Prostaglandin and fatty acid omega- and (omega-1)-oxidation in rabbit lung. Acetylenic fatty acid mechanism-based inactivators as specific inhibitors

Terminal acetylenic fatty acid mechanism-based inhibitors (Ortiz de Montellano, P. R., and Reich, N. O. (1984) J. Biol. Chem. 259, 4136-4141) were used as probes in determining the substrate specificity of rabbit lung cytochrome P-450 isozymes of pregnant animals in both microsomes and reconstituted systems. Lung microsomal and reconstituted P-450 form 5-catalyzed lauric acid omega- and (omega-1)-hydroxylase activities were inhibited by a 12-carbon terminal acetylenic fatty acid, 11-dodecynoic acid (11-DDYA), and an 18-carbon terminal acetylenic fatty acid, 17-octadecynoic acid (17-ODYA). Rabbit lung microsomal lauric acid omega-hydroxylase activity was more sensitive to inhibition by 11-DDYA than was (omega-1)-hydroxylase activity. In reconstituted systems containing purified P-450 form 5, both omega- and (omega-1)-hydroxylation of lauric acid were inhibited in parallel when either 11-DDYA or 17-ODYA was used. These data suggest the presence of at least two P-450 isozymes in rabbit lung microsomes capable of lauric acid omega-hydroxylation. This is the first report indicating the multiplicity of lauric acid hydroxylases in lung microsomes. Lung microsomal prostaglandin omega-hydroxylation, mediated by the pregnancy-inducible P-450PG-omega (Williams, D. E., Hale, S. E., Okita, R. T., and Masters, B. S. S. (1984) J. Biol. Chem. 259, 14600-14608) was subject to inhibition by 17-ODYA only, whereas 11-DDYA acid was not an effective inhibitor of this hydroxylase. We have recently developed a new terminal acetylenic fatty acid, 12-hydroxy-16-heptadecynoic acid (12-HHDYA), that contains a hydroxyl group at the omega-6 position. We show that 12-HHDYA possesses a high degree of selectivity for the inactivation of rabbit lung microsomal prostaglandin omega-hydroxylase activity which cannot be obtained with the long chain acetylenic inhibitor, 17-ODYA. In addition, 12-HHDYA has no effect on lauric acid omega- or omega-1-hydroxylation or on benzphetamine N-demethylation. The development of this new terminal acetylenic fatty acid inhibitor provides us with a useful tool with which to study the physiological role of prostaglandin omega-hydroxylation in the rabbit lung during pregnancy.     

Mechanism-based in vivo inactivation of lauric acid hydroxylases

The hepatic cytochrome P-450 isozymes that catalyze omega- and (omega - 1)-hydroxylation of lauric acid are specifically inactivated in vitro but not in vivo by 10-undecynoic acid. The lack of in vivo activity may result from rapid degradation of the inhibitor by beta-oxidation. Strategies for the construction of fatty acid analogues that retain the ability to inactivate fatty acid hydroxylases but are resistant to metabolic degradation have therefore been sought. Fatty acid analogues in which the carboxylic acid group is replaced by a sulfate moiety, or in which two methyl groups are placed vicinal to the carboxylic acid group, have been found to inactivate lauric acid hydroxylases in vitro and in vivo without causing time-dependent inhibition of ethoxycoumarin O-deethylation or N-methyl-p-chloroaniline N-demethylation.     

