P450 oxidoreductase deficiency: a new disorder of steroidogenesis affecting all microsomal P450 enzymes

Combined partial deficiency of 17alpha-hydroxylase and 21-hydroxylase activities was first described in 1985; however the genes for P450c17 and P450c21 in these patients lack mutations. In 1986 we postulated that this disorder might be due to mutations in P450 oxidoreductase (POR), the flavoprotein that donates electron to these and all other microsomal P450 enzymes, but this hypothesis was not tested until the POR gene sequence became available through the genome database. We found five POR missense mutations in our first four patients. In vitro assays of the activities of these mutations showed that the standard assay of POR activity, reduction of cytochrome c, correlated poorly with the patients' phenotypes, but that assays of POR-supported 17alpha-hydroxylase and 17,20 lyase activities correlated well. POR deficiency is a new disorder of adrenal and gonadal steroidogenesis that affects all microsomal cytochrome P450 enzymes, hence may have important implications for genetic differences in drug metabolism.

Angiotensin I-converting enzyme-gene-polymorphism: relationship to albumin excretion and blood pressure in pediatric patients with type-I-diabetes mellitus

The purpose of this study was the evaluation of a predictive genetic marker for nephropathy and hypertension in patients with type-I-diabetes mellitus (IDDM). The study was performed on 247 pediatric patients with IDDM. The mean age was 15.5 years (range 3.1-29.3), the mean duration of diabetes was 7.6 years (range 0.1-25.7). Age-related blood pressure and nocturnal albumin excretion rate were compared with the insertion/deletion-(I/D) polymorphism of the angiotensin-I converting enzyme gene. The genotype distribution did not differ significantly between IDDM patients (ID 48%, D 28%, I 24%) and the control group (ID 44%, D 37%, I 19%). Neither in the entire group, nor in patients with IDDM for more than 5 years, was a correlation found bet-ween allele distribution and albumin excretion rate. No correlation was found between genotype and blood pressure. When patients with a chronological age above 12 years were analysed separately, the genotype distribution between the groups with normal and elevated blood pressure showed no significant difference. The previously reported association of the I/D-polymorphism with nephropathy could not be confirmed in this study. The development of microalbuminuria, nephropathy and hypertension will be followed in our pediatric patients.

Dimerization of β-site β-amyloid precursor protein-cleaving enzyme

Cleavage of the beta-amyloid precursor protein (APP) by the aspartyl protease beta-site APP-cleaving enzyme (BACE) is the first step in the generation of the amyloid beta-peptide, which is deposited in the brain of Alzheimer's disease patients. Whereas the subsequent cleavage by gamma-secretase was shown to originate from the cooperation of a multicomponent complex, it is currently unknown whether in a cellular environment BACE is enzymatically active as a monomer or in concert with other proteins. Using blue native gel electrophoresis we found that endogenous and overexpressed BACE has a molecular mass of 140 kDa instead of the expected mass of 70 kDa under denaturing conditions. This suggests that under native conditions BACE exists as a homodimer. Homodimerization was confirmed by co-immunoprecipitation of full-length BACE carrying different epitope tags. In contrast, the soluble active BACE ectodomain was exclusively present as a monomer both under native and denaturing conditions. A domain analysis revealed that the BACE ectodomain dimerized as long as it was attached to the membrane, whereas the cytoplasmic domain and the transmembrane domain were dispensable for dimerization. By adding a KKXX-endoplasmic reticulum retention signal to BACE, we demonstrate that dimerization of BACE occurs already before full maturation and pro-peptide cleavage. Furthermore, kinetic analysis of the purified native BACE dimer revealed a higher affinity and turnover rate in comparison to the monomeric soluble BACE. Dimerization of BACE might, thus, facilitate binding and cleavage of physiological substrates.

Polymorphisms, linkage and mapping of four enzyme loci in the fish genus Xiphophorus (Poeciliidae).

