Isoelectric focusing analysis of detergent extracted renal 11 beta-hydroxysteroid dehydrogenase.
11 beta-hydroxysteroid dehydrogenase (11-HSD, EC 1.1.1.146) from rat renal cortex microsomes was solubilized using several detergents, the most effective being Zwittergent 3-10 and Triton X-100. The activity ratio oxidation/reduction of the reversible reaction corticosterone in equilibrium 11-dehydrocoticosterone varied depending on the detergent used. We attribute this variation to direct effects of different detergents on enzyme kinetics. In contrast, comparable results obtained with liver 11-HSD have been attributed to the possibility of spatially separated 11-oxidase and 11-reductase activities. In order to test whether renal 11-HSD represents a uniform oxido-reductase as generally assumed, or a dual enzyme system as has been recently proposed an attempt was made to characterize 11-HSD solubilized from renal microsomal fractions using isoelectric focusing (IEF). When 11-HSD was extracted with 1% Triton X-100 (= partially solubilized fraction) a heterogenous peak pattern was obtained. In contrast, IEF of 11-HSD extracted with 10% Triton X-100 (= delipidated fraction) resulted in a single peak at about pH 5.9 with both oxidative and reductive activity at practically identical positions within the gels. From this observation we conclude that the degree of detergent solubilization of a membrane bound protein affects its amphoteric properties and that removal of membranous lipids is a prerequisite for the analysis of its behaviour. Since the more delipidated fraction of 11-HSD revealed only one activity peak the data are compatible with the uniform enzyme concept since oxidative and reductive activities of renal cortical 11-HSD could not be separated.
Schulz
W
W
Institut für Klinische Physiologie, Klinikum Steglitz, Free University of Berlin, F.R.G.
Lichtenstein
I
I
Siebe
H
H
Hierholzer
K
K
D042842:11-beta-Hydroxysteroid Dehydrogenases; D000818:Animals; D003902:Detergents; D006913:Hydroxysteroid Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D007525:Isoelectric Focusing / Q000379:methods; D007672:Kidney Cortex / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains
D003902:Detergents; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases