Characterization of the adrenal 11 beta-hydroxylase in inbred salt-sensitive and resistant rats.
Rats have been bred for susceptibility and resistance to the hypertensive effect of dietary salt (S/JR & R/JR). S/JR have an abnormal adrenal steroid 11 beta,18-hydroxylase activity resulting in increased production of 18-OH-DOC. S/JR also produce increased quantities of 19-nor-DOC, which may be related, since the 11 beta,18-hydroxylase also catalyzes the 19-hydroxylation of DOC, a pivotal step in 19-nor-DOC biosynthesis. The purpose of the present studies was to further characterize the mutant S/JR adrenal steroid 11 beta,18-hydroxylase. Preliminary studies are also presented on assessing the renal 19-desmolase, the last step in 19-nor-DOC biosynthesis. Adrenal glands were harvested from R/JR and S/JR and prepared for incubation studies, protein immunoblotting, and RNA analysis. Kidneys from Sprague-Dawley rats were also used for isolated renal perfusion studies. Both S/JR and R/JR strains had a single immunostaining band for 11 beta,18-hydroxylase at 51,000 molecular weight which were equal in intensity. Both strains had a single RNA transcript at 4.3 kilobases which hybridized with equal intensity to the bovine cDNA (pB11-9). The Km for 11 beta- and 18-hydroxylation was identical within strains but was different between strains. The Km for 19-hydroxylation was different between S/JR and R/JR, and was much greater than 11 beta- and 18-hydroxylation in both strains. This suggests that the catalytic site for 19-hydroxylation is different than that for 11 beta- and 18-hydroxylation and that the S/JR enzyme binds the substrate with higher affinity than the R/JR enzyme. In the isolated perfusion studies the rat kidneys converted 80% of 19-oxo-DOC to either 19-oic-DOC or 19-nor-DOC. These data demonstrate that the difference in S/JR enzyme activity is probably due to a point mutation in the enzyme or to a change in a regulatory protein.
Griffing
G T
GT
Section of Endocrinology and Metabolism, Evans Memorial Department of Clinical Research, Boston University Medical Center, MA 02118.
Holbrook
M
M
Azar
S
S
Orme-Johnson
N R
NR
Melby
J C
JC
D000311:Adrenal Glands / Q000201:enzymology*; D000818:Animals; D015152:Blotting, Northern; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008297:Male; D012313:RNA / Q000032:analysis / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012044:Regression Analysis; D012964:Sodium / Q000494:pharmacology*; D013252:Steroid 11-beta-Hydroxylase / Q000235:genetics / Q000378:metabolism*
D012313:RNA; D012964:Sodium; D013252:Steroid 11-beta-Hydroxylase