Blood biomarkers associated with inflammation predict poor prognosis in cerebral venous thrombosis:: a multicenter prospective observational study.

BACKGROUND AND PURPOSE: Experimental studies suggest inflammation can contribute to blood barrier disruption and brain injury in cerebral venous thrombosis (CVT). We aimed to determine whether blood biomarkers of inflammation were associated with the evolution of brain lesions, persistent venous occlusion or functional outcome in patients with CVT. METHODS: Pathophysiology of Venous Infarction-Prediction of Infarction and Recanalization in CVT (PRIORITy-CVT) was a multicenter prospective cohort study of patients with newly diagnosed CVT. Evaluation of neutrophil-to-lymphocyte ratio (NLR) and C-reactive protein (CRP) concentrations in peripheral blood samples was performed at admission in 62 patients. Additional quantification of interleukin (IL)-6 was performed at day 1, 3 and 8 in 35 patients and 22 healthy controls. Standardized magnetic resonance imaging was performed at day 1, 8 and 90. Primary outcomes were early evolution of brain lesion, early recanalization and functional outcome at 90 days. RESULTS: Interleukin-6 levels were increased in patients with CVT with a peak at baseline. IL-6, NLR and CRP levels were not related with brain lesion outcomes or early recanalization but had a significant association with unfavourable functional outcome at 90 days (IL-6: OR = 1.28, 95% CI: 1.05-1.56, P = 0.046; NLR: OR = 1.39, 95% CI: 1.4-1.87, P = 0.014; CRP: OR = 1.756, 95% CI: 1.010-3.051, P = 0.029). Baseline IL-6 had the best discriminative capacity, with an area under the receiver operating characteristic curve to predict unfavourable functional outcome of 0.74 (P = 0.031). CONCLUSIONS: Increased baseline levels of NLR, CRP and IL-6 may serve as new predictive markers of worse functional prognosis at 90 days in patients with CVT. No association was found between inflammatory markers and early evolution of brain lesion or venous recanalization.

A Single Bout of Arm-crank Exercise Promotes Positive Emotions and Post-Exercise Hypotension in Patients with Symptomatic Peripheral Artery Disease.

OBJECTIVE/BACKGROUND: The objective was to analyze the acute effects of a single bout of arm cranking exercise on affective and cardiovascular parameters in patients with symptomatic peripheral artery disease (PAD). METHODS: This was a prospective, controlled, crossover study. Eleven men with symptomatic PAD underwent two experimental sessions in a random order: control or arm crank exercise (15 × 2 minutes bouts of arm crank exercise interrupted by 2 minutes rest intervals). During exercise, ratings of perceived exertion (Borg scale) and affective responses (pleasure/displeasure) were obtained at the first, fifth, tenth, and fifteenth bouts. Before and after the experimental sessions, cardiovascular parameters (blood pressure and heart rate) were obtained. Data were analysed by a two-way repeated measure analysis of variance with significance achieved at p < .05. RESULTS: During the arm crank exercise, patients reported positive feelings of pleasure. During exercise, heart rate (HR) remained within 80-90% of peak HR. Additionally, patients performed arm crank exercise with moderate levels of perceived exertion (Borg rating of 11-13) and with pleasant affective scores (Feeling Scale of +1 to +5). Blood pressure (systolic, diastolic, and mean) increase was lower after arm crank exercise than for control (greatest net effect: -15 ± 11 mmHg [p < .001]; -9 ± 5 mmHg [p < .001]; -9 ± 6 mmHg [p < .001], respectively), while HR increased (greatest net effect: +9 ± 6 beats per minute; p < .001). CONCLUSION: A single bout of arm crank exercise promotes pleasurable feelings while reducing blood pressure in patients with symptomatic PAD.

Vascular Mechanisms of Post-exercise Blood Pressure Responses in Peripheral Artery Disease.

