Rapid characterization of the major drug-metabolizing human hepatic cytochrome P-450 enzymes expressed in Escherichia coli.

The major drug-metabolizing human hepatic cytochrome P-450s (CYPs; CYP1A2, 2C9, 2C19, 2D6, and 3A4) coexpressed functionally in Escherichia coli with human NADPH-P-450 reductase have been validated as surrogates to their counterparts in human liver microsomes (HLM) using automated technology. The dealkylation of ethoxyresorufin, dextromethorphan, and erythromycin were all shown to be specific reactions for CYP1A2, CYP2D6, and CYP3A4 that allowed direct comparison with kinetic data for HLM. For CYP2C9 and CYP2C19, the kinetics for the discrete oxidations of naproxen and diazepam were compared to data obtained using established, commercial CYP preparations. Turnover numbers of CYPs expressed in E. coli toward these substrates were generally equal to or even greater than those of the major commercial suppliers [CYP1A2 (ethoxyresorufin), E. coli 0.6 +/- 0.2 min(-1) versus B lymphoblasts 0.4 +/- 0.1 min(-1); CYP2C9 (naproxen), 6.7 +/- 0.9 versus 4.9 min(-1); CYP2C19 (diazepam), 3.7 +/- 0.3 versus 0.2 +/- 0.1 min(-1); CYP2D6 (dextromethorphan), 4.7 +/- 0.1 versus 4.4 +/- 0.1 min(-1); CYP3A4 (erythromycin), 3 +/- 1.2 versus 1.6 min(-1)]. The apparent K(m) values for the specific reactions were also similar (K(m) ranges for expressed CYPs and HLM were: ethoxyresorufin 0.5-1.0 microM, dextromethorphan 1.3-5.9 microM, and erythromycin 18-57 microM), indicating little if any effect of N-terminal modification on the E. coli-expressed CYPs. The data generated for all the probe substrates by HLM and recombinant CYPs also agreed well with literature values. In summary, E. coli-expressed CYPs appear faithful surrogates for the native (HLM) enzyme, and these data suggest that such recombinant enzymes may be suitable for predictive human metabolism studies.

Effects of human cytochrome b5 on CYP3A4 activity and stability in vivo.

Cytochrome P450s (P450) form a superfamily of membrane-bound proteins that play a key role in the primary metabolism of both xenobiotics and endogenous compounds such as drugs and hormones, respectively. To be enzymically active, they require the presence of a second membrane-bound protein, NADPH P450 reductase, which transfers electrons from NADPH to the P450. Because of the diversity of P450 enzymes, much of the work on individual forms has been carried out on purified proteins, in vitro, which requires the use of complex reconstitution mixtures to allow the P450 to associate correctly with the NADPH P450 reductase. There is strong evidence from such reconstitution experiments that, when cytochrome b5 is included, the turnover of some substrates with certain P450s is increased. Here we demonstrate that allowing human P450 reductase, CYP3A4, and cytochrome b5 to associate in an in vivo-like system, by coexpressing all three proteins together in Escherichia coli for the first time, the turnover of both nifedipine and testosterone by CYP3A4 is increased in the presence of cytochrome b5. The turnover of testosterone was increased by 166% in whole cells and by 167% in preparations of bacterial membranes. The coexpression of cytochrome b5 also resulted in the stabilization of the P450 during substrate turnover in whole E. coli, with 109% of spectrally active CYP3A4 remaining in cells after 30 min in the presence of cytochrome b5 compared with 43% of the original P450 remaining in cells in the absence of cytochrome b5.

11Beta-hydroxysteroid dehydrogenase 1 in adipocytes: expression is differentiation-dependent and hormonally regulated.

11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD-1) catalyses the reversible metabolism of physiological glucocorticoids (cortisol, corticosterone) to inactive metabolites (cortisone, 11-dehydrocorticosterone), thus regulating glucocorticoid access to receptors. 11Beta-HSD-1 expression is regulated during development and by hormones in a tissue specific manner. The enzyme is highly expressed in liver, where it may influence glucocorticoid action on fuel metabolism, processes also important in adipose tissue. Here we show that 11beta-HSD-1 is expressed in white adipose tissue, in both the adipocyte and stromal/vascular compartments, and in the adipocyte cell lines 3T3-F442A and 3T3-L1. In these cells, 11beta-HSD-1 expression is induced upon differentiation into adipocytes and is characteristic of a 'late differentiation' gene, with maximal expression 6-8 days after confluence is reached. In intact 3T3-F442A adipocytes the enzyme direction is predominantly 11beta-reduction, activating inert glucocorticoids. The expression of 11beta-HSD-1 mRNA is altered in fully differentiated 3T3-F442A adipocytes treated with insulin, dexamethasone or a combination of the hormones, in an identical manner to glycerol-3-phosphate dehydrogenase (GPDH) mRNA (encoding a key enzyme in triglyceride synthesis and a well-characterised marker of adipocyte differentiation). The demonstration of 11beta-HSD-1 expression in adipocytes and its predominant reductase activity in intact 3T3-F442A adipocytes suggests that 11beta-HSD-1 may play an important role in potentiating glucocorticoid action in these cells. 3T3-F442A and 3T3-L1 represent useful model systems in which to examine the factors which regulate 11beta-HSD-1 gene expression and the role of 11beta-HSD-1 in modulating glucocorticoid action in adipose tissue.