Subterminal hydroxylation of fatty acids by a cytochrome P-450-dependent enzyme system from a fungus, Fusarium oxysporum

The cell-free extract of a cytochrome P-450-producing fungus, Fusarium oxysporum, was found to catalyze the hydroxylation of fatty acids. Three product isomers were formed from a single fatty acid. The products from lauric acid were identified by mass-spectrometry as 9-, 10-, and 11-hydroxydodecanoic acids, and those from palmitic acid as 13-, 14-, and 15-hydroxyhexadecanoic acids. The ratio of the isomers formed was 50 : 36 : 14 in the case of laurate hydroxylation, and 37 : 47 : 16 in the case of palmitate. The reaction was dependent on both NADPH (or NADH) and molecular oxygen,and was strongly inhibited by carbon monoxide, menadione, or the antibody to purified Fusarium P-450. Further, lauric acid induced a type I spectral change in purified Fusarium P-450. Further, lauric acid induced a type I spectral change in purified Fusarium P-450 with an apparent Kd of 0.3 mM. The hydroxylase activity together with cytochrome P-450 could be detected in both the soluble and microsome fractions, and the activity was almost proportional to the amount of cytochrome P-450 reducible with NADPH. It can be concluded from these results that Fusarium P-450 reducible with NADPH. It can be concluded from these results that Fusarium P-450 is involved in the (omega-1)-, (omega-2)-, and (omega-3)-hydroxylation of fatty acids catalyzed by the cell-free extract of the fungus.     

Omega- and (omega-1)-hydroxylation of lauric acid and arachidonic acid by rat renal cytochrome P-450

We resolved four cytochrome P-450s, designated as P450 K-2, K-3, K-4, and K-5, from the renal microsomes of untreated male rats by high-performance liquid chromatography (HPLC) and investigated the lauric acid and arachidonic acid hydroxylation activities of these fractions. P450 K-4 and K-5 had high omega- and (omega-1)-hydroxylation activities toward lauric acid. The ratio of the omega-/(omega-1)-hydroxylation activity of P450 K-4 and K-5 was 3 and 6, respectively. Also, P450 K-4 and K-5 effectively catalyzed the omega- and (omega-1)-hydroxylation of arachidonic acid. P450 K-3 was not efficient in the hydroxylation of either lauric acid or arachidonic acid. P450 K-2 had low omega- and (omega-1)-hydroxylation activities toward arachidonic acid, and efficiently catalyzed the hydroxylation of lauric acid at the (omega-1)-position only, not at the omega-position.     

Purification and NH2-terminal sequence of cytochrome P-450 from kidney microsomes of untreated male rats

The major form of cytochrome P-450, P-450K-5, was purified from kidney microsomes of untreated male rats with high-performance liquid chromatography with anion-exchange and hydroxylapatite columns. The monomeric molecular weight of P-450K-5 was 52000 on SDS-polyacrylamide gel electrophoresis and the CO-reduced absorption maximum was at 452 nm. P-450K-5 catalyzed the omega- and (omega-1)-hydroxylation of lauric acid, but was inefficient in the N-demethylation of benzphetamine and the O-dealkylation of 7-ethoxycoumarine. The NH2-terminal sequence of P-450K-5 was quite different from cytochrome P-450s purified from rat hepatic microsomes.     

Regiospecificity in the hydroxylation of lauric acid by rainbow trout hepatic cytochrome P450 isozymes

The catalytic activity of two hepatic cytochrome P450 isozymes from untreated rainbow trout towards lauric acid was investigated. In a reconstituted system, cytochrome P450 LMC1 and P450 LMC2 were found to catalyze exclusively the omega- and (omega-1)-hydroxylation of lauric acid, respectively. Microsomal enzyme inhibition studies with polyclonal antibodies raised against the individual P450 isozymes showed that P450 LMC1 and LMC2, respectively, accounted for most if not all the omega- and (omega-1)-lauric acid hydroxylase activity of trout liver microsomes. The polyclonal antibodies were highly specific in that they only inhibited the enzyme activity of the P450 used as the immunogen. These results illustrate that as in mammals, omega- and (omega-1)-hydroxylation of lauric acid by trout liver microsomes can be carried out separately by distinct isozymes of cytochrome P450.     

cDNA cloning and expression of the mRNA for cytochrome P-450kd which shows a fatty acid omega-hydroxylating activity