This study aimed to analyze the vascular mechanisms involved in post-resistance decreases in blood pressure in patients with peripheral artery disease. 17 patients underwent 2 experimental sessions conducted in random order: resistance exercise (REx-3×10 reps in 8 exercises with intensity of ~ 60% of 1 RM) and control (C- resting on the exercise machines for 50 min). Before and after each experimental session, blood pressure, reflected wave indicators, pulse wave velocity, blood flow, blood flow post-reactive hyperemia and peripheral vascular resistance responses were obtained. Both sessions increased brachial systolic, diastolic and mean blood pressure (greatest increase REx: 11 mmHg; greatest increase C: 19 mmHg; P<0.01); however, the increases were greater after the C session (P<0.01). Reflected wave indicators increased only after the C session (P<0.06), while pulse wave velocity increased similarly after both sessions (P=0.66). Individual analyses indicated a large variability between patients in vascular variables responses. A single bout of REx decreased blood pressure in peripheral artery disease patients, and these responses were followed by changes in reflected wave indicators. The other factors presented high individual variability, and thus it was not possible to identify specific factors associated with blood pressure reduction in peripheral artery disease patients.

Heterologous expression and purification of a biologically active legume lectin from Cratylia mollis seeds (CRAMOLL 1).

CRAMOLL 1 is a mannose/glucose isolectin isolated from Cratylia mollis seeds. This lectin has 82% sequence identity with Con A and essentially the same quaternary structure. As with Con A, CRAMOLL 1 seems to undergo complex post-translational processing which makes it difficult to the use of traditional molecular cloning for heterologous expression. Here we report the expression and purification of functional recombinant CRAMOLL 1 (rCRAMOLL 1) in Escherichia coli. This was accomplished by introducing a chemically synthesized DNA encoding the mature CRAMOLL 1 amino acid sequence into a bacterial expression vector under T7 promoter control. Most of the recombinant lectin was found in insoluble aggregates (inclusion bodies), but we were able to recover reasonable amounts of soluble lectin in the active form by decreasing the protein induction temperature. The recombinant lectin was purified to homogeneity with one-step affinity chromatography. The plant CRAMOLL 1 (pCRAMOLL 1) and rCRAMOLL 1 share several physicochemical properties such as molecular mass, charge density and secondary and tertiary structures. However, pCRAMOLL 1 has a lower thermodynamic stability than rCRAMOLL 1 when probed by acidification, high temperature or high hydrostatic pressure, and this is probably caused by the presence of tetramers composed of fragmented monomers, which are formed in the plant cotyledon but absent from the recombinant protein. rCRAMOLL 1 behaves identically to its plant counterpart with respect to its specificity for monosaccharides, and to its agglutinating activities against rabbit erythrocytes and Trypanosoma cruzi epimastigote cells.

Phosphorylation of native and heme-modified CYP3A4 by protein kinase C: a mass spectrometric characterization of the phosphorylated peptides.

As an initial approach toward the characterization of the phosphorylation of cumene hydroperoxide (CuOOH)-inactivated cytochrome P450 (CYP3A4, the major human liver drug-metabolizing enzyme) and its role in the degradation of the inactivated protein, we have identified one of the major participating cytosolic kinase(s) as rat liver cytosolic protein kinase C (PKC) with the use of specific and general kinase inhibitors. Accordingly, we employed a model phosphorylation system consisting of purified PKC, gamma-S-[(32)P]ATP, and either native or CuOOH-inactivated purified recombinant His(6)-tagged CYP3A4. Lysylendoprotease (Lys)-C digestion of the phosphorylated CuOOH-inactivated CYP3A4(His)(6) followed by HPLC-peptide mapping and mass spectrometric (LC/MS/MS) analyses led to the isolation and the unambiguous identification of two PKC-phosphorylated CYP3A4 peptides: E(258)SRLEDT(p)QK(266) and F(414)LPERFS(p)K(421). Similar analyses of the PKC-phosphorylated native enzyme predominantly yielded E(258)SRLEDT(p)QK(266) as the phosphorylated peptide. Studies are currently in progress to determine whether phosphorylation of any or both of these peptides is required for the Ub-dependent 26S proteasomal degradation of CuOOH-inactivated CYP3A4.