The in vivo regulation of liver and kidney glucose-6-phosphatase by dexamethasone.

The microsomal glucose-6-phosphatase enzyme is situated with its active site inside the lumen of the endoplasmic reticulum and for normal enzyme activity in vivo, transport systems are needed for the substrates and products of the enzyme. Most studies of glucose-6-phosphatase have been carried out on the liver enzyme and relatively little is known about the regulation of the kidney glucose-6-phosphatase enzyme system. Here we demonstrate that the liver and kidney glucose-6-phosphatase systems are regulated differently by dexamethasone and that dexamethasone acts on both the glucose-6-phosphatase enzyme and T1 its associated glucose-6-phosphate transport protein.

The sequence of 5' flanking DNA from the mouse 11 beta-hydroxysteroid dehydrogenase type 1 gene and analysis of putative transcription factor binding sites.

11 beta-Hydroxysteroid dehydrogenase type 1 (E.C. 1.1.1.146) (11 beta-HSD 1) is a key enzyme in the metabolism of glucocorticoids, catalysing their interconversion with physiologically inert 11-keto metabolites. To identify transcription factors which may be involved in the regulation of expression of mouse 11 beta-HSD 1 we have isolated and sequenced the 5' flanking DNA to 900 bp upstream from the major transcription start site.

11 beta-hydroxysteroid dehydrogenase type 1 expression in 2S FAZA hepatoma cells is hormonally regulated: a model system for the study of hepatic glucocorticoid metabolism.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) is a key enzyme in glucocorticoid metabolism, catalysing the conversion of active glucocorticoids into their inactive 11-keto metabolites, thus regulating glucocorticoid access to intracellular receptors. The type 1 isoform (11 beta-HSD 1) (EC 1.1.1.146) is widely distributed, with particularly high levels in liver, where accumulating evidence suggests that it acts as an 11 beta-reductase, regenerating active glucocorticoids. Investigation of the function and regulation of 11 beta-HSD 1 in liver has been hampered by the lack of hepatic cell lines which express 11 beta-HSD 1. Here, we describe 11 beta-HSD 1 mRNA expression and activity in 2S FAZA cells, a continuously cultured rat liver cell line. In intact 2S FAZA cells 11 beta-HSD 1 acts predominantly as a reductase, with very low dehydrogenase activity. In 2S FAZA cells 11 beta-HSD 1 activity and mRNA expression are regulated by hormones, with dexamethasone increasing activity and insulin, forskolin and insulin-like growth factor 1 decreasing it. Transfection of 2S FAZA cells with a luciferase reporter gene driven by the proximal promoter of the rat 11 beta-HSD 1 gene demonstrates that sequences which can mediate the responses to insulin, dexamethasone and forskolin all lie within 1800 bp of the transcription start site.

A comparison of the renal and hepatic microsomal glucose-6-phosphatase enzymes.

The liver glucose-6-phosphatase enzyme has been extensively characterized and relatively little is known about the renal microsomal glucose-6-phosphatase enzyme. The reason for lack of study of the renal glucose-6-phosphatase enzyme is that it has been assumed to be the same as the liver enzyme. Immunoblotting with antibodies raised against the liver enzyme revealed differences in apparent molecular weight and antigenicity between the liver and kidney glucose-6-phosphatase enzyme proteins. Characterization of the activity of the renal glucose-6-phosphatase enzyme also showed that it is regulated differently to the liver enzyme in some metabolic states. This implies that the renal and liver glucose-6-phosphatase enzymes may have different roles.

The in vivo regulation of hepatic and renal glucose-6-phosphatase by thyroxine.

The hepatic and renal microsomal glucose-6-phosphatase enzymes are situated with their active site in the lumen of the endoplasmic reticulum and for normal enzyme activity in vivo transport systems are needed for the substrates and products of the enzyme. We have shown that thyroxine activates the kidney glucose-6-phosphatase enzyme and the liver glucose 6-phosphate transport systems. In contrast, in hypophysectomised and adrenalectomised animals, thyroxine activates the transport systems and the enzyme in both liver and kidney.

The human adrenal microsomal glucose-6-phosphatase system.

Microsomal glucose-6-phosphatase (EC 3.1.3.9) is a multicomponent enzyme system traditionally thought only to be present in gluconeogenic tissues. The enzyme is associated with transport systems, for its substrate glucose-6-phosphate, and its products phosphate and glucose. It has been shown, using immunohistochemical methods and monospecific antibodies, that the component proteins of the enzyme system are present in human embryonic and fetal adrenal gland and are predominantly located in the fetal zone with lesser reactivities in the definitive zone. In addition, specific glucose-6-phosphatase activity was shown, and the rates of entry of glucose-6-phosphate, phosphate, and glucose into microsomes isolated from human fetal adrenals were measured. Although the complete enzyme system is present, the ratio of the component activities and comparison with human fetal and adult liver indicate that the regulation of the adrenal and liver glucose-6-phosphatase systems is different. In the human postnatal adrenal, immunoreactivies to the protein components decrease dramatically and are confined predominantly to the zona reticularis, suggesting a specialized role for adrenal glucose-6-phosphatase in fetal life.

Effects of hypophysectomy and thyroxine on the expression of hepatic oestrogen, hydroxysteroid and phenol sulphotransferases.

Sulphation in rats, and other mammals, is carried out by a family of sulphotransferase isoenzymes, which can be further subdivided into oestrogen, hydroxysteroid and phenol sulphotransferases. We have examined the effects of hypophysectomy on the activity and expression of representative members of the three major sulphotransferase sub-families in male Wistar rat liver cytosols, and have found that the different sub-families are subject to differential regulation by pituitary hormones. Our data show that in male rat liver hydroxysteroid sulphotransferases activity was increased, oestrogen sulphotransferases activity was not altered and phenol sulphotransferases activity was reduced. Further, we have studied the effect on sulphotransferase expression of administration of thyroxine and dexamethasone to hypophysectomized rats. Treatment of hypophysectomized rats with thyroxine virtually abolished oestrogen sulphotransferase activity in male rat liver but had no effect on hydroxysteroid sulphotransferase or phenol sulphotransferase activity. Treatment of hypophysectomized rats with dexamethasone had no effect on sulphotransferase activities. Quantitative immunoblot analysis of liver cytosols showed that these changes in enzyme activity were related to changes in levels of the respective enzyme proteins.

Cloning and expression of a hepatic microsomal glucose transport protein. Comparison with liver plasma-membrane glucose-transport protein GLUT 2.

Antibodies raised against a 52 kDa rat liver microsomal glucose-transport protein were used to screen a rat liver cDNA library. Six positive clones were isolated. Two clones were found to be identical with the liver plasma-membrane glucose-transport protein termed GLUT 2. The sequence of the four remaining clones indicates that they encode a unique microsomal facilitative glucose-transport protein which we have termed GLUT 7. Sequence analysis revealed that the largest GLUT 7 clone was 2161 bp in length and encodes a protein of 528 amino acids. The deduced amino acid sequence of GLUT 7 shows 68% identity with the deduced amino acid sequence of rat liver GLUT 2. The GLUT 7 sequence is six amino acids longer than rat liver GLUT 2, and the extra six amino acids at the C-terminal end contain a consensus motif for retention of membrane-spanning proteins in the endoplasmic reticulum. When the largest GLUT 7 clone was transfected into COS 7 cells the expressed protein was found in the endoplasmic reticulum and nuclear membrane, but not in the plasma membrane. Microsomes isolated from the transfected COS 7 cells demonstrated an increase in their microsomal glucose-transport capacity, demonstrating that the GLUT 7 clone encodes a functional endoplasmic-reticulum glucose-transport protein.

Cross-reacting antigens between Mycoplasma ovipneumoniae and other species of mycoplasma of animal origin, shown by ELISA and immunoblotting with reference antisera.

Using enzyme-linked immunosorbent assays with Mycoplasma ovipneumoniae as antigen, the cross-reactivity of antigens between this species and 22 other mycoplasma species was examined using reference polyclonal antisera. Significant cross-reactivity with M. ovipneumoniae was demonstrated by five species, only, viz. M. bovoculi, M. dispar, M. flocculare, M. hyopneumoniae and M. hyorhinis. Using one-dimensional SDS-PAGE and immunoblotting techniques with homologous and heterologous antisera, cross-reacting antigens of M. dispar, M. flocculare, M. hyopneumoniae and M. ovipneumoniae were further investigated. Cross-reacting antigens with apparent molecular weights of 64, 44 and 32 kDa were common to all and a 184 kDa cross-reacting antigen occurred in all except M. ovipneumoniae. Further cross-reacting antigens (one-way and two-way) between two of the four species are reported. Four monoclonal antibodies against different antigens of M. ovipneumoniae did not recognise any antigen in the other three species examined.