Date of Award

1974

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry

College

College of Graduate Studies

First Advisor

Billy Baggett

Second Advisor

Rupert L. Green

Third Advisor

A. R. Krall

Fourth Advisor

D. Priest

Abstract

Interactions between five different cardiac glycosides, digitoxin, cymarol, convallatoxol, digoxin and ouabain, and Na+K+-ATPase from guinea pig heart, kidney, brain and rat heart and brain have been studied. Concentrations of glycosides required for 50% inhibition of the enzyme (I50) have been determined at various concentrations of K+. The binding of the glycosides to the enzyme preparations was studied using tritiated glycosides. ATP, Mg2+, and Na+ were required for specific binding. Through the use of Scatchard plots, a high affinity binding site was demonstrated for each guinea pig enzyme preparation, and the association constants (Ka) were measured for each of the glycosides. Although rat brain was demonstrated to have Na+K+-ATPase with a high affinity binding site for glycosides, this was not found to be true of rat heart Na+K+-ATPase. The initial concentration of each glycoside required to half saturate the high affinity site (B50) was calculated. The effects of varying the concentration of K+ on Ka and B50 was ascertained. In the presence of a low concentration of K+ (0.625 mM), binding and inhibition were studied under identical conditions. The B50 and I50 values were similar to each other for each guinea pig enzyme and each glycoside. This established that with the guinea pig enzymes the binding observed was to the inhibitory site on the enzyme. Digitoxin, cymarol and convallatoxol were bound more tightly and were more potent inhibitors than digoxin and ouabain with all enzyme preparations. The enzymes from the three guinea pig organs did not differ appreciably in their interactions with the glycosides. Inorganic phosphate plus Mg2+ could replace ATP, Mg2+ plus Na+ in supporting the binding of the glycosides, but in this case Na+ as well as K+ inhibited the binding. These data support the concept that Na+­ K+-ATPase is the receptor for cardiac glycosides in the guinea pig and that the binding occurs reversibly to the inhibitory site on the enzyme when it is phosphorylated. The data also support the concept that the binding process and the hydrolytic reaction, promoted by K+, compete for the phosphorylated enzyme in guinea pig Na+K+-ATPase. The B50 and I50 values for rat brain ATPase were different, with 50% inhibition requiring 100 fold more glycoside than the amount required to obtain 50% saturation of the high affinity site. The B50 concentrations were consistent with guinea pig brain B50 concentrations not only in terms of relative order but also in magnitude. Since 50% saturation of this binding site was at a much lower cardiac glycoside concentration than the I50 concentration, it appears that this binding site is not related directly to the inhibitory site. Rat heart Na+K+-ATPase was inhibited by glycosides, but the I50 concentrations required were 30-1000 fold greater than those required with guinea pig heart ATPase. This observation, coupled with the fact that no high affinity binding to this enzyme could be demonstrated, indicates that the rat heart contains a Na+K+-ATPase which is extremely glycoside insensitive (lacks a high affinity inhibitory site). This may explain the relative lack of pharmacological action of glycosides on rat heart as compared to guinea pig heart.

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