Ubiquitin-dependent 26S proteasomal pathway: a role in the degradation of native human liver CYP3A4 expressed in Saccharomyces cerevisiae?

Cytochrome P450, CYP3A4, is the dominant human liver endoplasmic reticulum (ER) hemoprotein enzyme, responsible for the metabolism of over 60% of clinically relevant drugs. We have previously shown that mechanism-based suicide inactivation of CYP3A4 and its rat liver ER orthologs, CYPs 3A, via heme-modification of their protein moieties, results in their ubiquitin (Ub)-dependent 26S proteasomal degradation (Korsmeyer et al. (1999) Arch. Biochem. Biophys. 365, 31; Wang et al. (1999) Arch. Biochem. Biophys. 365, 45). This is not surprising given that the heme-modified CYP3A proteins are structurally damaged. To determine whether the turnover of the native enzyme similarly recruited this pathway, we heterologously expressed this protein in wild-type Saccharomyces cerevisiae and mutant strains (hrd1Delta, hrd2-1, and hrd3Delta) previously shown to be deficient in the Ub-dependent 26S proteasomal degradation of the polytopic ER protein 3-hydroxy-3-methylglutaryl-CoA reductase (isoform Hmg2p), the rate-limiting enzyme in sterol biosynthesis, as well as in strains deficient in ER-associated Ub-conjugating enzymes, Ubc6p and/or Ubc7p (Hampton et al. (1996) Mol. Biol. Cell 7, 2029; Hampton and Bhakta (1997) Proc. Natl. Acad. Sci. USA 94, 12,944). Our findings reveal that in common with the degradation of Hmg2p, that of native CYP3A4 also requires Hrd2p (a subunit of the 19S cap complex of the 26S proteasome) and Ubc7p, and to a much lesser extent Hrd3p, a component of the ER-associated Ub-ligase complex. In contrast to Hmg2p-degradation, that of native CYP3A4 does not appear to absolutely require Hrd1p, another component of the ER-associated Ub-ligase complex. Furthermore, studies in a S. cerevisiae pep4Delta strain proven to be deficient in the vacuolar degradation of carboxypeptidase Y indicated that CYP3A4 degradation is also largely independent of vacuolar (lysosomal) proteolytic function. The degradation of two other native ER proteins, Sec61p and Sec63p, normal components of the ER translocon, were also examined in parallel and found to be stabilized to some extent in HRD2- and UBC7-deficient strains. Together these findings attest to the remarkable mechanistic diversity in the normal degradation of ER proteins.

Structure--function relationships of rat hepatic tryptophan 2,3-dioxygenase: identification of the putative heme-ligating histidine residues.

The liver cytosolic enzyme tryptophan 2,3-dioxygenase (TDO) catalyzes the oxidation of L-tryptophan to formylkynurenine and controls the physiological flux of tryptophan into both the serotonergic and kynureninic pathways. This hemoprotein enzyme is composed of four noncovalently bound subunits of equivalent mass and contains two heme moieties per molecule. Electron paramagnetic resonance analyses have indicated that a histidyl nitrogen is involved in heme ligation [Henry et al., (1976) J. Biol. Chem. 251, 1578], but the identity of the His residue(s) is unknown. In an attempt to characterize the active site of the enzyme we have substituted each of the 12 His residues in the rat TDO subunit with Ala, to determine their relative importance in heme binding. Sequence alignment of the rat liver protein with that of known or putative TDO sequences from other organisms reveals that four of the His residues are conserved in eukaryotes, two of which are also conserved in prokaryotes. Our findings indicate that replacement of the evolutionarily conserved His 76 and 328 residues resulted in a dramatic reduction of TDO activity, whereas that of the eukaryotically conserved His70 resulted in a significant reduction relative to that of the wild-type enzyme. On the other hand, replacement of the other eukaryotically conserved His273 residue, while affecting the relative expression of the enzyme, had little effect on its specific activity. Size-exclusion analyses revealed that the His76Ala and His328Ala mutants retained little or no heme, suggesting that these may be key residues in ligating the prosthetic heme moieties. Whether these His residues are both provided by the same TDO subunit or a different TDO subunit remains to be determined.