Electrophoretic variants at four additional enzyme loci--two esterases (Est-2, Est-3), retinal lactate dehydrogenase (LDH-1) and mannose phosphate isomerase (MPI)--among three species and four subspecies of fish of the genus Xiphophorus were observed. Electrophoretic patterns in F1 hybrid heterozygotes confirmed the monomeric structures of MPI and the esterase and the tetrametric structure of LDH in these fishes. Variant alleles of all four loci displayed normal Mendelian segregation in backcross and F2 hybrids. Recombination data from backcross hybrids mapped with Haldane's mapping function indicate the four loci to be linked as Est-2--0.43--Est3--0.26--LDH-1--0.19--MPI. Significant interference was detected and apparently concentrated in the Est-3 to MPI region. No significant sex-specific differences in recombination were observed. This group (designated linkage group II) was shown to assort independently from the three loci of linkage group I (adenosine deaminase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase) and from glyceraldehyde-3-phosphate dehydrogenase and two isocitrate dehydrogenase loci. Evidence for conservation of the linkage group, at least in part, in other vertebrate species is presented.

Three linked enzyme loci in fishes: implications in the evolution of vertebrate chromosomes.

A three-point linkage group comprised of loci coding for adenosine deaminase (ADA), glucose-6-phosphate dehydrogenase (G6PDH), and 6-phospho-gluconate dehydrogenase (6PGD) is described in fish of the genus Xiphophorus (Poeciliidae). The alleles at loci in this group were shown to assort independently from the alleles at three other loci--isocitrate dehydrogenase 1 and 2, and glyceraldehyde-3-phosphate dehydrogenase 1. Alleles at the latter three loci also assort independently from each other. Data were obtained by observing the segregation of electrophoretically variant alleles in reciprocal backcross hybrids derived from crosses between either X. helleri guentheri or X. h. strigatus and X. maculatus. The linkage component of chi2 was significant (less than 0.01) in all crosses, indicating that the linkage group is conserved in all populations of both species of Xiphophorus examined. While data from X. h. guentheri backcrosses indicate the linkage relationship ADA--6%--G6PDH--24%--6PGD, and ADA--29%--6PGD (30% when corrected for double crossovers), data from backcrosses involving strigatus, while supporting the same gene order, yielded significantly different recombination frequencies. The likelihood of the difference being due to an inversion could not be separated from the possibility of a sex effect on recombination in the present data. The linkage of 6PGD and G6PDH has been shown to exist in species of at least three classes of vertebrates, indicating the possibility of evolutionary conservation of this linkage.

Intracellular phosphorylation of ornithine decarboxylase: Regulation of enzyme activity

Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis exists as two major and one minor ionic form in the macrophage cell line, RAW 264. The forms have the same molecular weight, 55,000, but differ in their isoelectric points, 5.2, 5.1, and 4.9-5.0. The hypothesis that phosphorylation accounts for the differences in the two major ionic forms and that phosphorylation is involved in the regulation of enzyme activity was investigated. Metabolic-radiolabeling of cells with $\sp{32}$P-orthophosphate indicated that only one of the major forms of the protein can be explained by phosphorylation: treatment of purified ODC with alkaline phosphatase resulted in the loss of the phosphorylated form of the protein, pl 5.1, with a concomitant increase in the unphosphorylated, pl 5.2, form of the protein. Characterization of the phosphorylation sites showed that serine was the present. Tryptic digests of $\sp{32}$P-labeled ODC, analyzed by either two dimensional tryptic peptide mapping or reverse-phase HPLC, contained only one major radiolabeled peptide.^ The role phosphorylation plays in the regulation of enzyme activity was also investigated. Treatment of purified ODC with alkaline phosphatase resulted in the loss of enzyme activity. A positive linear correlation exists between enzyme activity and the amount of phosphorylated form of the protein present.^ To ascertain if the two major forms of the protein were also found in animal cells, ODC was immunoprecipitated from various rat tissues, fractionated by isoelectric focusing, and detected by immunoblotting. ODC was present in rat tissues in a single major form, which comigrated with the pl 5.1, phosphorylated form of ODC present in RAW 264 cell.^ This study concludes that ODC exists as a phosphorylated form, pl 5.1, and an unphosphorylated form, pl 5.2 in RAW 264 cells. The amount of the phosphorylated form of ODC correlates well with the enzyme activity. ^