We have recently purified three distinct forms of fatty acid omega-hydroxylase cytochrome P-450 (P-450), designated P-450ka-1, P-450ka-2 and P-450kd, from rabbit kidney cortex microsomes, and isolated and sequenced cDNA clones corresponding to P-450ka-1 and P-450ka-2 [Yokotani, N., Bernhardt, R., Sogawa, K., Kusunose, E., Gotoh, M., Kusunose, M. & Fujii-Kuriyama, Y. (1989) J. Biol. Chem. 264, 21,665-21,669]. The present paper describes cloning, sequencing and expression of a cDNA for the third fatty acid, omega-hydroxylase, P-450kd, from a rabbit kidney cDNA library. The cDNA for P-450kd encodes a polypeptide of 511 amino acids with sequence similarity of 87% to P-450ka-1. Its deduced NH2-terminal sequence of amino acids 5-24 is in complete agreement with the NH2-terminal sequence of P-450kd. The identity of the cDNA was further confirmed by its expression in COS-7 cells. When 14C-labeled lauric acid was added to the culture medium of COS-7 cells transfected with the cDNA, significant amounts of radioactive dodecanedioic acid, together with omega- and (omega-1)-hydroxylauric acids, were produced. Microsomes prepared from the transfected cells also efficiently catalyzed the omega- and (omega-1)-hydroxylation of lauric acid without formation of dodecanedioic acid. RNA blot analysis demonstrated that the mRNA for P-450kd gave a single band at the approximately 2.6-kb position. The mRNA for P-450kd was expressed in the liver and kidney, but not in many other tissues examined. Treatment of rabbits with clofibrate resulted in a elevated level of mRNA for P-450kd in both liver and kidney. Furthermore, the mRNA was remarkably increased in the kidney by the administration of cyclosporin A.     

Hepatic and renal cytochrome P-450s in spontaneously hypertensive rats

The differences in the levels of cytochrome P-450s in hepatic and renal microsomes between spontaneously hypertensive rats (SHR) and normotensive control rats (Wistar Kyoto rats, WKY) were investigated by Western blotting with a specific antibody. Differences in the metabolic activity of the microsomes were also studied. In hepatic microsomes, the content of P450 PB-1 (IIIA2) was 140% higher in SHR than in WKY and the content of P450 IF-3 (IIA1) in SHR was one-seventh that in WKY. The differences reflected the increase in testosterone 6 beta-hydroxylation activity and decrease in testosterone 7 alpha-hydroxylation activity in hepatic microsomes of SHR. The level of P450 K-5 (IVA2) in hepatic microsomes of SHR was 4-times that in microsomes of WKY. The levels of other cytochrome P-450s in SHR were not very different from those in WKY. In renal microsomes, the levels of three renal cytochrome P-450s, P450 K-2, K-4, and K-5, were measured. The level of P450 K-5 (fatty acid omega-hydroxylase) in SHR was 50% higher than that in WKY and the difference reflected the increase in lauric acid omega- and (omega-1)-hydroxylation activities of the renal microsomes of SHR. The levels of P450 K-2 and K-4 did not differ in both rats.     

Induction of renal cytochrome P-450 in hepatic microsomes of diabetic rats

We purified two forms of cytochrome P-450 which was induced in hepatic microsomes of diabetic male rates treated with streptozotocin. One of these corresponded to P-450j. The other form, designated P450 DM-2, had a minimum molecular weight 53000 and a CO-reduced absorption maximum at 452 nm. The P450 DM-2 efficiently catalyzed the omega- and (omega-1)-hydroxylation of lauric acid, but was not efficient in metabolizing aminopyrine, 7-ethoxycoumarin, aniline, N-nitrosodimethylamine, or testosterone. The NH2-terminal sequence of P450 DM-2 was identical to that of P450 K-5, the major renal cytochrome P-450. Both forms gave very similar electrophoretic patterns of proteolytic digests. P450 DM-2 and P450 K-5 are closely related forms.     

