[Treatment of fractures of the distal femur on total knee arthroplasty]. / Traitement des fractures du fémur distal sur prothèse totale de genou.

PURPOSE OF THE STUDY: Fractures of the distal femur near a total knee arthroplasty are usually observed in patients aged over 70 years victims of low-energy trauma. The rate of complications is high: in the literature 25-75 %, even when treated by experienced surgeons. The purpose of this study was to detail therapeutic modalities used for the treatment of these fractures with their advantages and disadvantages and to determine the morbidity of complications which do occur. MATERIAL AND METHODS: Twenty distal fracture of the femur were treated between 1990 and 2000 in twenty patients who had a total knee arthroplasty. Mean patient age at surgery was 72 years (range 69-77). The fracture was a short oblique fracture in four, transversal in five, long and spiral in four and comminuted in the others. In four fractures, the preoperative radiographic analysis suggested concomitant loosening. Therapeutic modalities included suspension traction for two, femorotibial external fixation for two. Retrograde nailing was possible in two patients. Fixation with a screw plate or a blade plate was used for nine fractures. The prosthesis was revised systematically if loosening was observed. This was done in three cases with preservation of the tibial piece and in two cases, changing the complete prosthesis, using a long femoral stem in all. RESULTS: Mean follow-up was two to ten years. Immediate weight bearing was possible for four of the five patients for whom only the femoral piece was changed with a long stem. For the others, weight bearing was deferred until fracture healing was obtained after a mean five months (range 4-42). Bone healing required one, or two, revisions in three patients. Three patients died within one year of their fracture of the distal femur. There were no cases of infection or late healing. At last follow-up, the knee and function score regressed after treatment of the fracture. The two scores dropped 20 points compared with the pre-operative score (78 +/- 15 to 56 +/- 19 for the knee score and 72 +/- 16 to 54 +/- 18 for the function score). The knee score fell because of difficulty going up and down stairs and use of crutches. Treatments which stiffened the knee were traction suspension and external fixation. Loss of joint motion was about the same with or without prosthesis revision. The tibiofemoral angle measured before the fractures (postoperative goniometry) then after fracture healing or after changing the femoral prosthesis, showed an alignment of 183 degrees before the fracture and 178 degrees after healing or revision of the femoral piece. Three patients experienced another fracture of the same femur after healing of the initial fracture or after changing the knee prosthesis. DISCUSSION: Fracture of the distal femur on a total knee prosthesis is a serious complications because of the mortality and the difficulty in achieving functional recovery after treatment. Therapeutic difficulties involve the type of skin incision, the position of the screws in relation to the femoral component, the possible need for bone graft, and finally the requirement to change the femoral piece with a centro-medullary stem.

Reduction of nilutamide by NO synthases: implications for the adverse effects of this nitroaromatic antiandrogen drug.

Nitric oxide synthases (NOSs) are flavohemeproteins that catalyze the oxidation of l-arginine to l-citrulline with formation of the widespread signal molecule NO. Beside their fundamental role in NO biosynthesis, these enzymes are also involved in the formation of reactive oxygen species and in the interactions with some xenobiotic compounds. Nilutamide is a nonsteroidal antiandrogen that behaves as a competitive antagonist of the androgen receptors and is proposed in the treatment of metastatic prostatic carcinoma. However, therapeutic effects of nilutamide are overshadowed by the occurrence of several adverse reactions mediated by toxic mechanism(s), which remain(s) poorly investigated. Here, we studied the interaction of NOSs with nilutamide. Our results show that the purified recombinant neuronal NOS reduced the nitroaromatic nilutamide to the corresponding hydroxylamine. The reduction of nilutamide catalyzed by neuronal NOS proceeded with intermediate formation of a nitro anion free radical easily observed by EPR, was insensitive to the addition of the usual heme ligands and l-arginine analogues, but strongly inhibited by O(2) and a flavin/NADPH binding inhibitor. Involvement of the reductase domain of nNOS in the reduction of nilutamide was confirmed by (i) the ability of the isolated reductase domain of nNOS to catalyze the reaction and (ii) the stimulating effect of Ca(2+)/calmodulin on the accumulation of hydroxylamine and nitro anion radical. In a similar manner, the recombinant inducible and endothelial NOS isoforms also displayed nitroreductase activity, albeit with lower yields. The selective reduction of nilutamide to its hydroxylamino derivative by the NOSs could explain some of the toxic effects of this drug.

