Abstract
We have expressed in Escherichia coli functionally active proteins encoded by two human cDNAs that were isolated previously by using rat 3 alpha-hydroxysteroid dehydrogenase cDNA as the probe. The expressed proteins catalyzed the interconversion between 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha,17 beta-diol. Therefore, we name these two enzymes type I and type II 3 alpha-hydroxysteroid dehydrogenases. The type I enzyme has a high affinity for dihydrotestosterone, whereas the type II enzyme has a low affinity for the substrate. The tissue-specific distribution of these two enzymes was determined by reverse transcription polymerase chain reaction using gene-specific oligonucleotide primers. The mRNA transcript of the type I enzyme was found only in the liver, whereas that of the type II enzyme appeared in the brain, kidney, liver, lung, placenta, and testis. The structure and sequence of the genes encoding these two 3 alpha-hydroxysteroid dehydrogenases were determined by analysis of genomic clones that were isolated from a lambda EMBL3 SP6/T7 library. The genes coding for the type I and type II enzymes were found to span approximately 20 and 16 kilobase pairs, respectively, and to consist of 9 exons of the same sizes and boundaries. The exons range in size from 77 to 223 base pairs (bp), whereas the introns range in size from 375 bp to approximately 6 kilobase pairs. The type I gene contains a TATA box that is located 27 bp upstream of multiple transcription start sites. In contrast, the type II gene contains two tandem AP2 sequences juxtaposed to a single transcription start site.
Highlights
From the Wepartment of Pediatrics, Cornell University Medical College, New York, New York 10021 and the §Department of Drug Metabolism, Merck, Sharp & Dohme Research Laboratories, Rahway, New Jersey 07065
We have expressed in Escherichia coli functionally active proteins encoded by two human cDNAs that were isolated previously by using rat 3a-hydroxysteroid dehydrogenase cDNA as the probe
The structure and sequence ofthe genes encoding these two 3a-hydroxysteroid dehydrogenases were determined by analysis of genomic clones that were isolated from a A EMBL3 SP6ff7library
Summary
3a-HSD, 3a-hydroxysteroid dehydrogenase; PCR, polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction; kb, kilobase pair(s); bp, base pairs; BABP, bile acid-binding protein. Teins recently identified in rats and humans in our laboratory [7] These enzymes catalyze the conversion of aldehydes and ketones to alcohols by utilizing NADH and/or NADPH as the cofactor and exist in cellular cytoplasm as monomeric 34-36kDa proteins. The 3ahydroxy A-ring-reduced pregnane steroids (allopregnanolone) have been shown to exert sedative, hypnotic, and anesthetic effects when they were administered to animals [13] These tetrahydrosteroids produce a number of other behavioral effects, such as anticonflict, anticonvulsant, and analgesic actions [14]. Administration ofprogesterone to healthy humans induced changes in fatigue and in delayed verbal recall; these changes correlate with the levels of 3a-tetrahydro-metabolites These studies support the hypothesis that 3a-tetrahydrosteroids are endogenous modulators ofthe gammaaminobutyric acid receptor [16]. We compare the enzyme characteristics and describe the gene structure of these steroid hormone-metabolizing enzymes
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