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Experimental Studies |
Department of Endocrinology, Klinikum Benjamin Franklin, Freie Universität Berlin, Germany
Address all correspondence and requests for reprints to: Sven Diederich, Department of Endocrinology, Klinikum Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany.
Recently, two distinct isoenzymes of 11ß-hydroxysteroid-dehydrogenase (11ß-HSD) have been cloned and characterized in several species: The isoenzyme 11ß-HSD-I is widely distributed, bidirectional, prefers NADP(H) and has a low substrate affinity. The isoenzyme 11ß-HSD-II seems to exclusively oxidize physiological glucocorticoids, uses NAD as cosubstrate, has high substrate affinity, and is only found in mineralocorticoid target tissues and the placenta. Synthetic steroids fluorinated in position 9, however, are rapidly reduced by human kidney cortex slices. We attempted to find out which isoenzyme is responsible for this unexpected reductase activity. We studied the 11ß-HSD activity towards cortisol (F)/cortisone (E) and dexamethasone (D)/11-dehydro-dexamethasone (DH-D) in microsomes prepared from human kidney cortex. For the reaction E to F (not for DH-D to D!), glucose-6-phosphate and glucose-6-phosphate-dehydrogenase had to be added as a NADH/NADPH-regenerating system.
Oxidation of F to E: NAD was the exclusively used cosubstrate; the affinity [Michael’s constant (Km) for F = 25.5 nmol/L] and the maximum velocity (Vmax = 22.9 nmol/mg/min) were high. Reduction of E to F: Without the NADH/NADPH-regenerating system, this reaction was very slow. With this system, the Km value for E was in the nanomolar range (80.6 nmol/L) and the Vmax value was very low (0.88 nmol/mg/min). The reaction was clearly NADH-preferring. For the steroid pair F/E, the quotient Vmaxoxidation/Vmaxreduction (=26) demonstrates an equilibrium far on the 11-keto side. Oxidation of D to DH-D: With NAD as the only used cosubstrate, the kinetic analysis is compatible with the existence of two different NAD-dependent isoenzymes: Km for D = 327 nmol/L, Vmax = 53.5 nmol/mg/min and Km for D = 81.2 nmol/L; Vmax = 20.4 nmol/mg/min. Reduction of DH-D to D: The maximum velocity was higher than that of all other reactions tested: Vmax = 226.0 nmol/mg/min. The reaction was exclusively NADH-dependent; the Km value for DH-D was 68.4 nmol/L. For D/DH-D, the ratio Vmaxoxidation/Vmaxreduction was 0.24, demonstrating a shift to reductase activity with the reaction equilibrium far on the 11-hydroxy side. The reaction F to E was inhibited by E, DH-D, and D in a concentration-dependent manner.
In conclusion, the cosubstrate dependence, the Km value of the oxidation of F and the product inhibition are in good correspondence with data for the cloned human 11ß-HSD-II. The NADH-dependent 11ß-reduction of E and especially of DH-D are inconsistent with the dogma of an unidirectional 11ß-HSD-II. The preference of D for the reductase reaction in human kidney slices is probably caused by the fluor atom in position 9, is catalyzed by 11ß-HSD-II, and leads to an activation of 11-DH-D to D in the human kidney.
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