Interaction of new sulfaphenazole derivatives with human liver cytochrome p450 2Cs: structural determinants required for selective recognition by CYP 2C9 and for inhibition of human CYP 2Cs.

A series of new derivatives of sulfaphenazole (SPA), in which the NH(2) and phenyl substituents of SPA are replaced by various groups or in which the sulfonamide function of SPA is N-alkylated, were synthesized in order to further explore CYP 2C9 active site and to determine the structural factors explaining the selectivity of SPA for CYP 2C9 within the human P450 2C subfamily. Compounds in which the NH(2) group of SPA was replaced with R(1) = CH(3), Br, CH = CH(2), CH(2)CH = CH(2), and CH(2)CH(2)OH exhibited a high affinity for CYP 2C9, as shown by the dissociation constant of their CYP 2C9 complexes, K(s), which was determined by difference visible spectroscopy (K(s) between 0.1 and 0.4 microM) and their constant of CYP 2C9 inhibition (K(i) between 0.3 and 0.6 microM). This indicates that the CYP 2C9-iron(III)-NH(2)R bond previously described to exist in the CYP 2C9-SPA complex does not play a key role in the high affinity of SPA for CYP 2C9. Compounds in which the phenyl group of SPA was replaced with various aryl or alkyl R(2) substituents only exhibited a high affinity for CYP 2C9 if R(2) is a freely rotating and sufficiently electron-rich aryl substituent. Finally, compounds resulting from a N-alkylation of the SPA sulfonamide function (R(3) = CH(3), C(2)H(5), or C(3)H(7)) did not retain the selective inhibitory properties of SPA toward CYP 2C9. However, they are reasonably good inhibitors of CYP 2C8 and CYP 2C18 (IC(50) approximately 20 microM). These data allow one to better understand the structural factors that are important for selective binding in the CYP 2C9 active site. They also provide us with clues towards new selective inhibitors of CYP 2C8 and CYP 2C18.

New inhibitors of iron-containing nitrile hydratases.

There is growing evidence in the literature emphasizing the significance of the post-translational modification of cysteine thiols to sulfenic acids (SOH), which have been found in a number of proteins. Crystallographic and mass spectrometric evidence has shown the presence of this group in an inactive form of the industrially important enzyme nitrile hydratase (NHase). This oxidized cysteine is unique in that it forms part of the coordination sphere of the low-spin iron III at the active site of the enzyme. The presence of this unstable sulfenic group in the active form of NHase is the subject of some controversy. To try to detect this function in NHase, we have studied the inhibitory effect on nitrile hydration of reagents known to react with sulfenic acids. Two NHases were studied, namely, Rhodococcus rhodochrous R312 NHase and Comamonas testosteroni NI1 NHase, and the reagents used were meta-chlorocarbonyldicyano-phenylhydrazone (m-ClCP), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), and 2-nitro-5-thiocyanato-benzoic acid (NTBA). Following this approach we report three novel inhibitors of NHases. In addition, we report thiocyanate reagents that can be used to monitor NHase activity spectroscopically.

Recognition of alpha-amino acids bearing various C=NOH functions by nitric oxide synthase and arginase involves very different structural determinants.