Influence of P450 3A4 SRS-2 residues on cooperativity and/or regioselectivity of aflatoxin B(1) oxidation.

The major human liver drug-metabolizing cytochrome P450 enzymes P450 3A4 and P450 3A5 share >85% amino acid sequence identity yet exhibit different regioselectivity toward aflatoxin B(1) (AFB(1)) biotransformation [Gillam et al. (1995) Arch. Biochem. Biophys. 317, 74-384]. P450 3A4 prefers AFB1 3alpha-hydroxylation, which detoxifies and subsequently eliminates the hepatotoxin, over AFB1 exo-8,9-oxidation. P450 3A5, on the other hand, is a relatively sluggish 3alpha-hydroxylase and converts AFB(1) predominantly to the genotoxic exo-8,9-epoxide. Using a combination of approaches (sequence alignment, homology modeling and site-directed mutagenesis), we have previously identified several divergent residues in four of the six putative substrate recognition sites (SRSs) of P450 3A4, which when replaced individually with the corresponding amino acid of P450 3A5, resulted in a significant switch of the characteristic P450 3A4 AFB(1) regioselectivity toward that of P450 3A5 [Wang et al. (1998) Biochemistry 37, 12536-12545]. In particular, residues N206 and L210 in SRS-2 were found to be critical for AFB(1) detoxification via 3alpha-hydroxylation, and the corresponding mutants N206S and L210F most closely mimicked P450 3A5, not only in its regioselectivity of AFB(1) metabolism but also in its overall functional capacity. We have now further explored the plausible reasons for such relative inactivity of the SRS-2 mutants by examining N206S and additional mutants (L210A, L211F, L211A, and N206E) and found that the dramatically lowered activities of the N206S mutant are accompanied by a loss of cooperativity of AFB(1) oxidation. Molecular dynamics analyses with an existing P450 3A4 homology model [Szklarz and Halpert (1997) J. Comput. Aided Mol. Des. 11, 265] suggested that N206 (helix F) interacts with E244 (helix G), creating a salt bridge that stabilizes the protein structure and/or defines the active site cavity. To examine this possibility, several E244 mutants (E244A, V, N, S) were tested, of which E244S was the most notable for its relatively greater impairment of P450 3A4-dependent AFB(1) 3alpha-hydroxylation. However, the results with these E244 mutants failed to validate the N206-E244 interaction predicted from these molecular dynamics analyses. Collectively, our findings to date have led us to reconsider our original interpretations and to reexamine them in the light of AFB(1) molecular modeling analyses with a newly refined P450 3A4 homology model. These analyses predicted that F304 in SRS-4 (I-helix) plays a pivotal role in AFB(1) binding at the active site in either orientation leading to 3alpha- or exo-8,9-oxidation. Consistent with this prediction, conversion of F304 to Ala abolished P450 3A4-dependent AFB(1) 3alpha-hydroxylation and exo-8,9-oxidation.

Heme: a regulator of rat hepatic tryptophan 2,3-dioxygenase?

The hepatic cytosolic hemoprotein tryptophan 2,3-dioxygenase (TDO) is the rate-limiting enzyme in tryptophan catabolism and thus plays a key role in regulating the physiological flux of tryptophan into relevant metabolic pathways. The TDO protein is induced by corticosteroids such as dexamethasone (DEX) and is stabilized by its prosthetic heme. In rats, acute chemically induced hepatic heme depletion reduces the functional hepatic TDO levels to 25-30% of basal levels within 1 h, and this decrease persists beyond 28 h of heme depletion at which time only 25-30% of the protein is available for heme incorporation. Since this could stem from impaired de novo synthesis and/or instability of the newly synthesized apoTDO protein in the absence of heme, we examined the specific role of heme in these events in a previously validated rat model of acute hepatic heme depletion triggered by the P450 suicide substrate 3, 5-dicarbethoxy 2,6-dimethyl-4-ethyl-1,4-dihydropyridine. We now show that exogenous heme can reverse the functional impairment of the enzyme observed during hepatic heme depletion and fully restore the impaired DEX-mediated induction of the enzyme to normal. Furthermore, through Northern/slot blot analyses coupled with nuclear run-on studies, we now document that this heme regulation of TDO is exerted primarily at the transcriptional level. Immunoblotting analyses also reveal corresponding changes in the TDO protein, thereby establishing that heme is necessary for DEX-inducible TDO mRNA transcription and subsequent translation. Thus, the TDO gene may contain heme-regulatory elements in addition to the reported glucocorticoid-responsive elements. Together, these findings suggest that clinically, hepatic heme deficiency may enhance the tryptophan flux into synthetic (serotonergic) pathways, not only by depriving prosthetic heme for a functionally competent TDO hemoprotein, its primary catabolic enzyme, but also by impairing the de novo synthesis of this enzyme.

