Analysis of phosphoryl transfer mechanism and catalytic centre geometries of transport ATPase by means of spin-labelled ATP.

Spin-labelled ATP [3'-O-(1-oxyl-2,2,5,5-tetramethyl-3-carbonyl pyrrolidine)-adenosine 5'-triphosphate], abbreviated SL-ATP, is used to study firstly the occurrence of an associative phosphorane mechanism for the phosphoryl transfer from ATP to the transport-ATPase protein, and secondly the presence of two geometrically unequal catalytic centres in the two catalytic peptide chains deduced to explain the existence of two KD'(ATP) values under equilibrium conditions and two Km(ATP) values under turnover conditions. 1. In the presence of Na+, K+ and Mg2+, SL-ATP is not hydrolysed by transport-ATPase from three different sources. In the presence of Na+ and Mg2+, SL-ATP reacts initially like ATP with the enzyme, as indicated by the production of a similar ouabain-binding protein conformation. With both nucleotides, this initial reaction includes the formation of the covalent enzyme-nucleotide complex through nucleophilic attack of the aspartate carboxyanion of the catalytic centre on the terminal phosphorus atom of the triphosphate chain. This produces the ouabain-binding conformation of the enzyme. Unlike ATP, the covalent enzyme-SL-ATP complex resists further transformation. 2. In the presence of Na+, K+ and Mg2+, the influence of SL-ATP on ATP hydrolysis by transport-ATPase depends on the ATP concentration chosen. At low ATP concentration, when the enzyme works as Na+-ATPase, SL-ATP does not affect the rate of ATP cleavage. At high ATP concentration, however, when the enzyme works as (Na+ + K+)-ATPase, SL-ATP reduces the rate of ATP hydrolysis to the level of Na+-ATPase activity, apparently due to the formation of the covalent enzyme-SL-ATP complex. 3. SL-ATP in the covalent enzyme-SL-ATP complex shows an ESR spectrum which is indistinguishable regarding the overall shape, the rotational correlation time, tau, and the hyperfine coupling constant, aN, from the ESR spectrum of free SL-ATP. Consequently, the dimensions of the catalytic centre cleft of transport-ATPase provide the labelled group of SL-ATP, opposite to its 3'-O-esterification site at the ribose moiety, in a wide-cleft groove, enough free space for an essentially unhindered rotational mobility within an aqueous environment like that of the bulk medium. Judged from literature data, similarly wide grooves exist in the catalytic centre clefts of mitochondrial and myosin ATPases. 4. In the framework of present knowledge, the idea is put forward that the structural unit forming the binding site for the AMP moiety of ATP in ATPases is similar to the structural unit forming the binding site for the AMP moiety of NAD and ADP in several dehydrogenases and kinases.

Electron microscopic visualization of the arrangement of the two protein components of (Na+ + K+)-ATPase.

The information obtained by electron microscopic examination of highly purified membrane preparations of (Na+ + K+)-ATPase after freeze-fracturing or negative staining suggests the following conclusions. The catalytic 100 000 dalton protein component penetrates with its greater 'globular' mass the plasma membrane and protudes with its smaller mass from the protoplasmic surface by a stalked knob carrying the catalytic centre. The 40 000 dalton glycoprotein component is anchored in the membrane interior by a non-pom the outer membrane surface forming a surface coat of ill-definable substructure.

Mathematical modelling of ATP, K+ and Na+ interactions with (Na+ + K+)-ATPase occurring under equilibrium conditions.

The controlling effect of ATP, K+ and Na+ on the rate of (Na+ + K+)-ATPase inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) is used for the mathematical modelling of the interaction of the effectors with the enzyme under equilibrium conditions. 1. Of a series of conceivable interaction models, designed without conceptual restrictions to describe the effector control of inactivation kinetics, only one fits the experimental data described in a preceding paper. 2. The model is characterized by the coexistence of two binding sites for ATP and the coexistence of two separate binding sites for K+ and Na+ on the enzyme-ATP complex. On the basis of this model, the effector parameters fitting the experimental data most closely are estimated by means of nonlinear least-squares fits. 3. The apparent dissociation constants for ATP fo the enzyme-ATP complex or of the enzyme-(ATP)2 complex are computed to lie near 0.0024 mM and 0.34 mM, respectively, irrespective of whether K+ and Na+ were absent or K+ and K+ plus Na+, respectively, were present in the experiments. 4. The origin of the high and the low affinity site for binding of ATP to the (Na+ + K+)-ATPase molecule is traced back to the coexistence of two catalytic centres which, although primarily equivalent as to the reactivity of their thiol groups with NBD-C1, are induced into anticooperative communication by ATP binding and thus show an induced geometric asymmetry. 5. On the basis of the interaction model outlined under item 2 the apparent dissociation constant for K+ or Na+ in the (K+ + Na+)-liganded enzyme-ATP complex are computed to be 1.7 mM and 3.5 mM, respectively. 6. The conclusions concerning the coexistence of two primarily equivalent but anticooperatively interacting catalytic centres and the coexistence of two separate ionophoric centres for Na+ and K+ correspond to the appropriate basic postulates of the flip-flop concept of (Na+ + K+)-ATPase mechanism.

