme protein in energy exchange processes and the particular reactivities of phosphorus that make it an outstanding element for functionally variable work assignments in enzymatic systems.

Modeling of the three-dimensional structure of the digitalis intercalating matrix in Na+/K(+)-ATPase protodimer.

Based on the knowledge that the digitalis receptor site in Na+/K(+)-ATPase is the interface between two interacting alpha-subunits of the protodimer (alpha beta)2, the present review makes an approach towards modeling the three-dimensional structure of the digitalis intercalating matrix by exploiting the information on: the primary structure and predicted membrane topology of the catalytic alpha-subunit; the determinants of the secondary, tertiary and quaternary structure of the membrane-spanning protein domains; the impact of mutational amino acid substitutions on the affinity of digitalis compounds, and the structural characteristics in potent representatives. The designed model proves its validity by allowing quantitative interpretations of the contributions of distinct amino acid side chains to the special bondings of the three structural elements of digitalis compounds.

Potential suitability of Na+/K(+)-transporting ATPase in pre-screens for anti-cancer agents.

Twenty-five compounds [digitalis (generic name for cardenolides, bufadienolides and their glycosides) representatives and derivatives, various steroids as well as some customary carcinostatics] have been compared in terms of their potency to suppress the proliferation of Ehrlich mouse ascites carcinoma (EMAC) cells and to inhibit the activity of Na/K-ATPase from EMAC cells and from human cardiac muscle. The inhibitor susceptibilities of the Na/K-ATPase isoforms of EMAC and cardiac muscle are very different, in favour of the cardiac muscle with the digitalis-like acting steroids, whereas they are quite similar with the digitalis-unlike acting compounds. Whereas the K0.5 values for the inhibition of EMAC Na/K-ATPase display the expected dependence on steroid structure, the IC50 values for the suppression of EMAC cell proliferation all lie within a narrow concentration range. With ouabain, the IC50 value for the suppression of proliferation of oestrogen receptor-negative, human mammary carcinoma (MCA) cells is four orders of magnitude higher than the K0.5 value for inhibition of the activity of human cardiac muscle Na/K-ATPase. In contrast to this effectivity order, some synthetic derivatives of digitalis steroids develop primarily antiproliferative potency.

Differentiation between isoforms of Na+/K+-transporting atpase from human and guinea-pig muscle through use of digitalis derivatives as analytical probes.

The aims of the study included: to explore the protein structure basis for the differences in digitalis sensitivity between isoforms of Na/K-ATPase from human and guinea-pig cardiac muscle; to determine the relative significance of the constituents of tripartite digitalis compounds in their inhibitory action on these Na/K-ATPase isoforms; to evaluate the potential significance of the receptor kinetics for pharmacological characteristics. The analytical method has been the recording of the inhibitory interaction of various digitalis derivatives with the Na/K-ATPase isoforms. The protein structure basis for the isoform differences in digitalis susceptibility has been explored by analysing in free-energy plots the kinetics of their inhibitory interaction with 53 digitalis derivatives of grossly different structure. The slope of the regression line and the parameters of the regression equation proved to be similar for the two isoforms in spite of the great difference in their digitalis susceptibilities. This surprising uniformity indicates that a uniform "macroscopic" mechanism underlies the inhibitory effect of the various derivatives on the two isoforms. On the other hand, the differences in the positions of delta G*on and delta G*off values for particular inhibitors relative to the regression line reveal differences in the "microscopic" interaction energy surfaces of the two isoforms. In conclusion, the origin of the isoform distinctions in their susceptibility towards inhibition by various digitalis derivatives is essentially confined to differences in the chemotopology of the digitalis recognition matrix and binding cleft. Specific observations allowed to disentangle the impact of various steroid derivatizations at carbon atoms 3, 17, and diverse other positions on the kinetics of their interaction with the enzyme isoforms. The steroid nucleus of the cardiac glycosides, 5 beta, 14 beta-androstane, proves to be the basal structural element for discrimination of Na/K-ATPase isoforms. This discrimination becomes much enlarged by steroid glycosidation at C3 beta-OH and/or by steroid substitution of C17 beta-H by a lactone ring. The higher inhibitory sensitivity of the human isoform is based either on an increased association rate or a decreased dissociation rate, depending on the nature of derivatization.(ABSTRACT TRUNCATED AT 400 WORDS)

Location and properties of the digitalis receptor site in Na+/K(+)-ATPase.