Isolation of a new form of cytochrome P-450 with prostaglandin A and fatty acid omega-hydroxylase activities from rabbit kidney cortex microsomes

Two different forms of cytochrome P-450, highly active in the omega-hydroxylation of prostaglandin A, and the omega- and (omega-1)-hydroxylation of fatty acids (P-450ka-1 and P-450ka-2), have been purified from kidney cortex microsomes of rabbits treated with di(2-ethylhexyl)-phthalate. On the basis of the peptide map patterns and NH2-terminal amino acid sequence, P-450ka-1 was determined to be a new form of omega-hydroxylase cytochrome P-450, whereas P-450ka-2 is identical to P-450ka reported earlier. The first 20 NH2-terminal amino acid sequence (ALNPTRLPGSLSGLLQVAGL) and (ALSPTRLPGSFSGFLQAAGL) of P-450ka-1 and P-450ka-2 showed 90 and 80% homology with that of the lung prostaglandin omega-hydroxylase, respectively, suggesting that these three cytochromes P-450 are members of the same omega-hydroxylase cytochrome P-450 gene family.     

Circulating C-terminal propeptide of type I procollagen is cleared mainly via the mannose receptor in liver endothelial cells.

The cytochrome P-450 enzyme which catalyses 25-hydroxylation of vitamin D3 (cytochrome P-450(25] from pig kidney microsomes [Postlind & Wikvall (1988) Biochem. J. 253, 549-552] has been further purified. The specific content of cytochrome P-450 was 15.0 nmol.mg of protein-1, and the protein showed a single spot with an apparent isoelectric point of 7.4 and an Mr of 50,500 upon two-dimensional isoelectric-focusing/SDS/PAGE. The 25-hydroxylase activity towards vitamin D3 was 124 pmol.min-1.nmol of cytochrome P-450-1 and towards 1 alpha-hydroxyvitamin D3 it was 1375 pmol.min-1.nmol-1. The preparation also catalysed the 25-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha-diol at a rate of 1000 pmol.min-1.nmol of cytochrome P-450-1 and omega-1 hydroxylation of lauric acid at a rate of 200 pmol.min-1.nmol of cytochrome P-450-1. A monoclonal antibody raised against the 25-hydroxylating cytochrome P-450, designated mAb 25E5, was prepared. After coupling to Sepharose, the antibody was able to bind to cytochrome P-450(25) from kidney as well as from pig liver microsomes, and to immunoprecipitate the activity for 25-hydroxylation of vitamin D3 and 5 beta-cholestane-3 alpha,7 alpha-diol when assayed in a reconstituted system. The hydroxylase activity towards lauric acid was not inhibited by the antibody. By SDS/PAGE and immunoblotting with mAb 25E5, cytochrome P-450(25) was detected in both pig kidney and pig liver microsomes. These results indicate a similar or the same species of cytochrome P-450 in pig kidney and liver microsomes catalysing 25-hydroxylation of vitamin D3 and C27 steroids. The N-terminal amino acid sequence of the purified cytochrome P-450(25) from pig kidney microsomes differed from those of hitherto isolated mammalian cytochromes P-450.     

Differential expression of cytochrome P-450 1 and related forms in rabbit liver and kidney

