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)
Schön
R
R
Max Delbrück Center for Molecular Medicine, Berlin-Buch, Germany.
Weiland
J
J
Megges
R
R
Repke
K R
KR
D000818:Animals; D004071:Digitalis Glycosides / Q000494:pharmacology*; D006168:Guinea Pigs; D006801:Humans; D007527:Isoenzymes / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000737:chemistry; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology*; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000737:chemistry; D013045:Species Specificity; D013816:Thermodynamics
D004071:Digitalis Glycosides; D007527:Isoenzymes; D000254:Sodium-Potassium-Exchanging ATPase