Since 1985, several research groups have shown that a number of amino acids in the catalytic alpha-subunit of Na+/K(+)-ATPase more or less strongly modulate the affinity of a digitalis compound like ouabain to the enzyme. However, scrutiny of these findings by means of chimeric Na+/K(+)-ATPase constructs and monoclonal antibodies has recently revealed that the modulatory effect of most of these amino acids does not at all result from direct interaction with ouabain, but rather originates from long-range effects on the properties of the digitalis binding matrix. Starting from this knowledge, the present review brings together the various pieces of evidence pointing to the conclusion that the interface between two interacting alpha-subunits in the Na+/K(+)-ATPase protodimer (alpha beta)2 provides the cleft for inhibitory digitalis intercalation.

Synthesis of a self-contained concept of the molecular mechanism of energy interconversion by H(+)-transporting ATP synthase.

The original aim of the review has been to probe into the validity of the paradigm on the high energy-carrier function of ATP. It seemed to be called into question on the basis of findings with H(+)-transporting ATP synthase suggesting the formation of ATP from ADP and Pi without energy input. Thus, ATP appeared as a low-energy compound. Starting from the current, rich knowledge of the molecular structure and the inviting thinking on the mechanism of H(+)-transporting ATP synthase, we have endeavoured to freshly interpret and integrate the pertinent observations in the light of the comprehensively derived model of the molecular mechanism of energy interconversion by Na+/K(+)-transporting ATPase. In this way, we have uncovered the common mechanistic elements of the two energy-interconverting enzymes. The emerging purpose of the present paper has been the 'synthesis' of a self-contained concept of the molecular mechanism of the interconversion of electrochemical and chemical Gibbs energies by H(+)-transporting ATP synthase. The outcome is reflected in the following tentative evaluations. 1. In ATP hydrolysis, the great Gibbs energy change which is observed in solution, is largely conserved by the F1 sector of ATP synthase as mechanical Gibbs energy in the enzyme's protein fabric, so that it can be utilized in the resynthesis of ATP from enzyme-bound ADP and Pi. The plainly measured low Gibbs energy change results from large compensating enthalpy and entropy changes that reflect the underlying changes in protein conformation. 2. In stoichiometric ATP synthesis by F1 sector from ADP and Pi bound to the catalytic centre, their intrinsic binding energy brings about a loss of peptide chain entropy that makes possible an entropy-driven ATP formation. 3. The driving force for ATP synthesis cannot be the high Gibbs energy change on binding of product ATP; the tight ATP-enzyme complex rather is a low Gibbs energy intermediate from which escape is difficult. 4. The catalytic centre exists either in an open state unable to firmly bind the substrate-product couple, or in a closed state protecting formed ATP from facile hydrolysis by ambient water. 5. The cleft closure, induced by binding of Pi and ADP or ATP, does not necessarily need external energy supply, because the cleft closure proceeds from rigid domain rotations which can occur rather spontaneously. In further analogy to adenylate kinase, the driving force of this domain movement presumably comes from the electrostatic interactions between phosphate moieties and arginine side chains in the catalytic centre.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunologic identification of Na+,K(+)-ATPase isoforms in myocardium. Isoform change in deoxycorticosterone acetate-salt hypertension.