Monoclonal antibodies developed to cytochrome P-450 1, some of which react with proteins in addition to P-450 1, were used to investigate the differential expression of P-450 1 dependent 21-hydroxylase activity in renal tissue of rabbits exhibiting differences in hepatic 21-hydroxylase activity. Using immunohistochemical techniques, the monoclonal antibodies, 2F5 and 3C3, localized protein in the S2 and S3 segments of the proximal tubule in the renal cortex. These two monoclonal antibodies, 2F5 and 3C3, reacted with a kidney protein that migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a relative electrophoretic mobility that did not correspond to known rabbit hepatic isozymes and was termed P-450 K. Antibodies specific for P-450 1 and 3b, 1F11 and 8-27, respectively, produced no staining in kidney. The protein recognized by the 2F5 and 3C3 antibodies is immunologically distinct from cytochrome P-450s 1, 2, and 3b. The rate of 21-hydroxylation of progesterone was shown to be approximately 100-fold less in kidney than liver microsomes where this pathway is largely catalyzed by P-450 1. The activity of the kidney microsomes was not inhibited by antibodies directed to P-450 1. In addition, the variation observed for the 21-hydroxylase activity in the hepatic microsomal fraction of outbred New Zealand white rabbits was not evident in kidney microsomes from these same animals. The 2F5 antibody was found, however, to be inhibitory (about 50%) of the 11-hydroxylation of lauric acid in kidney microsomes. This suggests that P-450 K participates in lauric acid 11-hydroxylase activity. The treatment of rabbits with phenobarbital, but not 2,3,7,8-tetrachlorodibenzo-p-dioxin, was found to induce the levels of P-450 K.     

Biochemical and ultrastructural studies on the interaction of basic proteins with mitochondria: a primary effect on membrane configuration

The cytochrome P-450_K containing monooxygenase system of rat kidney cortex microsomes catalyzes the hydroxylation of various saturated fatty acids of medium chain length to the corresponding ω- and (ω-1)-hydroxy derivatives. The hydroxylation activity, as well as the ratio between the two hydroxylated products, vary with the carbon chain length of the fatty acid. Optimal hydroxylation activity is observed with myristic acid which yields the 13- and 14-hydroxylated products at a ratio of about 1. The ω/(ω-1)-hydroxylation ratio decreases with increasing carbon chain length of the fatty acid. On the other hand, with lauric acid as a substrate the ratio between ω- and (ω-1)-hydroxylation does not change significantly with varying time of incubation or substrate concentration, or incubation in a medium containing D₂O or after induction of enhanced hydroxylation activity by starvation of the animals. Furthermore, 12-hydroxylauric acid and capric acid—which is almost exclusively ω-hydroxylated by rat kidney cortex microsomes—inhibit both 11- and 12-hydroxylation of lauric acid to a similar extent whereas 11-hydroxylauric acid does not seem to inhibit either 11- or 12-hydroxylation.C₁₀-C₁₆ fatty acids produce the type I spectral change upon addition to rat kidney cortex microsomes and seem to interact with similar amounts of the cytochrome P-450_K present in these particles. In agreement with the metabolic studies, 12-hydroxylauric acid interacts with cytochrome P-450_K giving rise to a reverse type I spectral change, whereas 11-hydroxylauric acid does not produce an observable spectral change. Finally, results of binding experiments with a series of derivatives of dodecane suggest that type I binding to cytochrome P-450_K requires, besides a proper chain length, the presence of a carbonyl group together with an electron pair on a neighboring atom at the end of the carbon chain. A chain length of 14 carbon atoms seems to be optimal and it is suggested that this chain length may correspond to the distance between a possible binding site and the catalytic site of cytochrome P-450_K     

Differential induction of peroxisomal and microsomal fatty-acid-oxidising enzymes by peroxisome proliferators in rat liver and kidney. Characterisation of a renal cytochrome P-450 and implications for peroxisome proliferation