Several alpha-amino acids bearing a C=NOH function separated from the Calpha carbon by two to five atoms have been synthesized and tested as substrates or inhibitors of recombinant nitric oxide synthases (NOS) I and II and as inhibitors of rat liver arginase (RLA). These include four N-hydroxyguanidines, N(omega)-hydroxy-L-arginine (NOHA) and its analogues homo-NOHA, nor-NOHA, and dinor-NOHA, two amidoximes bearing the -NH-C(CH(3))=NOH group, and two amidoximes bearing the -CH(2)-C(NH(2))=NOH group. Their behavior toward NOS and RLA was compared to that of the corresponding compounds bearing a C=NH function instead of the C=NOH function. The results obtained clearly show that efficient recognition of these alpha-amino acids by NOS and RLA involves very different structural determinants. NOS favors molecules bearing a -NH-C(R)=NH motif separated from Calpha by three or four CH(2) groups, such as arginine itself, with the necessary presence of delta-NH and omega-NH groups and a more variable R substituent. The corresponding molecules with a C=NOH function exhibit a much lower affinity for NOS. On the contrary, RLA best recognizes molecules bearing a C=NOH function separated from Calpha by three or four atoms, the highest affinity being observed in the case of three atoms. The presence of two omega-nitrogen atoms is important for efficient recognition, as in the two best RLA inhibitors, N(omega)-hydroxynorarginine and N(omega)-hydroxynorindospicine, which exhibit IC(50) values at the micromolar level. However, contrary to what was observed in the case of NOS, the presence of a delta-NH group is not important. These different structural requirements of NOS and RLA may be directly linked to the position of crucial residues that have been identified from crystallographic data in the active sites of both enzymes. Thus, binding of arginine analogues to NOS particularly relies on strong interactions of their delta-NH and omega-NH(2) groups with glutamate 371 (of NOS II), whereas binding of C=NOH molecules to RLA is mainly based on interactions of their terminal OH group with the binuclear Mn(II).Mn(II) cluster of the enzyme and on possible additional bonds between their omega-NH(2) group with histidine 141, glutamate 277, and one Mn(II) ion. The different modes of interaction displayed by both enzymes depend on their different catalytic functions and give interesting opportunities to design useful molecules to selectively regulate NOS and arginase.

Active site topology of artificial peroxidase-like hemoproteins based on antibodies constructed from a specifically designed ortho-carboxy-substituted tetraarylporphyrin.

The topology of the binding site has been studied for two monoclonal antibodies 13G10 and 14H7, elicited against iron(III)-alpha,alpha,alpha,beta-meso-tetrakis(ortho-carboxyphenyl)porph yrin [alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin]], and which exhibit in the presence of this alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] cofactor a peroxidase activity. A comparison of the dissociation constants of the complexes of 13G10 and 14H7 with various tetra-aryl-substituted porphyrin has shown that: (a) the central iron(III) atom of alpha,alpha,alpha,beta-Fe[(o-COOHPh)4-porphyrin] is not recognized by either of the two antibodies; and (b) the ortho-carboxylate substituents of the meso-phenyl rings of alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] are essential for the recognition of the porphyrin by 13G10 and 14H7. Measurement of the dissociation constants for the complexes of 13G10 and 14H7 with the four atropoisomers of (o-COOHPh)4-porphyrinH2 as well as mono- and di-ortho-carboxyphenyl-substituted porphyrins suggests that the three carboxylates in the alpha, alpha, beta position are recognized by both 13G10 and 14H7 with the two in the alpha, beta positions more strongly bound to the antibody protein. Accordingly, the topology of the active site of 13G10 and 14H7 has roughly two-thirds of the alpha,alpha,alpha,beta-Fe[(o-COOHPh)4-porphyrin] cofactor inserted into the binding site of the antibodies, with one of the aryl ring remaining outside. Three of the carboxylates are bound to the protein but no amino acid residue acts as an axial ligand to the iron atom. Chemical modification of lysine, histidine, tryptophan and arginine residues has shown that only modification of arginine residues causes a decrease in both the binding of alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] and the peroxidase activity of both antibodies. Consequently, at least one of the carboxylates of the hapten is bound to an arginine residue and no amino acids such as lysine, histidine or tryptophan participate in the catalysis of the heterolytic cleavage of the O-O bond of H2O2. In addition, the amino acid sequence of both antibodies not only reveals the presence of arginine residues, which could be those involved in the binding of the carboxylates of the hapten, but also the presence of several amino acids in the complementary determining regions which could bind other carboxylates through a network of H bonds.