Evidence against parallel operation of sodium/calcium antiport and ATP-driven calcium transport in plasma membrane vesicles from kidney tubule cells.

The present study aimed to clarify the existence of a Na+/Ca2+ antiport device in kidney tubular epithelial cells discussed in the literature to represent the predominant mechanistic device for Ca2+ reabsorption in the kidney. Inside-out oriented plasma membrane vesicles from tubular epithelial cells of guinea-pig kidney showed an ATP-driven Ca2+ transport machinery similar to that known to reside in the plasma membrane of numerous cell types. It was not affected by digitalis compounds which otherwise are well-documented inhibitors of Ca2+ reabsorption. The vesicle preparation contained high, digitalis-sensitive (Na+ + K+)-ATPase activities indicating its origin from the basolateral portion of plasma membrane. The operation of a Na+/Ca2+ antiport device was excluded by the findings that steep Ca2+ gradients formed by ATP-dependent Ca2+ accumulation in the vesicles were not discharged by extravesicular Na+, and did not drive 45Ca2+ uptake into the vesicles via a Ca2+-45Ca2+ exchange. The ATP-dependent Ca2+ uptake into the vesicles became increasingly depressed with time by extravesicular Na+. This was not due to an impairment of the Ca2+ pump itself, but caused by Na+/Ca2+ competition for binding sites on the intravesicular membrane surface shown to be important for high Ca2+ accumulation in the vesicles. Earlier observations on Na+-induced release of Ca2+ from vesicles pre-equilibrated with Ca2+, seemingly favoring the existence of a Na+/Ca2+ antiporter in the basolateral plasma membrane, were likewise explained by the occurrence of Na+/Ca2+ competition for binding sites. The weight of our findings disfavors the transcellular pathway of Ca2+ reabsorption through tubule epithelium essentially depending on the operation of a Na+/Ca2+ antiport device.

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.

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.

Interaction of cardiac glycosides and Na,K-ATPase is regulated by effector-controlled equilibrium between two limit enzyme conformers.

The paper describes the dissociation parameters of the complexes between [3H]-digitoxin and Na,K-ATPase (Na+ + K+-activated, Mg2+-dependent ATP phosphohydrolase, E.C. 3.6.1.3) from pig cardiac muscle and brain cortex formed and dissociated in the presence of different combinations and concentrations of the enzyme effectors ATP, Mg2+, Na+ and K+. Systematic variation of effector-ligation of Na,K-ATPase allowed production of glycoside complexes with two enzyme conformers only, which showed either rapid or slow dissociation kinetics. Appropriate changes of enzyme ligation allowed the interconversion of the two conformer types. Biphasic, rapid and slow glycoside release was not bound with the presence of two Na,K-ATPase isozymes, but caused by the enzyme ligation-determined coexistence of the two conformers of Na,K-ATPase. The rate constants for the rapid and slow glycoside release were within the complexes of each dissociation type much alike indicating uniform isomerization kinetics of the two conformers even when differently liganded. Taken together, the observations indicated the effector-controlled isomerizations of two conformers of Na,K-ATPase possessing different geometries of the glycoside binding domain. Present findings and relevant literature data were integrated in a circular, consecutive and simultaneous model for induced conformation changes that accounted for the regulation of the interaction of cardiac glycosides and Na,K-ATPase through an effector-controlled equilibrium between two limit enzyme conformers.