The induction of renal fatty-acid-oxidising enzymes has been investigated following short-term exposure to a group of structurally diverse peroxisome proliferators and compared to the more extensively documented hepatic responses in the rat. There was a marked compound dependence on induction of both cytochrome P-450-IVA1-dependent omega-hydroxylation of lauric acid and enzymes of the peroxisomal fatty acid beta-oxidation pathway (measured as cyanide-insensitive palmitoyl-CoA oxidation and enoyl-CoA hydratase). Cytochrome P-450 IVA1 (or a very closely related isoenzyme in the same gene family) was a major constitutive haemoprotein in rat kidney microsomes and actively supported the omega-hydroxylation of lauric acid. This activity was induced 2-3-fold by peroxisome proliferators such as clofibrate, di-(2-ethylhexyl)phthalate, bezafibrate and nafenopin. By using a cDNA probe to the cytochrome P-450 IVA1 gene in Northern blot analysis, we have shown that increased renal and hepatic omega-hydroxylation of lauric acid, after treatment with peroxisome proliferators is a consequences of a substantial increase in the mRNA coding for this haemoprotein. In addition, programming of an in vitro rabbit reticulocyte translation system with both renal and hepatic RNA resulted in the synthesis of similar (if not identical) cytochrome-P-450-IVA1-related polypeptides. Furthermore, we have provided Western blot evidence that both rat liver and kidney microsomes contain two closely related cytochrome P-450 IVA1 polypeptides, the major one characterised by a monomeric molecular mass of 51.5 kDa (identical to authentic, purified hepatic cytochrome P-450 IVA1) and a minor one of 52 kDa. The kidney-supported fatty acid omega-hydroxylase activity was refractory to inhibition by a polyclonal antibody to liver cytochrome P-450 IVA1, which may be related to the existence of two closely related (but immunochemically distinct) fatty acid hydroxylases in this tissue. Our studies have also demonstrated that certain of the compounds tested (including clofibrate, bezafibrate and nafenopin) induced renal fatty acid beta-oxidation, mirroring the increased omega-hydroxylase activity in the endoplasmic reticulum. Our studies have also indicated that the kidney was more refractory to induction of the endoplasmic reticulum and peroxisomal fatty-acid-oxidising enzymes than the liver. Taken collectively, our data is strongly suggestive of a possible linkage of the renal fatty acid oxidative enzymes in these two organelles, a situation that also occurs in the liver. In addition, our studies have provided a possible conceptual framework that may rationalise the decreased susceptibility of the k     

25-Hydroxylation of vitamin D3 by a cytochrome P-450 from rabbit liver mitochondria.

A cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 was purified from liver mitochondria of untreated rabbits. The enzyme fraction contained 9 nmol of cytochrome P-450/mg of protein and showed only one protein band with an apparent Mr of 52,000 upon SDS/polyacrylamide-gel electrophoresis. The preparation showed a single protein spot with an apparent isoelectric point of 7.8 and an Mr of approx. 52,000 upon two-dimensional isoelectric-focusing-polyacrylamide-gel electrophoresis. The purified cytochrome P-450 catalysed 25-hydroxylation of vitamin D3 up to 5000 times more efficiently than did the mitochondria. The cytochrome P-450 required both ferredoxin and ferredoxin reductase for catalytic activity. Microsomal NADPH-cytochrome P-450 reductase could not replace ferredoxin and ferredoxin reductase. The cytochrome P-450 catalysed, in addition to 25-hydroxylation of vitamin D3, the 25-hydroxylation of 1 alpha-hydroxyvitamin D3 and the 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. The enzyme did not catalyse side-chain cleavage of cholesterol, 11 beta-hydroxylation of deoxycorticosterone, 1 alpha-hydroxylation of 25-hydroxyvitamin D3, hydroxylations of lauric acid and testosterone or demethylation of benzphetamine. The results raise the possibility that the 25-hydroxylation of vitamin D3 and the 26-hydroxylation of C27 steroids are catalysed by the same species of cytochrome P-450 in liver mitochondria. The possible role of the liver mitochondrial cytochrome P-450 in the metabolism of vitamin D3 is discussed.     