Detection of a nitric oxide synthase possibly involved in the regulation of the Rhodococcus sp R312 nitrile hydratase.

Crude homogenates from Rhodococcus sp 312 catalyze the conversion of L-arginine into L-citrulline and NO2-, the usual oxidation product of NO under aerobic conditions. They also catalyze the conversion of N omega-hydroxy-L-arginine (NOHA) into L-citrulline and NO2- with similar rates (10-15 and 100-150 nmol of product.min-1.(mg of protein)-1 respectively for the crude homogenate and for a fraction obtained from ammonium sulfate precipitation). L-citrulline formation is strongly inhibited by classical inhibitors of mammalian nitric oxide synthases (NOSs) such as N omega-methyl-L-arginine (NMA) and thio-L-citrulline (TC). Finally, the lack of inhibitory effects of EGTA, a classical inhibitor of constitutive mammalian NOSs, and the specific immunodetection of a 100 kD protein from Rhodococcus cytosol by an antibody raised against human inducible NOS, is in favor of the presence of a NOS similar to inducible mammalian NOSs in Rhodococcus sp 312. This NOS should be responsible for the NO-dependent inactivation of Rhodococcus Nitrile Hydratase (NHase) in the absence of light; it could regulate the activity of the latter enzyme.

Substrate specificity of NO synthases: detailed comparison of L-arginine, homo-L-arginine, their N omega-hydroxy derivatives, and N omega-hydroxynor-L-arginine.

A detailed comparison of the oxidation of five compounds closely related to L-arginine (Arg) by purified recombinant neuronal and macrophage NO synthases (NOS I and NOS II) was performed. Homo-L-arginine (homo-Arg) is oxidized by both NOSs in the presence of NADPH with major formation of NO and homo-L-citrulline, with a molar ratio of close to 1, and minor formation of N omega-hydroxyhomo-L-arginine (homo-NOHA). Oxidation of homo-NOHA by the two NOSs also leads to NO and homocitrulline in a 1:1 molar ratio. On the contrary, N omega-hydroxynor-L-arginine (nor-NOHA) is a very poor substrate of NOS I and II, which fails to produce significant amounts of nitrite. The catalytic efficiency of both NOSs markedly decreases in the order Arg > NOHA > homo-Arg > homo-NOHA, as shown by the 20- and 10-fold decrease of kcat/Km observed for NOS I and NOS II, respectively, when comparing Arg to homo-NOHA. The greater loss of catalytic efficiency for homo-Arg, when compared to that for Arg, appears to occur at the first step (N-hydroxylation) of the reaction. In that regard, it is noteworthy that the Vm values for NOHA and homo-NOHA oxidation are very similar (about 1 and 2 micromol of NO min-1 mg of protein-1 for NOS I and II, respectively). In fact, lengthening of the Arg chain by one CH2 leads not only to markedly decreased kcat/Km but also to clear disturbances in NOS functioning. This is shown by a greater accumulation of the N omega-hydroxyguanidine intermediate (homo-NOHA:homocitrulline ratio between 0.2 and 0.4) and an increased consumption of NADPH for NO formation (between 2.0 and 2.6 mol of NADPH consumed for the formation of 1 mol of NO in the case of homo-Arg, instead of 1.5 mol in the case of Arg). Most of the above results could be interpreted by comparing the possible positionings of the various substrates relative to the two NOS active oxygen species which are believed to be responsible for the two steps of the reaction.

Formation of nitric oxide synthase-iron(II) nitrosoalkane complexes: severe restriction of access to the iron(II) site in the presence of tetrahydrobiopterin.