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 3.6.1.37) from human cardiac muscle, human brain cortex and guinea-pig cardiac muscle was compared to probe the complementary chemotopology of the inhibitor binding site areas on the three enzyme variants. The changes of potency, resulting from systematic variations of the geometry of steroid skeleton and the character as well as the structure of side chains at C3 or/and C17 of steroid backbone, allowed the following major conclusions. With the human cardiac and cerebral enzyme forms, the paired K0.5 (K'D) values for 77 steroid derivatives, covering seven orders of ten, were highly correlated. On an average, the total of compounds showed a 1.5-fold higher affinity to the cardiac enzyme. This tiny differentiation did not appear to be connected with an important difference in the chemotopology of the complementary subsites for steroid nucleus binding on the two enzyme forms. With the human and guinea-pig cardiac enzyme variants, the K0.5 values for 69 steroid derivatives, covering six orders of ten, were determined. For 41 5 beta, 14 beta-androstane derivatives only, the paired K0.5 values showed a close correlation. Here, the human enzyme variant exhibited 27-fold higher affinity. However, the paired K0.5 values determined on both enzymes for 28 steroid derivatives of differing structural features were but poorly correlated. Essentially, the geometries of the steroid nucleus determined the differential contributions of the side chains at C3 and C17 to the integral inhibitory potency on the two enzyme variants. Thus, the species differences in the potency of cardiac glycosides were traced to species differences in the complementarity of the steroid binding subsites. Hence, estimates of the potency of new steroidal compounds obtained on the guinea-pig cardiac enzyme can be neither quantitatively nor qualitatively easily extrapolated to the human cardiac enzyme. The extrathermodynamic analysis of the data opened major new insights in the structure-activity relationships concerning the role of C14 beta-OH, the character of the lead structure in cardioactive steroid lactones, and the significance of the configuration of A/B ring junction.

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)

The lead structure in cardiac glycosides is 5 beta, 14 beta-androstane-3 beta 14-diol.

The purpose of the present study was to determine the lead structure in cardiac glycosides at the receptor level, i.e. the minimal structural requirement for specific and powerful receptor recognition. Accordingly 73 digitalis-like acting steroids were characterized as to the concentration effecting half-maximum inhibition of Na,K-ATPase from human cardiac muscle under standardized turnover conditions. Since the Ki value equaled the apparent KD value, K'D was expressed in terms of the apparent standard Gibbs energy change delta G degrees' of steroid interaction with Na,K-ATPase. This allowed the use of the extrathermodynamic approach as a rational way of correlating in a quantitative manner, the potency and structure of the various steroidal compounds. The results of the present analysis taken in conjunction with relevant findings reported in the literature, favour the following conclusions. Cassaine, canrenone, prednisolone- and progesterone-3,20-bisguanylhydrazone, and chlormadinol acetate are compounds that are not congeneric with digitalis. The butenolide ring of cardenolides or the analogous side-chains at C17 beta of 5 beta, 14 beta-androstane-3 beta, 14-diol are not pharmacophoric substructures, but merely amplifiers of the interaction energy of the steroid lead. All modifications of the structure, geometry and spatial relationship between the steroid nucleus and butenolide side chain of digitoxigenin all at once weaken the close fit interaction with the steroid and butenolide binding subsites of the enzyme in such way that the cardenolide derivatives interact with the receptor binding site area in whatever orientation that will minimize the Gibbs energy of the steroid-receptor-solvent system. The "butenolide carbonyl oxygen distance model" (Ahmed et al. 1983) for the interpretation of the differences in potency of the cardenolide derivatives describes the change in interaction energy through structural modification as a function of the entire molecule. 5 beta, 14 beta-androstane-3 beta, 14-diol, the steroid nucleus of cardiac glycosides of the digitalis type, is the minimum structure for specific receptor recognition and the key structure for inducing protein conformational change and thus Na,K-ATPase inhibition. It is also the structural requirement for maximum contributions of the butenolide substituent at C17 beta and the sugar substituent at C3 beta-OH to the overall interaction energy, i.e. this steroid nucleus is the lead structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Penta-acetyl-gitoxin: the prototype of a prodrug in the cardiac glycoside series.

Penta-acetyl-gitoxin is a suitable prodrug of gitoxin since it shows--side-effect latentiation, due to its inactive application form;--bioavailability, due to its improved solubility and thus good enteral absorption and--bioactivation, due to rapid de-acetylation in the body after absorption.