Cytochrome P450 in mammalian brain: Purification, anatomical distribution and regulation of the cytochrome P450 monooxygenase system in rat brain

The cytochrome P450 (P450) monooxygenase system plays a major role in metabolizing a wide variety of xenobiotic as well as endogenous compounds. In performing this function, it serves to protect the body from foreign substances. However, in a number of cases, P450 activates procarcinogens to cause harm. In most animals, the highest level of activity is found in the liver. Virtually all tissues demonstrate P450 activity, though, and the role of the P450 monooxygenase system in these other organs is not well understood. In this project I have studied the P450 system in rat brain; purifying NADPH-cytochrome P450 reductase (reductase) from that tissue. In addition, I have examined the distribution and regulation of expression of reductase and P450 in various anatomical regions of the rat brain.^ NADPH-cytochrome P450 reductase was purified to apparent homogeneity and cytochrome P450 partially purified from whole rat brain. Purified reductase from brain was identical to liver P450 reductase by SDS-PAGE and Western blot techniques. Kinetic studies utilizing cerebral P450 reductase reveal Km values in close agreement with those determined with enzyme purified from rat liver. Moreover, the brain P450 reductase was able to function successfully in a reconstituted microsomal system with partially purified brain cytochrome P450 and with purified hepatic P4501A1 as measured by 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation. These results indicate that the reductase and P450 components may interact to form a competent drug metabolism system in brain tissue.^ Since the brain is not a homogeneous organ, dependent upon the well orchestrated interaction of numerous parts, pathology in one nucleus may have a large impact upon its overall function. Hence, the anatomical distribution of the P450 monooxygenase system in brain is important in elucidating its function in that organ. Related to this is the regulation of P450 expression in brain. In order to study these issues female rats--both ovariectomized and not--were treated with a number of xenobiotic compounds and sex steroids. The brains from these animals were dissected into 8 discrete regions and the presence and relative level of message for P4502D and reductase determined using polymerase chain reaction. Results of this study indicate the presence of mRNA for reductase and P4502D isoforms throughout the rat brain. In addition, quantitative PCR has allowed the determination of factors affecting the expression of message for these enzymes. ^

The angiotensin-converting enzyme insertion/deletion polymorphism and its associations with carotid artery wall thickness and coronary heart disease: The atherosclerosis risk in communities study

Coronary heart disease (CHD) is the leading cause of death in the United States. Recently, renin-angiotensin system (RAS) was found associated with atherosclerosis formation, with angiotensin II inducing vascular smooth muscle cell growth and migration, platelet activation and aggregation, and stimulation of plasminogen activator inhibitor-1. Angiotensin II is converted from angiotensin I by angiotensin I-converting enzyme (ACE) and this enzyme is mainly genetically determined. The ACE gene has been assigned to chromosome 17q23 and an insertion/deletion (I/D)polymorphism has been characterized by the presence/absence of a 287 bp fragment in intron 16 of the gene. The two alleles form three genotypes, namely, DD, ID and II and the DD genotype has been linked to higher plasma ACE levels and cell ACE activity.^ In this study, the association between the ACE I/D polymorphism and carotid artery wall thickness measured by B-mode ultrasound was investigated in a biracial sample, and the association between the gene and incident CHD was investigated in whites and if the gene-CHD association in whites, if any, was due to the gene effect on atherosclerosis. The study participants are from the prospective Atherosclerosis Risk in Communities (ARIC) Study, including adults aged 45 to 65 years. The present dissertation used a matched case-control design for studying the associations of the ACE gene with carotid artery atherosclerosis and an unmatched case-control design for the association of the gene with CHD. A significant recessive effect of the D allele on carotid artery thickness was found in blacks (OR = 3.06, 95% C.I: 1.11-8.47, DD vs. ID and II) adjusting for age, gender, cigarette smoking, LDL-cholesterol and diabetes. No similar associations were found in whites. The ACE I/D polymorphism is significantly associated with coronary heart disease in whites, and while stratifying data by carotid artery wall thickness, the significant associations were only observed in thin-walled subgroups. Assuming a recessive effect of the D allele, odds ratio was 2.84 (95% C.I:1.17-6.90, DD vs. ID and II) and it was 2.30 (95% C.I:1.22-4.35, DD vs. ID vs. II) assuming a codominant effect of the D allele. No significant associations were observed while comparing thick-walled CHD cases with thin-walled controls. Following conclusions could be drawn: (1) The ACE I/D polymorphism is unlikely to confer appreciable increase in the risk of carotid atherosclerosis in US whites, but may increases the risk of carotid atherosclerosis in blacks. (2) ACE I/D polymorphism is a genetic risk factor for incident CHD in US whites and this effect is separate from the chronic process of atherosclerosis development. Finally, the associations observed here are not causal, since the I/D polymorphism is in an intron, where no ACE proteins are encoded. ^

CYTOCHROME P450 MEDIATED REDUCTIVE DEHALOGENATION OF HEXACHLOROBENZENE

The role of the cytochrome (CYT) P-450 mixed-function oxidase (MFO) in the biotransformation of hexachlorobenzene (HCB) was investigated, since in vivo interaction between this enzyme and chemical is very probable. HCB is a type I substrate with (Fe('3+)) CYT P-450 isozymes present in untreated, b-naphthoflavone (BNF) and phenobarbital (PB) induced rat liver microsomes. HCB dependent and saturable type I binding titrations yield spectral dissociation constants (K(,s)) of 180 and 83 uM for the isozymes present in untreated and PB induced microsomes, respectively. Purified CYT P-450b, the major isozyme induced by PB, produces HCB dependent and saturable type I spectra with a K(,s) of 0.38 uM.^ CYT P-450 mediated reductive dehalogenation occurs in microsomes and purified/reconstituted MFO systems and produces pentachlorobenzene (PCB) as the initial and major metabolite under both aerobic and anaerobic conditions. In microsomal reactions secondary metabolism of PCB occurs in the presence of oxygen. Pentachlorophenol (PCP) is produced only in aerobic reactions with PB induced microsomes with a concomitant decrease in PCB production. PCP is not detected in aerobic reactions with BNF induced microsomes, although PCB production is decreased compared to anaerobic conditions. A reaction scheme for the production of phenolic metabolities from PCB is deduced.^ CYT P-450 dependent and NADPH independent modes of PCB production occur with purified/reconstituted MFO systems and are consistent with dehalogenation pathways observed with microsomal experiments. The NADPH independent production of PCB requires native microsomal or purified MFO protein components and may be the result of nucleophilic displacement of a chlorine atom from HCB mediated or coupled with redox active functions (primary, secondary, tertiary and quarternary structures) of the proteins. CYT P-450 dependent production of PCB from HCB is isozyme dependent: CYT P-450c = CYT P-450d > CYT P-450a > CYT 450b. The low apparent specific activity may be due to non-optimal reconstitution conditions (e.g., isozyme choice and requirement of other microsomal elecron transport components) and secondary metabolism of PCB and the phenols derived from PCB. CYT P-450 mediated dehalogenation may be catalyzed through attack, by the iron oxene (postulated intermediate of CYT P-450 monooxygenations), at the chlorines of HCB instead of the aromatic nucleus. (Abstract shortened with permission of author.) ^

IN VITRO METABOLISM OF 6-THIOGUANINE IN RAT SUBCELLULAR FRACTIONS

The metabolism of the antitumor agent 6-thioguanine (TG, NSC-752) by rat liver was studied in vitro. Livers from adult male Sprague-Dawley rats were homogenized and the "liver homogenate" was subjected to differential centrifugation to obtain the "10,000 x g pellet", the "post-mitochondrial fraction", the "cytosol fraction", and the "microsomes". The homogenity of each fraction was estimated by appropriate marker enzyme assays. To delineate the in vitro metabolism of TG by rat liver, 0.2 mM of {8-('14)C}TG was incubated with different subcellular fractions in KCl-Tris-MgCl(,2) buffer, pH 7.4 at 37(DEGREES). The metabolites formed were identified by chromatography, UV spectrometry, as well as mass spectrometry. After a 1 hr incubation, TG was metabolized by the liver homogenate, the 10,000 x g pellet and the post-mitochondrial fraction mainly to 6-thioguanosine (TGR), accompanied by varying lesser amounts of 6-thiouric acid (TUA), allantoin, guanine-6-sulfinic acid (G-SO(,2)H) and an unknown product. In comparison, the cytosal fraction converted TG almost entirely to TGR and TUA in equal amounts. The formation of TGR from TG was limited by the endogenous supply of ribose-1-phosphate. With the microsomal fraction, however, TG was metabolized significantly to G-SO(,2)H and the unknown, accompanied with some TGR. After a 5 hr incubation the metabolism of TG was changed to favor the catabolic route, yielding mostly TUA in the post-mitochondrial and cytosol fractions; but mainly allantoin in the liver homogenate fraction. The kinetic studies of TG metabolism by the subcellar fractions indicated that the formation of TGR served as a depot form of TG. The level of TGR decreased when the catabolism of TG became prominent. The oxidation of TG to GSO(,2)H mediated by the hepatic microsomes represented a new catabolic pathway of TG. This GSO(,2)H, under acidic conditions, readily decomposes to guanine and inorganic sulfate. In the presence of reduced glutathione in Tris buffer, pH 7.8 at 25(DEGREES), GSO(,2)H is adducted to glutathione chemically to form S-(2-amino-purin-6-yl) glutathione and conceivably, inorganic sulfate. Therefore, the formation of GSO(,2)H from TG might have implication in the desulfuration mechanism of TG. On the other hand, the unknown formed from TG by the action of the microsomal enzymes appeared to be a TG conjugate. However, it is neither a glutathione, a glucuronide, nor a ribose conjugate. Additionally, the deamination of TG by guanine deaminase (E.C.3.5.4.3) isolated from rat liver was also investigated. TG is a poorer substrate (Km = 4.8 x 10('-3)M) for guanine deaminase than that of guanine (Km = 4.7 x 10('-6)M) at pH 7.25, optimal pH for TG as a substrate. TG is also a competitive inhibitor of guanine for guanine deaminase, with a ki of 2.2 x 10('-4)M. ^

PREPARATION AND CHARACTERIZATION OF COVALENTLY LINKED ENZYME-ANTIBODY CONJUGATES AND THEIR USE IN MEASURING MINUTE QUANTITIES OF PROTEIN ANTIGENS

The discovery and characterization of oncofetal proteins have led to significant advances in early cancer diagnosis and therapeutic monitoring of patients undergoing cancer chemotherapy. These tumor-associated antigens are presently measured by sensitive, specific immunoassay techniques based on the detection of minute amounts of labeled antigen or antibody incorporated into immune complexes, which must be isolated from free antigen and antibody.^ Since there are several disadvantages with using radioisotopes, the most common immunolabel, one major objective was to prepare covalently coupled enzyme-antibody conjugates and evaluate their use as a practical alternative to radiolabeled immune reagents. An improved technique for the production of enzyme-antibody conjugates was developed that involves oxidizing the carbohydrate moieties on a glycoprotein enzyme, then introducing antibody in the presence of polyethylene glycol (PEG). Covalent enzyme-antibody conjugates involving alkaline phosphatase and amyloglucosidase were produced and characterized.^ In order to increase the sensitivity of detecting the amyloglucosidase-antibody conjugate, an enzyme cycling assay was developed that measures glucose, the product of maltose cleavage by amyloglucosidase, in the picomole range. The increased sensitivity obtained by combined usage of the amyloglucosidase-antibody conjugate and enzyme cycling assay was then compared to that of conventional enzyme immunoassay (EIA).^ For immune complex isolation, polystyrene tubes and protein A-bearing Staphylococcus aureus were evaluated as solid phase matrices, upon which antibodies can be immobilized. A sandwich-type EIA, using antibody-coated S. aureus, was developed that measures human albumin (HSA) in the nanogram range. The assay, using an alkaline phosphatase-anti-HSA conjugate, was applied to the determination of HSA in human urine and evaluated extensively for its clinical applicability.^ Finally, in view of the clinical significance of alpha-fetoprotein (AFP) as an oncofetal antigen and the difficulty with its purification for use as an immunogen and assay standard, a chemical purification protocol was developed that resulted in a high yield of immunochemically pure AFP. ^