Nitric oxide synthases (NOS) are heme proteins, closely related to cytochromes P450, that catalyze oxidation of l-arginine (l-Arg) to nitric oxide (NO) and citrulline. To get further insight into their active site, we have studied the ability of recombinant mouse inducible NOS (iNOS) and rat brain neuronal NOS (nNOS), and of their oxygenase domains (iNOSoxy and nNOSoxy), to form Fe(II)-nitrosoalkane complexes. In the absence of BH4, iNOSoxy, nNOSoxy, and full-length iNOS readily form complexes characterized by Soret peaks around 448 nm, after reaction with various nitroalkanes and sodium dithionite. These complexes displayed physicochemical characteristics very similar to those of previously reported microsomal cytochrome P450-Fe(II)-nitrosoalkane complexes: (i) a Soret peak around 450 nm, (ii) a clear stability in the presence of CO, and (iii) a fast destruction upon oxidation of the iron by ferricyanide. Thus, in the absence of l-Arg and BH4, NOSs Fe(II) appear to be largely opened to even large R-NO ligands with R = cyclohexyl or p-Cl-C6H4-CH2CH(CH3) for instance, in a manner similar to microsomal P450s Fe(II). As expected, the presence of l-Arg inhibits the formation of NOSs Fe(II)-RNO complexes. More surprisingly, the presence of BH4 also strongly inhibits the formation of the NOSs Fe(II) complexes even with the smallest nitrosoalkane ligand, CH3NO (IC50 values of 0.5 and 4 microM for nNOSoxy and iNOSoxy, respectively). Accordingly, recombinant full-length nNOS containing BH4 and l-Arg is completely unable to form Fe(II)-nitrosoalkane complexes, even with CH3NO. These results suggest that, in the absence of l-Arg and BH4, the distal pocket of NOSs Fe(II) is largely opened even to bulky ligands, in a manner similar to that of microsomal cytochromes P450. On the contrary, the distal heme pocket of iNOS and nNOS seems to be closed after binding of l-Arg and BH4, particularly in the Fe(II) state. This results in a highly restricted access for Fe(II) ligands, except very small ones such as CO, NO, and O2. Such effects of BH4 in controlling the size of the distal heme pocket of NOS Fe(II) correspond to a new role of biopterins in biological systems.

Bromocriptine is a strong inhibitor of brain nitric oxide synthase: possible consequences for the origin of its therapeutic effects.

The ergot alkaloid bromocriptine (BKT) was found to act as a strong inhibitor of purified neuronal nitric oxide synthase (NOS) (IC50 = 10 +/- 2 microM) whereas it was poorly active towards inducible macrophage NOS (IC50 > 100 microM). BKT affects the activation of NOS by calmodulin, as it not only inhibits L-arginine oxidation to NO and L-citrulline but also NADPH oxidation and calmodulin-dependent cytochrome c reduction catalyzed by neuronal NOS. These results suggest that BKT could exert some of its therapeutic effects by interfering with the NOS-dependent formation of nitric oxide and/or superoxide ion in various tissues.

Strong inhibition of neuronal nitric oxide synthase by the calmodulin antagonist and anti-estrogen drug tamoxifen.

The anti-estrogen drug tamoxifen (TMX) was found to act as a strong inhibitor of purified neuronal nitric oxide synthase (nNOS) (IC50 = 2 +/- 0.5 microM), whereas it was inactive toward inducible macrophage NOS (IC50 > 100 microM). TMX affected the activation of NOS by calmodulin, as it not only inhibited L-arginine oxidation to nitric oxide and L-citrulline but also NADPH oxidation and calmodulin-dependent cytochrome c reduction catalyzed by nNOS. These results suggest that TMX could exert some of its biological effects by interfering with constitutive NOS-dependent formation of nitric oxide and/or superoxide ion in various tissues.

Expression in yeast and purification of functional macrophage nitric oxide synthase. Evidence for cysteine-194 as iron proximal ligand.