Purification and characterization of rat pulmonary cytochrome P-450

The pulmonary cytochrome P-450, P450 L-2, was purified 460-fold from pulmonary microsomes of untreated male rats. Its specific content was 10.6 nmol/mg of protein. The monomeric molecular weight was 54,000 on SDS-polyacrylamide gel electrophoresis. The CO-reduced absorption maximum of P450 L-2 was at 451 nm, and the oxidized heme iron appeared to be in the low-spin state, as deduced from the Soret maximum at 421 nm. P450 L-2 had high lauric acid omega- and (omega-1)-hydroxylation activities, but low prostaglandin A1 omega- and (omega-1)-hydroxylation activities. It catalyzed the O-dealkylation of 7-ethoxycoumarin, but was not efficient in the hydroxylation of testosterone or the N-demethylation of aminopyrine. The NH2-terminal amino acid sequence of P450 L-2 was V-L-N-F-L-X-P-X-L (X being an unidentified residue). The catalytic properties of P450 L-2 resembled those of P450 K-5, the major rat renal cytochrome P-450. However, anti-P450 K-5 antibody did not cross-react with P450 L-2, and these forms had different NH2-terminal sequences. To judge from the results of NH2-terminal sequence analysis, P450 L-2 seems to be placed in the IVB gene family. Also, P-450 IIB1 was detected by immunoblotting in one of the peaks on ion-exchange HPLC during the purification of P450 L-2, suggesting the presence of P-450 IIB1 in rat pulmonary microsomes.     

Purification and characterization of two forms of fatty acid omega-hydroxylase cytochrome P-450 from rabbit kidney cortex microsomes

We have previously reported the isolation of two forms of cytochrome P-450 (P-450) with omega-hydroxylase activities toward prostaglandin A (PGA) and fatty acids, designated as P-450ka-1 and P-450ka-2, from kidney cortex microsomes of rabbits treated with di(2-ethylhexyl)phthalate [Kusunose, E. et al. (1989) J. Biochem. 106, 194-196]. In the present work, we have purified and characterized two additional forms of rabbit kidney fatty acid omega-hydroxylase, designated as P-450kc and P-450kd. The purified P-450kc and P-450kd had specific contents of 13 and 16 nmol of P-450/mg of protein, with apparent molecular weights of 52,000 and 55,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Both the forms showed absorption maxima at 450 nm in the carbon monoxide-difference spectra for their reduced forms. These P-450s efficiently catalyzed the omega- and (omega-1)-hydroxylation of fatty acids such as caprate, laurate, myristate, and palmitate, in a reconstituted system containing P-450, NADPH-P-450 reductase, and phosphatidylcholine. Cytochrome b5 stimulated the reactions to only a slight extent. They had no detectable activity toward PGA and several xenobiotics tested. The two P-450s showed different peptide map patterns after limited proteolysis with papain or Staphylococcus aureus V8 protease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autocatalytic inactivation of plant cytochrome P-450 enzymes: selective inactivation of the lauric acid in-chain hydroxylase from Helianthus tuberosus L. by unsaturated substrate analogs

Lauric acid in-chain hydroxylation is inhibited in microsomes from Jerusalem artichoke tubers (Helianthus tuberosus L.) incubated with 9-decenoic, 11-dodecenoic, or 11-dodecynoic acids. 9-Decenoic acid is at best a weak competitive inhibitor of the in-chain hydroxylase, but inactivates the enzyme in a time-dependent, pseudo-first-order process with a rate constant of approximately 1.1 X 10(-3) s-1. In contrast, 11-dodecenoic acid causes a slower, time-dependent loss of the hydroxylase activity, but is a potent competitive inhibitor of the enzyme (Ki = 2 microM). Neither agent decreases the microsomal concentration of cytochrome b5, NADH-cytochrome b5 reductase, or NADPH cytochrome P-450 reductase. Cinnamic acid 4-hydroxylation, catalyzed by a cytochrome P-450 enzyme, is not affected by concentrations of 9-decenoic acid that suppress lauric acid hydroxylation. 11-Dodecenoic acid is much less specific and, at higher concentrations, markedly reduces the microsomal cytochrome P-450 content, and the hydroxylation of both lauric and cinnamic acids.     

Species differences in ciprofibrate induction of hepatic cytochrome p450 4A1 and peroxisome proliferation

Six species (CD-1 mouse, Fischer 344 rat, Syrian golden hamster, Duncan-Hartley guinea pig, half-lop rabbit and marmoset monkey) were treated orally with ciprofibrate, a potent oxyisobutyrate hypolipidaemic drug for 14 days. A dosedependent liver enlargement was observed in the mouse and rat and at the high dose level in the hamster. A marked dose-dependent increase in the 12-hydroxylation of lauric acid was observed in the treated mouse, hamster, rat, and rabbit, associated with a concomitant elevation in the specific content of cytochrome P-450 4A1 apoprotein, determined by an ELISA technique. Similarly, in these responsive species, an increase in mRNA levels coding for cytochrome P450 4A1 was observed. Lauric acid 12-hydroxylation was unchanged in the guinea pig and marmoset after ciprofibrate pre-treatment, and cytochrome P-450 4A1 was not detected immunochemically in liver microsomes from these latter species. In the untreated mouse, hamster, rat, and rabbit, the 12-hydroxylation of lauric acid was more extensive than the 11-hydroxylation, whereas in the guinea pig and marmoset the activity ratios were reversed, with 11-hydroxylation predominating. Peroxisomal fatty acid β-oxidation was markedly induced in the mouse, hamster, rat, and rabbit on treatment at the higher dose level (39-, 3-, 13- and 5-fold, respectively) and was slightly increased in the marmoset (2-fold), yet was unchanged in the guinea pig following treatment. In the marmoset the increase in peroxisomal β-oxidation was 3- to 4-fold at the high dose level; however, the dose levels used in the marmoset were 20 and 100 mg/kg as opposed to 2 and 20 mg/kg in the other species. The differences in the foregoing hepatic enzyme responses to ciprofibrate between the species examined in our studies indicate a specific pattern of enzyme changes in responsive species. In the responsive species (rat, mouse, hamster, and rabbit), cytochrome P-450 4A1 specific content and related enzyme activity were increased concomitant with elevated peroxisomal β-oxidation. By contrast, the marmoset and guinea pig lack the coordinate hepatic induction of peroxisomal and microsomal parameters and may be categorized as less responsive species. Accordingly, the rat hepatic responses to peroxisome proliferators cannot confidently be used to predict biological responses in primates, with obvious implications for the extrapolation of animal data to man.     

Biochemical characterization of hepatic microsomal leukotriene B4 hydroxylases

omega-Hydroxylation of leukotriene B4 (LTB4) has been reported in human and rodent polymorphonuclear leukocytes; preliminary information indicates that this metabolism is cytochrome P-450 dependent. Therefore, these studies were initiated to characterize the cytochrome P-450-dependent metabolism of LTB4 in other tissues. LTB4 was metabolized by rat hepatic microsomes to two products, 20-hydroxy(omega)-LTB4 and 19-hydroxy(omega-1)-LTB4. The formation of these metabolites was both oxygen and NADPH dependent indicating that a monooxygenase(s) was responsible for these reactions. The apparent Km and Vmax for LTB4 omega-hydroxylase were 40.28 microM and 1202 pmol/min/mg of protein, respectively. In contrast, the apparent Km and Vmax for LTB4 (omega-1)-hydroxylase were 61.52 microM and 73.50 pmol/min/mg of protein, respectively. Both LTB4 omega- and (omega-1)-hydroxylases were inhibited by metyrapone in a concentration-dependent fashion. However, SK&F 525A inhibited LTB4 (omega-1)- but not omega-hydroxylase. In contrast, alpha-naphthoflavone decreased LTB4 omega- but not (omega-1)-hydroxylase activities. The differences in the Km apparent for substrate as well as the differential inhibition by inhibitors of cytochrome P-450 suggest that the omega- and (omega-1)-hydroxylations of LTB4 in hepatic microsomes are mediated by different isozymes of P-450. Furthermore, several additional characteristics of LTB4 hydroxylases indicate that these isozymes of P-450 may be different from those which catalyze similar reactions on medium-chain fatty acids, such as laurate and prostaglandins.