In pneumococcal meningitis a novel water-soluble inhibitor of matrix metalloproteinases and TNF-alpha converting enzyme attenuates seizures and injury of the cerebral cortex

Matrix metalloproteinases (MMPs) and TNF-alpha converting enzyme (TACE) contribute to the pathophysiology of bacterial meningitis. To date, MMP-inhibitors studied in models of meningitis were compromised by their hydrophobic nature. We investigated the pharmacokinetics and the effect of TNF484, a water-soluble hydroxamate-based inhibitor of MMP and TACE, on disease parameters and brain damage in a neonatal rat model of pneumococcal meningitis. At 1 mg/kg q6h TNF484 reduced soluble TNF-alpha and the collagen degradation product hydroxyproline in the cerebrospinal fluid. Clinically, TNF484 attenuated the incidence of seizures and was neuroprotective in the cortex. Water-soluble MMP-inhibitors may hold promise in the therapy of bacterial meningitis.

The role of cytochromes P450 and peroxidases in the detoxification of sulphonated anthraquinones by rhubarb and common sorrel plants cultivated under hydroponic conditions

Sulphonated anthraquinones are precursors of many synthetic dyes and pigments, recalcitrant to biodegradation and thus not eliminated by classical wastewater treatments. In the development of a phytotreatment to remove sulphonated aromatic compounds from dye and textile industrial effluents, it has been shown that rhubarb (Rheum rabarbarum) and common sorrel (Rumex acetosa) are the most efficient plants. Both species, producing natural anthraquinones, not only accumulate, but also transform these xenobiotic chemicals. Even if the precise biochemical mechanisms involved in the detoxification of sulphonated anthraquinones are not yet understood, they probably have cross talks with secondary metabolism, redox processes and plant energy metabolism. The aim of the present study was to investigate the possible roles of cytochrome P450 monooxygenases and peroxidases in the detoxification of several sulphonated anthraquinones. Both plant species were cultivated in a greenhouse under hydroponic conditions, with or without sulphonated anthraquinones. Plants were harvested at different times and either microsomal or cytosolic fractions were prepared. The monooxygenase activity of cytochromes P450 toward several sulphonated anthraquinones was tested using a new method based on the fluorimetric detection of oxygen consumed during cytochromes P450-catalysed reactions. The activity of cytosolic peroxidases was measured by spectrophotometry, using guaiacol as a substrate. A significant activity of cytochromes P450 was detected in rhubarb leaves, while no (rhizome) or low (petioles and roots) activity was found in other parts of the plants. An induction of this enzyme was observed at the beginning of the exposition to sulphonated anthraquinones. The results also indicated that cytochromes P450 were able to accept as substrate the five sulphonated anthraquinones, with a higher activity toward AQ-2,6-SS (0.706 nkat/mg protein) and AQ-2-S (0.720 nkat/mg protein). An activity of the cytochromes P450 was also found in the leaves of common sorrel (1.212 nkat/mg protein (AQ-2,6-SS)), but no induction of the activity occurred after the exposition to the pollutant. The activity of peroxidases increased when rhubarb was cultivated in the presence of the five sulphonated anthraquinones (0.857 nkat/mg protein). Peroxidase activity was also detected in the leaves of the common sorrel (0.055 nkat/mg protein), but in this plant, no significant difference was found between plants cultivated with and without sulphonated anthraquinones. Results indicated that the activity of cytochromes P450 and peroxidases increased in rhubarb in the presence of sulphonated anthraquinones and were involved in their detoxification mechanisms. These results suggest the existence in rhubarb and common sorrel of specific mechanisms involved in the metabolism of sulphonated anthraquinones. Further investigation should be performed to find the next steps of this detoxification pathway. Besides these promising results for the phytotreatment of sulphonated anthraquinones, it will be of high interest to develop and test, at small scale, an experimental wastewater treatment system to determine its efficiency. On the other hand, these results reinforce the idea that natural biodiversity should be better studied to use the most appropriate species for the phytotreatment of a specific pollutant.