John M. Trant

3β-Hydroxysteroid dehydrogenase/Δ5→4-isomerase expression in rat and characterization of the testis isoform

Abstract The isolation, cloning and expression of a DNA insert complementary to mRNA encoding rat testis 3β-hydroxysteroid dehydrogenase/Δ5→4-isomerase (3β-HSD) is reported. The insert contains an open reading frame encoding a protein of 373 amino acids, which exhibits 73% and 78% identity to the cDNA encoding the human placental form at the amino acid and nucleotide levels respectively. Northern blot analysis of total RNA of rat tissues using as probe a specific radiolabeled cDNA insert encoding rat testis 3β-HSD demonstrated high levels of 1.6 kb mRNA species in ovary, adrenal and Leydig tumor, with lower but detectable message in testis and adult male liver, while the probe also hybridized to a 2.1 kb mRNA species in liver. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to 3β-HSD present in H540 Leydig tumor cell homogenate and human placental microsomal 3β-HSD, as detected by immunoblot analysis, and catalyzed the conversion of pregnenolone to progesterone, 17α-hydroxypregnenolone to 17α-hydroxyprogesterone, and dehydroepiandrosterone to androstenedione. Transfected COS cell homogenates, supplemented with NAD+, but not NADP+, converted pregnenolone to progesterone and dehydroepiandrosterone to androstenedione with apparent Km values of 0.13 and 0.09 μM, respectively. Immunoblot analysis of various rat tissues using a polyclonal antibody directed against human placental 3β-HSD, in addition to immunoreactivity in the adrenal and testis, demonstrated immunoreactive 3β-HSD protein in adult male liver, but not in adult female or fetal liver. We conclude that while one gene product is highly expressed in testicular Leydig cells, and probably adrenal and ovary, accounting for their 3β-HSD content, a 3β-HSD is also expressed in liver in a sex-specific manner.

Expression of a full-length cDNA encoding bovine adrenal cytochrome P450C21

Two full-length cDNA clones encoding bovine adrenocortical P450C21 have been constructed in a eukaryotic expression vector using partial-length cDNAs whose structures have been previously reported. Following expression of these cDNAs in COS 1 cells, the substrate specificity of P450C21 was determined. Of the 18 steroids tested, progesterone, 17 alpha-hydroxyprogesterone, and 11 beta,17 alpha-dihydroxyprogesterone were found to be the only steroids to serve as substrates for this adrenal enzyme, a much higher degree of substrate specificity than has been reported for a hepatic 21-hydroxylase. The Vmax for 17 alpha-hydroxyprogesterone was 2.5 times greater than that for progesterone, whereas delta 5-steroids were unable to serve as substrate for this enzyme. A difference between the two cDNAs is located at amino acid 401 where one resultant enzyme contains tyrosine while the other contains histidine. This amino acid difference appears to have no effect on the kinetic properties of adrenal P450C21.

Incorporation of steroidogenic pathways which produce cortisol and aldosterone from cholesterol into nonsteroidogenic cells

Cortisol production from cholesterol requires the activity of four steroid hydroxylases: cholesterol side chain cleavage cytochrome P-450 (P-450scc), 17 alpha-hydroxylase cytochrome P-450 (P-45017 alpha), 21-hydroxylase cytochrome P-450 (P-450C21) and 11 beta-hydroxylase cytochrome P-450 (P-45011 beta). We have previously shown that transformed, nonsteroidogenic COS 1 cells derived from monkey kidney are a useful system for expression of various forms of cytochrome P-450. The present study shows that COS 1 cell cultures multiply transfected with six plasmids containing all four steroid hydroxylases, 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase (3 beta HSD) and adrenodoxin produce cortisol and aldosterone when 22(R)-hydroxycholesterol is supplied to the system. When pregnenolone is used as substrate, various intermediate metabolites are detected at different time points further establishing the incorporation of complete functional steroidogenic pathways into the nonsteroidogenic cell cultures. Since the first and the last reactions in these pathways take place in the mitochondrion, the movement of various intermediate metabolites from mitochondrion to endoplasmic reticulum and back to mitochondrion occurs in and between COS 1 cells.

Expression of P-450 enzyme activities in heterologous cells by transfection.

The product of the A gene of simian virus 40 (SV40), T antigen, is synthesized and accumulates in the nucleus of infected cells. This regulartory protein acts to initiate viral DNA replication as well as regulating SV40 gene transcription (Tjian 1981). Following transformation of CV-1 cells with an origin-defective mutant of SV40, the resultant monkey kidney cell line (called COS 1) produces T antigen (Gluzman 1981). Transfection of COS 1 cells with plasmid vectors which contain an SV40 origin of replication leads to replication of the plasmid DNA under the influence of T antigen. Accordingly the number of plasmid vectors is amplified in transfected COS 1 cells and the amplified plasmid DNA can then be transcribed leading to production of relatively large quantities of RNA derived from the plasmid. This RNA can, of course, be translated by the endogenous protein synthetic machinery in the COS 1 cells. When the plasmid vector contains a cDNA insert encoding a specific protein, readily detectable quantities of the protein encoded by the insert can be produced in COS 1 cells. A convenient vector for such expression studies is the pcD vector constructed by Okayama and Berg (1982) which contains an SV40 origin of replication and SV40 promotor sequence. A similar vector (pSVL) can be purchased from Pharmacia. pSVL contains a limited multiple cloning site which facilitates insertion of different cDNAs into this expression vector.

“Designer Membranes”: Construction of a Cell Containing Multiple Membrane-Bound Cytochromes P450

Publisher Summary This chapter discusses designer membranes and its construction of a cell containing multiple membrane-bound cytochromes p450. The techniques of molecular biology offer the opportunity to introduce foreign DNA into naive cells to express enzymatically active proteins that can be tested in situ . Indeed, one is able to introduce simultaneously the DNAs for a number of different soluble and membrane-bound proteins, and thereby construct a functional metabolic pathway in a cell not programmed for such activities. Cytochrome P450 is best characterized as the catalyst for many monooxygenase—mixed function oxidase—reactions. For these reactions, a P450 serves as the central agent for the binding of a molecule of substrate, the acceptance of reducing equivalents provided by electrons transferred from reduced pyridine nucleotide via an abbreviated electron transport chain, and the activation of a molecule of molecular oxygen. The use of heterologous expression of enzymes, in particular membrane-bound enzymes, at present offers the possibility to contribute new types of data needed for the better understanding the functioning of enzymes in the environment of a cell.