There are three isoforms of the catalytic (alpha) subunit of the Na+,K(+)-ATPase, each derived from a different gene, that differ in their sensitivity to inhibition by cardiac glycosides. Antibodies specific for the three isoforms were used to study Na+,K(+)-ATPase isoform expression in ventricular myocardium, where an understanding of digitalis receptor diversity is most important. In the rat heart, there is simultaneous expression of two isoforms in adult ventricle, and immunofluorescence studies demonstrated that both isoforms are expressed uniformly in cardiomyocytes. Hypertension and hypertrophy have been reported to selectively depress alpha 2 isoform mRNA levels, and we show in the present study that alpha 2 protein levels were correspondingly depressed in rats made hypertensive by uninephrectomy and treatment with deoxycorticosterone acetate and a high-salt diet. In the human heart, where mRNA for all three alpha isoforms has been reported, we detected all three isoform proteins (alpha 1, alpha 2, and alpha 3). Two isoforms (alpha 1 and alpha 3) predominated in the macaque heart; dissection of the heart showed uniformity of isoform expression in different ventricular regions but markedly less alpha 3 in the atrium. Finally, isoform-specific antibodies were used to detect which alpha isoforms were expressed in the ventricles of several commonly used experimental animals to test the correlation of isoform expression with cardiac glycoside-response heterogeneity. Two isoforms (alpha 1 and alpha 3) were found in canine myocardium, whereas only one (alpha 1) was found in sheep and guinea pig. Expression of Na+,K(+)-ATPase isoforms can thus be readily followed and related to the physiology of the digitalis receptor.

Role of protein conformation changes and transphosphorylations in the function of Na+/K(+)-transporting adenosine triphosphatase: an attempt at an integration into the Na+/K+ pump mechanism.

The particular aim of the review on some basic facets of the mechanism of Na+/K(+)-transporting ATPase (Na/K-ATPase) has been to integrate the experimental findings concerning the Na(+)- and K(+)-elicited protein conformation changes and transphosphorylations into the perspective of an allosterically regulated, phosphoryl energy transferring enzyme. This has led the authors to the following summarizing evaluations. 1. The currently dominating hypothesis on a link between protein conformation changes ('E1 in equilibrium with E2') and Na+/K+ transport (the 'Albers-Post scheme') has been constructed from a variety of partial reactions and elementary steps, which, however, do not all unequivocally support the hypothesis. 2. The Na(+)- and K(+)-elicited protein conformation changes are inducible by a variety of other ligands and modulatory factors and therefore cannot be accepted as evidence for their direct participation in effecting cation translocation. 3. There is no evidence that the 'E1 in equilibrium with E2' protein conformation changes are moving Na+ and K+ across the plasma membrane. 4. The allosterically caused ER in equilibrium with ET ('E1 in equilibrium with E2') conformer transitions and the associated cation 'occlusion' in equilibrium with 'de-occlusion' processes regulate the actual catalytic power of an enzyme ensemble. 5. A host of experimental variables determines the proportion of functionally competent ER enzyme conformers and incompetent ET conformers so that any enzyme population, even at the start of a reaction, consists of an unknown mixture of these conformers. These circumstances account for the occurrence of contradictory observations and apparent failures in their comparability. 6. The modelling of the mechanism of the Na/K-ATPase and Na+/K+ pump from the results of reductionistically designed experiments requires the careful consideration of the physiological boundary conditions. 7. Na+ and K+ ligandation of Na/K-ATPase controls the geometry and chemical reactivity of the catalytic centre in the cycle of E1 in equilibrium with E2 state conversions. This is possibly effected by hinge-bending, concerted motions of three adjacent, intracellularly exposed peptide sequences, which shape open and closed forms of the catalytic centre in lock-and-key responses. 8. The Na(+)-dependent enzyme phosphorylation with ATP and the K(+)-dependent hydrolysis of the phosphoenzyme formed are integral steps in the transport mechanism of Na/K-ATPase, but the translocations of Na+ and K+ do not occur via a phosphate-cation symport mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction of progesterone derivatives with the digitalis target enzyme: impact of glycosidation on inhibitory potency.

