Graduate Thesis Or Dissertation
 

Site-specific labeling of the cardiac glycoside receptor Na⁺,K⁺-ATPase : synthesis of a C-17 side group photoaffinity label ; Relationship of cardioactive steroid structure and conformation to biological activity

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  • In an effort to isolate and characterize cardiac glycosides C-17 binding site(s), we have synthesized different affinity and photoaffinity C-17 side chain probes. These probes were screened for their ability to inhibit Na⁺,K⁺-ATPase enzyme, a pharmacological receptor of cardiac glycosides. The enzyme was isolated from hog kidney. Screening of these compounds was undertaken in search of the most active and stable probe to be used in labeling studies. From all the compounds synthesized, affinity label compound 39, although having good activity (I₅₀ = 6.7 x 10⁻⁷M), was found to be a non-specific inactivator, binding at a site different from that of ouabain. Among photoaffinity label compounds (45, 46, 47, 48, 50, 53, 56, 59), 56 and 59 were the most active (I₅₀ = 3 x 10⁻⁷ and 1.07 x 10⁻⁷M respectively). Both compounds were made radioactive with tritium. Specific activities obtained were 2.20 Ci/mmole for 56 and 1.89 Ci/mmole for 59. Since 59 was the most active, it is currently being used to label C-17 side chain binding site(s) on Na⁺,K⁺-ATPase. Preliminary results obtained from the binding studies suggested that tritiated 59 binds at a site specific to and blocked by ouabain. However, there are some non-specific sites which ouabain cannot block. Research in progress is seeking the isolation and characterization of C-17 side chain binding site(s). Part II of this study shows that the carbonyl oxygen position and activity relationship initially demonstrated in rat brain enzyme applies to Na⁺,K⁺-ATPase derived from other tissues (cat heart, hog kidney, guinea pig heart); the relationship also holds in different binding conditions (Type I, Type II) of cardiac glycosides to the ATPase enzyme. Part II also demonstrates that the carbonyl oxygenactivity relationship can be used to explain digitalis-induced cardiac inotropism.
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