Mouse macrophage NO-synthase (mNOS) was expressed in a unique yeast-based system by using a three-step procedure which allows yeast growth and NOS expression to be uncoupled. Despite cytotoxic effects related to mNOS expression, levels of catalytically active enzyme up to 0.5 mg of protein per 5 L of culture was obtained after purification. Its electrophoretic, spectroscopic [lambda max = 446 nm for its Fe(II)-CO complex], and catalytic properties were similar to those previously reported for mNOS purified from macrophages. Recombinant mNOS catalyzed the NADPH-dependent oxidation of L-arginine to citrulline (Km = 7 +/- 3 microM) as well as the reduction of cytochrome C by NADPH [Km = 34 +/- 8 microM and Vm = 25 +/- 5 mumol min-1 (mg of protein-1)]. Two mutants of mNOS in which Cys 194 was replaced with either serine or histidine were constructed and expressed in the same yeast strain at a level higher than that of the wild type protein, as they appear less toxic for the host. Both mutants exhibited electrophoretic properties and activities toward cytochrome C reduction identical to those of wild type NOS. However, they were unable to catalyze the oxidation of L-arginine to citrulline and did not appear to bind heme (no appearance of peaks around 400 and 446 nm for the resting enzyme and its CO complex, respectively, in visible spectroscopy). These data provide the first experimental evidence in favor of previous suggestions that Cys 194 was the proximal iron ligand of mouse mNOS.

Expression of human cytochrome P450 enzymes in yeast and bacteria and relevance to studies on catalytic specificity.

Heterologous expression systems can be utilized to great advantage in the study of cytochrome P450 (P450) and other enzymes involved in the biotransformation of drugs and other xenobiotics. The list of studies made possible with the technology includes discernment of catalytic specificity, elucidation of structure-activity relationships, and various biophysical measurements. There are advantages and disadvantages to each of the vector systems and choices must be made on the basis of needs. Yeast expression systems were used to establish that different P450 2C enzymes are involved in the hydroxylations of tolbutamide and (S)-mephenytion. P450 3A4 was also expressed in yeast and its very broad catalytic specificity was confirmed. Recently, it has been possible to express P450 3A4 as well as other human and animal P450s in bacteria after slight modification of their 5'-coding sequences.

Structure-activity relationships for DNA photocleavage by cationic porphyrins.

The influence of molecular structure and DNA binding mode on the ability of cationic porphyrins to photosensitize DNA strand break formation has been studied for a series of meso-substituted pyridinium porphyrins using electrophoretic and DNA sequencing techniques. Porphyrins substituted with pyridyl groups in which the heterocyclic nitrogen is in the para or meta position vis-à-vis the substitution point are capable of intercalative binding and are considerably more efficient DNA photosensitizers than the corresponding non-intercalating ortho compounds. Within each group of porphyrins the photosensitizer efficiency increases with the number of positive charges. Using DNA sequencing experiments, we have demonstrated that photomodification occurs primarily at the guanine and thymine bases, and that alkali-labile sites produced by photo-oxidation are as important as direct cleavage events. The kinetics of strand degradation in aerated and degassed solution suggest that type II reactions (probably mediated by singlet oxygen) occur with significantly higher yield than type I reactions and are responsible for the formation of alkali-labile sites in aerated systems. These observations seem to confirm the hypothesis that those structural features which influence the strength and mode of binding also serve to establish favourable porphyrin-DNA interactions for photosensitization.

Oxidation of dihydropyridine calcium channel blockers and analogues by human liver cytochrome P-450 IIIA4.