As a function of the structural modification of the steroid nucleus, the inhibitory interaction of 11 progesterone derivatives with human Na/K-ATPase (Na+/K(+)-transporting ATPase, EC 3.6.1.37), through C3-O-rhamnosylation, is either much decreased or weakly up to strongly increased, so that the rhamnosyl residue contributes to the complementary Gibbs energy of interaction, at the most, the same Gibbs energy increment as realized in ouabain. After C3 beta-O-rhamnosylation, the activity of some progesterone derivatives considerably surpasses that of 3 beta-O-rhamnosyl-chlormadinolacetate, which has been known to elicit positive inotropy in cats. The progesterone derivatives (aglycons and glycosides), that have been analysed more closely, produce their effects by the same molecular mechanism of interaction with Na/K-ATPase as characteristic for digitalis aglycons and glycosides. The results promise to pave the way for the identification of the chemical nature of endogenous digitalis and for the design of novel inotropic drugs.

Chemical models for the chemical nature of endogenous digitalis.

The inability or the capacity to promote the phosphorylation of Na+/K(+)-transporting ATPase (Na/K-ATPase) from [32P]Pi is shown to differentiate between mechanistically digitalis-unlike and digitalis-like inhibitors of this enzyme known to be the receptor for all digitalis actions. A negative or positive response in the phosphorylation promotion assay introduced here appears thus to be suitable to diagnose the chemical species in the isolates of animal origin related to the putative endogenous digitalis. Various digitalis-congeneric C/D-cis steroids, progesterone-congeneric C/D-trans steroids and the Erythrophleum alkaloid cassaine promote the enzyme phosphorylation and show a similar pattern of discrimination between three Na/K-ATPase variants. Thus, their cyclopentanoperhydrophenanthrene or perhydrophenanthrene nuclei appear to serve as the minimal pharmacophoric lead structures for bimolecular recognition and to represent chemical models for the chemical nature of endogenous digitalis. Specifically, the hormonal C/D-trans steroids could provide the basic skeleton in endogenous digitalis biosynthesis.

The thermodynamic essence of the reversible inactivation of Na+/K+-transporting ATPase by various digitalis derivatives is relaxation of enzyme conformational energy.

This paper reports on the kinetic and thermodynamic parameters describing the interaction of selected digitalis derivatives with hog and guinea-pig cardiac (Na+ + K+)-ATPase (Na+/K+-transporting ATPase EC 3.6.1.37). 32 digitalis derivatives were characterized as to the values of the delta G0', delta G----not equal to, and delta G----not equal to quantities in their interaction with (Na+ + K+)-ATPase from hog cardiac muscle in the presence of ATP, Mg2+, Na+ and K+. Nine derivatives were additionally characterized as to the values of the delta H0', delta S0', delta H----not equal to, delta S----not equal to, delta H not equal to, and delta S not equal to quantities in their interaction with the hog enzyme promoted by ATP, Mg2+ and Na+ in the presence or absence of K+. The formation of the inhibitory complexes is in any case an endothermic, entropically driven process. The Gibbs energy barriers in the formation and dissociation of the complexes, delta G----not equal to and delta G----not equal to, are imposed by large, unfavourable delta H not equal to values. K+ decreases the delta G0' value by increasing the delta G----not equal to value more than the delta G----not equal to value. In comparison with hog (Na+ + K+)-ATPase, the interaction of three derivatives with guinea-pig cardiac enzyme in the presence of ATP, Mg2+, Na+ and K+ is characterized by lower delta G0' values caused by lower favourable delta S0' values, and is accompanied by lower delta G----not equal to values. The magnitude of the kinetic parameters and the characteristic of the thermodynamic quantities describing the interaction between various digitalis derivatives and (Na+ + K+)-ATPase, indicate the induction of substantial conformational changes in the enzyme protein. A large entropy gain in the enzyme protein, observed irrespective of enzyme origin and ligation, appears to be the common denominator of the inhibitory action of all digitalis derivatives studied, suggesting that the digitalis-elicited relaxation of high conformational energy (negentropy strain) of the enzyme protein is the thermodynamic essence of the reversible inactivation of (Na+ + K+)-ATPase.