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.

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.

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.

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.

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.

Regional variations in electrolytes related to resistivity during bovine lens maturation.

Little is known regarding the behavior of ions in protein-rich cytoplasm characteristic of lens fiber cells. Resistivity is dependent upon the electrolyte concentration available to conduct an applied current and the mobility of these electrolytes. In the present study, the relative importance of these factors in the increasing cortico-nuclear resistivity gradient reported for both calf and bovine lens homogenates was analysed.

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.

Synaptogenesis in the rat suprachiasmatic nucleus: a light microscopic immunocytochemical survey.

MabQ155, a monoclonal antibody against synaptophysin, has been used to conduct a light microscopic survey of synaptogenesis in the suprachiasmatic nucleus of the perinatal rat. Synaptophysin is an integral component of synaptic vesicle membranes which is expressed in growth cones and growth cone filopodia as well as in mature synapses. With the light microscope, mabQ155 immunoreactivity in growth cones can be distinguished from that in presynaptic terminals on the basis of the size of immunoreactive puncta.

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.

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.

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.

5 Beta,14 beta-androstane-3 beta,14-diol binds to the digitalis receptor site on Na/K-ATPase.

5 beta,14 beta-Androstane-3 beta,-14-diol, the lead (minimum) structure in digitalis compounds, shows the same characteristics of interaction with Na/K-ATPase as ordinary digitalis compounds judged by the following six criteria: (I) shape of the concentration-inhibition curves, (II) species differences in affinity for the enzyme, (III) apparent competition with K+, (IV) competition with digitoxigenin for binding to the enzyme, (V) stabilization of phosphoenzyme formed from ATP, and (VI) enhancement of phosphorylation from orthophosphate.

Glycosidation of chlormadinol acetate alters its actions on Na+/K+-transporting ATPase and cardiac contractility: a contribution to the endogenous digitalis problem.

Compared to the progesterone derivative chlormadinol acetate 1, the arabinofuranoside 2, rhamnoside 3 and glucoside 4 of 1 are less potent in the Na/K-ATPase assay, but evoke, contrary to 1, positive inotropy in vivo. In anaesthetized cats the circulation effects of 2 and 3 appear to be more favourable than those of the digitalis glycoside digitoxin. Hence, the progestin 1 is transformed through glycosidation into an interesting cardioactive steroid.

Origin of differences of inhibitory potency of cardiac glycosides in Na+/K+-transporting ATPase from human cardiac muscle, human brain cortex and guinea-pig cardiac muscle.

The inhibitory potency of altogether 95 steroidal compounds (including cardenolides, bufadienolides and their glycosides) on the Na/K-ATPases (Na+/K+-transporting ATPases, EC 3.6.1.