A series of 21 different 4-substituted 2,6-dimethyl-3-(alkoxycarbonyl)-1,4-dihydropyridines was considered with regard to oxidation to pyridine derivatives by human liver microsomal cytochrome P-450 (P-450). Antibodies raised against P-450 IIIA4 inhibited the microsomal oxidation of nifedipine and felodipine to the same extent, as did cimetidine and the mechanism-based inactivator gestodene. Gestodene was approximately 10(3) times more effective an inhibitor than cimetidine, on a molar basis. When rates of oxidation of the 1,4-dihydropyridines were compared to each other in different human liver microsomal preparations, all were highly correlated with each other with the exceptions of a derivative devoid of a substituent at the 4-position and an N1-CH3 derivative. A P-450 IIIA4 cDNA clone was expressed in yeast and the partially purified protein was used in reconstituted systems containing NADPH-cytochrome P-450 reductase and cytochrome b5. This system catalyzed the oxidation of all of the 1,4-dihydropyridines except the two for which poor correlation was seen in the liver microsomes. Principal component analysis supported the view that most of these reactions were catalyzed by the same enzyme in the yeast P-450 IIIA4 preparation and in the different human liver microsomal preparations, or by a closely related enzyme showing nearly identical properties of catalytic specificity and regulation. The results indicate that the enzyme P-450 IIIA4 is probably the major human catalyst involved in the formal dehydrogenation of most but not all 1,4-dihydropyridine drugs.

Catalytic activities of human liver cytochrome P-450 IIIA4 expressed in Saccharomyces cerevisiae.

A human liver cytochrome P-450 (P-450) IIIA4 cDNA clone was inserted behind an alcohol dehydrogenase promoter in the plasmid vector pAAH5 and expressed in Saccharomyces cerevisiae (D12 and AH22 strains). A cytochrome P-450 with typical spectral properties was expressed at a level of approximately 8 x 10(5) molecules/cell in either strain of yeast. The expressed P-450 IIIA4 had the same apparent monomeric Mr as the corresponding protein in human liver microsomes (P-450NF) and could be isolated from yeast microsomes. Catalytic activity of the yeast microsomes toward putative P-450 IIIA4 substrates was seen in the reactions supported by cumene hydroperoxide but was often lower and variable when supported by the physiological donor NADPH. The catalytic activity of purified P-450 IIIA4 was also poor in some systems reconstituted with rabbit liver NADPH-P-450 reductase and best when both the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and a lipid extract (from liver or yeast microsomes) or L-alpha-1,2-dilauroyl-sn-glycero-3-phosphocholine were present. Under these conditions the expressed P-450 IIIA4 was an efficient catalyst for nifedipine oxidation, 6 beta-hydroxylation of testosterone and cortisol, 2-hydroxylation of 17 beta-estradiol and 17 alpha-ethynylestradiol, N-oxygenation and 3-hydroxylation of quinidine, 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate, erythromycin N-demethylation, the 10-hydroxylation of (R)-warfarin, the formation of 9,10-dehydrowarfarin from (S)-warfarin, and the activation of aflatoxins B1 and G1, sterigmatocystin, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (both + and - diastereomers), 3,4-dihydroxy-3,4-dihydrobenz[a]anthracene, 3,4-dihydroxy-3,4-dihydro-7, 12-dimethylbenz[a]anthracene, 9,10-dihydroxy-9,10-dihydrobenzo[b]fluoranthene, 6-aminochrysene, and tris(2,3-dibromopropyl) phosphate to products genotoxic in a Salmonella typhimurium TA1535/pSK1002 system where a chimeric umuC' 'lacZ plasmid is responsive to DNA alkylation. Reaction rates were stimulated by 7,8-benzoflavone and inhibited by rabbit anti-P-450 IIIA (anti-P-450NF), troleandomycin, gestodene, and cimetidine. Evidence was obtained that rates of reduction of ferric P-450 IIIA4 in yeast microsomes and the reconstituted systems are slow and at least partially responsible for the lower rates of catalysis seen in these systems (relative to liver microsomes). The results of these studies with a defined protein clearly demonstrate the ability of P-450 IIIA4 to catalyze regio- and stereoselective oxidations with a diverse group of substrates, and this enzyme appears to be one of the most versatile catalysts in the P-450 family.

Studies on the expression and metabolic capabilities of human liver cytochrome P450IIIA5 (HLp3).