Ethynylestradiol-mediated induction of hepatic CYP3A9 in female rats: implication for cyclosporine metabolism.

Repeated treatment of female rats with the synthetic estrogen ethynylestradiol (EE(2)) increases the formation of the cyclosporine A (CyA) metabolites AM1c and AM9 by 3-fold, whereas the formation of AM1 and AM4N is not significantly enhanced. The formation of all four CyA metabolites was inhibited by greater than 80% by the CYP3A-selective substrate midazolam or polyclonal anti-rat CYP3A IgGs in liver microsomes of untreated and EE(2)-induced rats. In contrast, anti-rat CYP2C6 IgGs had little effect, indicating the involvement of a CYP3A but not 2C6 in this EE(2)-stimulated CyA metabolism. Semiquantitative reverse-transcriptase polymerase chain reaction was used to determine the mRNA content for four CYP3A genes (CYP3A2, CYP3A9, CYP3A18, and CYP3A23) in livers of control and EE(2)-treated female rats. EE(2) selectively induced CYP3A9 by 3.3-fold whereas the expression of CYP3A18 and CYP3A23 was slightly decreased; neither CYP3A2 mRNA nor CYP3A1 mRNA was detectable in these EE(2)-treated livers. To determine whether rat liver microsomal CYP3A9 was indeed responsible for the EE(2)-stimulated CyA metabolism, a recombinant CYP3A9 was heterologously expressed in Escherichia coli. When functionally reconstituted, this enzyme was active in metabolizing CyA preferentially to its AM9 and AM1c metabolites as compared with CYP3A4. These findings thus support the notion that the increased CyA-metabolizing capacity of EE(2)-treated female rat liver microsomes is due to the induction of the CYP3A9 enzyme.

Cytochrome P450 3A degradation in isolated rat hepatocytes: 26S proteasome inhibitors as probes.

Mechanism-based inactivation of liver microsomal cytochromes P450 3A (CYP 3A, P450s 3A) in vivo and/or in vitro, via heme modification of the protein, results in accelerated proteolytic degradation of the enzyme that is preceded by the ubiquitination of the protein, thereby implicating the ubiquitin-ATP-dependent 26S proteasomal system. In this study, this involvement is confirmed with the use of the proteasomal inhibitors aclarubicin and MG-132 as probes, in isolated rat hepatocytes treated with the P450 3A mechanism-based inactivator, 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1, 4-dihydropyridine (DDEP). In addition, the findings reveal that during the course of this proteolysis, the endoplasmic reticulum (ER)-anchored DDEP-inactivated P450 3A is translocated from the ER to the cytosol in a brefeldin A-insensitive manner.

Proteolytic degradation of heme-modified hepatic cytochromes P450: A role for phosphorylation, ubiquitination, and the 26S proteasome?

