A 3-hydroxy-⌬ 5 -C 27 -steroid dehydrogenase active in bile acid biosynthesis was purified from pig liver microsomes by solubilization with sodium cholate and by chromatography on DEAE-Sepharose, aminohexylSepharose, and blue Sepharose. The last step in the purification procedure was preparative isoelectric focusing in a Rotofor cell. The final enzyme preparation showed only one protein band upon SDS-polyacrylamide gel electrophoresis. The isoelectric point was estimated to about 7.0 and the apparent M r was 36,000.The purified enzyme catalyzed the conversion of 7␣-hydroxycholesterol, 7␣,25-dihydroxycholesterol, 7␣,27-dihydroxycholesterol, and 3,7␣-dihydroxy-5-cholestenoic acid into the corresponding 3-oxo-⌬ 4 compounds. The enzyme was inactive with C 19 and C 21 steroids as substrates. The enzyme was also inactive with C 27 steroids having the 7-hydroxy group in -instead of ␣-position. The K m was found to be 0.30 and 0.32 M with 7␣-hydroxycholesterol and 7␣,27-dihydroxycholesterol as substrates, respectively. NAD ؉ was the preferred cofactor. A monoclonal antibody raised against the 3-hydroxy-⌬ 5 -C 27 -steroid dehydrogenase was prepared. After coupling to Sepharose, the antibody was able to bind the dehydrogenase and to decrease the conversion of 7␣-hydroxycholesterol into 7␣-hydroxy-4-cholest-3-one by more than 90%. The N-terminal amino acid sequence was determined and found to be similar but not identical with those of known 3-hydroxy-⌬ 5 -steroid dehydrogenases active in steroid hormone biosynthesis. Thus, the purified enzyme active toward C 27 steroids in bile acid biosynthesis appears to represent a novel type of 3-hydroxy-⌬ 5 -steroid dehydrogenase.
More Swedish boys than girls used melatonin regularly. The boys started treatment earlier and more often combined regular use of melatonin with regular use of medication for ADHD. This indicates that girls and boys partly are prescribed melatonin for different reasons. About half of the younger children stayed on melatonin treatment for several years, while 90% of adolescents (15-19 years) concluded their treatment.
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