The human P450III family has been shown to be composed of at least four members, P450IIIA3 (HLp), P450IIIA4 (P450NF), P450IIIA5 (HLp3), and P450IIIA6 (HLp2). Due to the lack of probes that specifically recognize the individual members of this family, little is known about their relative expression. We prepared a form-specific antibody to P450IIIA5 by immunoabsorption of anti-P450IIIA5 IgG against Sepharose 4B upon which microsomes that did not contain P450IIIA5 or purified P450IIIA3 had been bound. Immunoblot analyses demonstrated that P450IIIA5 was expressed at detectable levels in only 19 of 66 (29%) human livers. The expression of P450IIIA5 was not influenced by the gender or medical history of the patients. When the expression of P450IIIA5 in different age groups was examined, it was observed that P450IIIA5 was detected in a statistically significantly higher percentage of children and adolescents (19 years old and under), as compared with the remaining population (8 of 17, 47%, versus 11 of 46, 24%, respectively). Furthermore, P450IIIA5 was detected in 1 of 10 human fetal livers. Of the large number of compounds identified as substrates of P450III family members, P450IIIA5 was found to actively metabolize nifedipine, testosterone, estradiol, dehydroepiandrosterone 3-sulfate, and cortisol, whereas it metabolized poorly or did not metabolize erythromycin, quinidine, 17 alpha-ethynylestradiol, and aflatoxins. The acetylenic steroid gestodene was found to be an effective mechanism-based inhibitor of both P450IIIA4 and P450IIIA5. Immunoblots of microsomes isolated from untreated and dexamethasone-, phenobarbital-, or 3-methylcholanthrene-treated HepG2 cells that were developed with an antibody that recognizes all the P450III family members demonstrated that no proteins in the P450III family were expressed by the HepG2 cells. In conclusion, our studies indicate that P450IIIA5 is polymorphically expressed at all stages of human development and is more limited in its metabolic capabilities than is P450IIIA4.

Interaction of cationic porphyrins with DNA: importance of the number and position of the charges and minimum structural requirements for intercalation.

Thirty-three porphyrins or metalloporphyrins corresponding to the general formula [meso-[N-methyl-4(or 3 or 2)-pyridiniumyl]n(aryl)4-nporphyrin]M (M = H2, CuII, or ClFeIII), with n = 2-4, have been synthesized and characterized by UV-visible and 1H NMR spectroscopy and mass spectrometry. These porphyrins differ not only in the number (2-4) and position of their cationic charges but also in the steric requirements to reach even temporarily a completely planar geometry. In particular, they contain 0, 1, 2, 3, or 4 meso-aryl substituents not able to rotate. Interaction of these porphyrins or metalloporphyrins with calf thymus DNA has been studied and their apparent affinity binding constants have been determined by use of a competition method with ethidium bromide which was applicable not only for all the free base porphyrins but also for their copper(II) or iron(III) complexes. Whatever their mode of binding may be, their apparent affinity binding constants were relatively high (Kapp between 1.2 x 10(7) and 5 x 10(4) M-1 under our conditions), and a linear decrease of log Kapp with the number of porphyrin charges was observed. Studies of porphyrin-DNA interactions by UV and fluorescence spectroscopy, viscosimetry, and fluorescence energy transfer experiments showed that not only the tetracationic meso-tetrakis[N-methyl-4(or 3)-pyridiniumyl]porphyrins, which both involved four freely rotating meso-aryl groups, but also the corresponding tri- and dicationic porphyrins were able to intercalate into calf thymus DNA. Moreover, the cis dicationic meso-bis(N-methyl-2-pyridiniumyl)diphenylporphyrin, which involved only two freely rotating meso-aryl groups in a cis position, was also able to intercalate. The other meso-(N-methyl-2-pyridiniumyl)n(phenyl)4-nporphyrins, which involved either zero, one, or two trans freely rotating meso-aryl groups, could not intercalate into DNA. These results show that only half of the porphyrin ring is necessary for intercalation to occur.