The resident integral hepatic endoplasmic reticulum (ER) proteins, cytochromes P450 (P450s), turn over in vivo with widely varying half-lives. We and others (Correia et al., Arch. Biochem. Biophys. 297, 228, 1992; and Tierney et al., Arch. Biochem. Biophys. 293, 9, 1992) have previously shown that in intact animals, the hepatic P450s of the 3A and 2E1 subfamilies are first ubiquitinated and then proteolyzed after their drug-induced suicide inactivation. Our findings with intact rat hepatocytes and ER preparations containing native P450s and P450s inactivated via heme modification of the protein have revealed that the proteolytic degradation of heme-modified P450s requires a cytosolic ATP-dependent proteolytic system rather than lysosomal or ER proteases (Correia et al., Arch. Biochem. Biophys. 297, 228, 1992). Using purified cumene hydroperoxide-inactivated P450s (rat liver P450s 2B1 or 3A and/or a recombinant human liver P450 3A4) as models, we now document that these heme-modified enzymes are indeed ubiquitinated and then proteolyzed by the 26S proteasome, but not by its 20S proteolytic core. In addition, our studies indicate that the ubiquitination of these heme-modified P450s is preceded by their phosphorylation. It remains to be determined whether, in common with several other cellular proteins, such P450 phosphorylation is indeed required for their degradation. Nevertheless, these findings suggest that the membrane-anchored P450s are to be included in the growing class of ER proteins that undergo ubiquitin-dependent 26S proteasomal degradation.

Identification of the heme-modified peptides from cumene hydroperoxide-inactivated cytochrome P450 3A4.

Cumene hydroperoxide-mediated (CuOOH-mediated) inactivation of cytochromes P450 (CYPs) results in destruction of their prosthetic heme to reactive fragments that irreversibly bind to the protein. We have attempted to characterize this process structurally, using purified, 14C-heme labeled, recombinant human liver P450 3A4 as the target of CuOOH-mediated inactivation, and a battery of protein characterization approaches [chemical (CNBr) and proteolytic (lysylendopeptidase-C) digestion, HPLC-peptide mapping, microEdman sequencing, and mass spectrometric analyses]. The heme-peptide adducts isolated after CNBr/lysylendopeptidase-C digestion of the CuOOH-inactivated P450 3A4 pertain to two distinct P450 3A4 active site domains. One of the peptides isolated corresponds to the proximal helix L/Cys-region peptide 429-450 domain and the others to the K-region (peptide 359-386 domain). Although the precise residue(s) targeted remain to be identified, we have narrowed down the region of attack to within a 17 amino acid peptide (429-445) stretch of the 55-amino acid proximal helix L/Cys domain. Furthermore, although the exact structures of the heme-modifying fragments and the nature of the adduction remain to be established conclusively, the incremental masses of approximately 302 and 314 Da detected by electrospray mass spectrometric analyses of the heme-modified peptides are consistent with a dipyrrolic heme fragment comprised of either pyrrole ring A-D or B-C, a known soluble product of peroxidative heme degradation, as a modifying species.

Structure-function relationships of human liver cytochromes P450 3A: aflatoxin B1 metabolism as a probe.

Cytochromes P450 3A4 and 3A5, the dominant drug-metabolizing enzymes in the human liver, share >85% primary amino acid sequence identity yet exhibit different regioselectivity toward aflatoxin B1 (AFB1) biotransformation [Gillam et al., (1995) Arch. Biochem. Biophys. 317, 374-384]. P450 3A4 apparently prefers AFB1 3alpha-hydroxylation, which results in detoxification and subsequent elimination of the hepatotoxin, over AFB1 exo-8,9-oxidation. In contrast, P450 3A5 is incapable of appreciable AFB1 3alpha-hydroxylation and converts it predominantly to the exo-8,9-oxide which is genotoxic. To elucidate the structural features that govern the regioselectivity of the human liver 3A enzymes in AFB1 metabolism and bioactivation, a combination of approaches including sequence alignment, homology modeling, and site-directed mutagenesis was employed. Specifically, the switch in AFB1 regioselectivity was examined after individual substitution of the divergent amino acids in each of the six putative substrate recognition sites (SRSs) of P450 3A4 with the corresponding amino acid of P450 3A5. Of the P450 3A4 mutants examined, P107S, F108L, N206S, L210F, V376T, S478D, and L479T mutations resulted in a significant switch of P450 3A4 regioselectivity toward that of P450 3A5. The results confirmed the importance of some of these residues in substrate contact in the active site, with residue N206 (SRS-2) being critical for AFB1 detoxification via 3alpha-hydroxylation. Moreover, the P450 3A4 mutant N206S most closely mimicked P450 3A5, not only in its regioselectivity of AFB1 metabolism but also in its overall functional capacity. Furthermore, the other SRS-2 mutant, L210F, also resembled P450 3A5 in its overall AFB1 metabolism and regioselectivity. These findings reveal that a single P450 3A5 SRS domain (SRS-2) is capable of conferring the P450 3A5 phenotype on P450 3A4. In addition, some of these P450 3A4 mutations that affected AFB1 regioselectivity had little influence on testosterone 6beta-hydroxylation, thereby confirming that each substrate-P450 active site fit is indeed unique.

