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.
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)
SynopsisThis paper probes the calculation of conformation-related basis spectra from infrared spectra (amide I' band) of reference proteins of known conformational composition and, with their aid, the computation of conformations from the amide I' band of globular proteins using in both approaches a least-squares, curve-fitting computer program for the analysis of the spectra. The following results were obtained. The infrared basis spectra for the a-helix conformation, the 0-(antiparallel-chain pleated sheet) conformation and the p-conformation were calculated and their physical reality was substantiated. The basis spectra were shown to be similar when the absorption contributions of the side chains of amino acids were either neglected or taken into account (uncorrected or corrected basis spectra). The mutual correlation of the basis spectra, quantified by the roots of the diagonal elements of the inverse matrix, was found to be low enough only for the @-conformation to allow a statistically reliable estimate of the 0-conformation content of proteins. The comparison of the percentages of the @-conformation derived from x-ray structural analysis or calculated from infrared spectra showed the suitability of the basis spectra for the rough estimate of the 0-conformation percentages of proteins. The results were not significantly different when using the uncorrected or corrected basis spectra. INTRODUCTIONThere is an obvious requirement for methodic procedures to estimate the conformational composition and the function-related conformational changes of globular proteins that are unavailable in the crystalline state or that must be studied in a dissolved or suspended condition. This requirement is partially fulfilled by the procedure of the computer curvefitting analysis of circular dichroic (CD) spectra which takes advantage of the application of protein-derived, conformation-specific CD basis spectra.1-4 Unfortunately, CD absorption is liable to optical interference^.^,^ This is not true for ir absorption. Hence, we have tried to develop a procedure for curve-fitting analysis of ir spectra suitable for determining the conformation of globular proteins. To this end, the absorption spectra of the proteins between 1720 and 1594 cm-l when dissolved in D20 were chosen since Timasheff et and Susi et a1.8 had shown that this amide I' band is well suited for the characterization of protein conformation and that the frequencies obtained for different conformations of globular proteins in H2O and D2O solution are internally consistent.In the present paper, we wish to show that it is possible to calculate from the amide I' bands of reference protein sets the basis spectra characterizing the a-helix conformation, the 0-(antiparallel-chain pleated sheet) conformation and the unordered conformation p(p = 1 -a -p), as well as to use the basis spectra for the curve-fitting analysis of amide I' bands of proteins revealing their 0-conformation content. In an earlier a p p r~a c h ,~ we succeeded in computing the basis spectra of 0-an...
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