Cytochrome P450 2C11: Escherichia coli expression, purification, functional characterization, and mechanism-based inactivation of the enzyme.

The male-specific P450 enzyme CYP 2C11, whose expression is developmentally and hormonally regulated, is the major steroid 16alpha-hydroxylase of the untreated rat liver. The enzyme metabolizes a host of substrates, including mechanism-based inactivators, such as 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) and spironolactone (SPL). Structural and functional characterization of the specific mode of such inactivation, however, requires sufficient quantities of the fully purified enzyme. Although several laboratories including our own have isolated and purified the enzyme from male rats, the yields are typically low and of the order of 1%. For these reasons, we chose to heterologously express the enzyme in Escherichia coli. The full-length cDNA was excised from the yeast vector pD2M1 and cloned into the plasmid vector pCW after appropriate modifications for optimal expression in E. coli. The enzyme was isolated and purified from E. coli membranes in relatively high yields (approximately 60%) and relatively high specific content (19 nmol/mg protein). The purified recombinant enzyme had spectral and functional characteristics comparable to those reported for the native rat liver enzyme, including mechanism-based inactivation by DDEP and SPL. Studies with 14C-heme-labeled enzyme indicated that the major mode of DDEP inactivation was via heme-N-ethylation. On the other hand, studies with radiolabeled SPL-SH (the proximal inactivating deacetylated metabolite of SPL) revealed that although both [22-14C]SPL-SH and SPL-35SH inactivated the enzyme, only SPL-35SH was found to irreversibly radiolabel the 2C11 protein. The latter findings thus suggest that during mechanism-based inactivation of 2C11, the thiol moiety of SPL-SH is oxidatively activated to a species that attacks the 2C11 protein during or after cleavage from the thiosteroid. Thus, these modes of mechanism-based 2C11 inactivation by DDEP and SPL-SH considerably differ from the corresponding modes of P450 3A inactivation by these agents, wherein heme modification of the protein predominates.

Secobarbital-mediated inactivation of cytochrome P450 2B1 and its active site mutants. Partitioning between heme and protein alkylation and epoxidation.

Secobarbital (SB) is a relatively selective mechanism-based inactivator of cytochrome P450 2B1, that partitions between epoxidation and heme and protein modification during its enzyme inactivation. The SB-2B1 heme adduct formed in situ in a functionally reconstituted system has been spectrally documented and structurally characterized as N-(5-(2-hydroxypropyl)-5-(1-methylbutyl)barbituric acid)protoporphyrin IX. The SB-protein modification has been localized to 2B1 peptide 277-323 corresponding to the active site helix I of cytochrome P450 101. The targeting of heme and this active site peptide suggests that the 2B1 active site topology could influence the course of its inactivation. To explore this possibility, the individual SB epoxidation, heme and protein modification, and corresponding molar partition ratios of the wild type and seven structural 2B1 mutants, site-directed at specific substrate recognition sites, and known to influence 2B1 catalysis were examined after Escherichia coli expression. These studies reveal that Thr-302 is critical for SB-mediated heme N-alkylation, whereas Val-367 is a critical determinant of 2B1 protein modification, and Val-363 is important for SB epoxidation. SB docking into a refined 2B1 homology model coupled with molecular dynamics analyses provide a logical rationale for these findings.