The importance of threonine-301 from cytochromes P-450 (laurate (omega-1)-hydroxylase and testosterone 16 alpha-hydroxylase) in substrate binding as demonstrated by site-directed mutagenesis

Threonine-301 from rabbit liver cytochromes P-450 (laurate (omega-1)-hydroxylase and testosterone 16 alpha-hydroxylase) has been replaced by histidine via site-directed mutagenesis. In the oxidized state the mutant P-450s exhibited typical low-spin type absorption spectra of P-450 and their reduced CO complexes showed a Soret peak at 450 nm. However, no spectral change was induced on addition of substrates for their wild-type counterparts. The mutant P-450s were also completely devoid of the hydroxylase activity. These findings suggest that threonine-301, which is highly conserved in P-450s and located at the distal heme surface, plays an important role in substrate binding.     

Point mutations at threonine-301 modify substrate specificity of rabbit liver microsomal cytochromes P-450 (laurate (omega-1)-hydroxylase and testosterone 16 alpha-hydroxylase)

Thr-301 of cytochrome P-450 (laurate (omega-1)-hydroxylase) was replaced by Ser, Val, Ile, or Asn via site-directed mutagenesis. The Ser-, Val-, and Asn-mutants had lower laurate (omega-1)-hydroxylase activities than the wild-type P-450. The mutation to Ser did not affect caprate (omega-1)-hydroxylase activity and rather increased caprate omega-hydroxylase activity, but the Val- and Asn-mutants could not hydroxylate caprate. The Ile-mutant was devoid of the hydroxylase activities. The mutation also led to changes in the affinities for the fatty acids and exogenous ligands. Replacement of Thr-301 of cytochrome P-450 (testosterone 16 alpha-hydroxylase) by Ser or Val also affected the activities toward testosterone and progesterone in different ways. These findings indicate that residue 301 of the P-450s plays an important role in determining their substrate specificities.     

Identification of regions functioning in substrate interaction of rabbit liver cytochrome P-450 (laurate (omega-1)-hydroxylase)

The nucleotide sequence of cDNA for rabbit liver cytochrome P-450 (laurate (omega-1) hydroxylase) was replaced with that for rabbit liver cytochrome P-450 (testosterone 16 alpha-hydroxylase) in various regions coding for the amino acid sequence between residues 43 and 261. Six chimeric cDNAs thus constructed were cloned into expression vector pAAH5, and expressed in Saccharomyces cerevisiae AH22 cells under the control of yeast ADH1 promoter. Chimeric P-450s synthesized in the transformed yeast cells were purified partially and their catalytic and spectral properties were examined and compared with those of the chimeric P-450 which is considered to possess the same catalytic properties as the wild-type P-450. In the oxidized state the chimeric P-450s exhibited a low-and high-spin mixed-type absorption spectrum of cytochrome P-450 and the spectrum was converted to a typical high-spin type on addition of laurate or caprate, indicating the binding of the fatty acids to the substrate site of the chimeric P-450s. However the affinities of the fatty acids for the chimeras devoid of the sequence of P-450 (laurate (omega-1)-hydroxylase) in either of the regions spanning residues 90-125 and 210-261 were 10 to 20 times lower than those for the chimeras containing the sequence of the wild-type P-450 in both regions. The latter chimeras have about the same affinities as the chimera which is essentially the wild-type P-450.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of two forms of cytochrome P-450 from rat kidney cortex microsomes

Two forms of cytochrome P-450 (P-450), designated P-450 k-1 and P-450 k-2, have been purified about 100-fold from rat kidney cortex microsomes. P-450 k-1 and P-450 k-2 have monomeric molecular weights of 51,500 and 52,000, respectively, on sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis. Absolute spectra of the oxidized forms indicate that P-450 k-1 is largely in the low-spin state and partly in the high-spin state, and that P-450 k-2 is essentially all in the former. The absorption maxima in reduced carbon monoxide difference spectra are at 450.5 and 451 nm with P-450 k-1 and P-450 k-2, respectively. The two P-450s catalyze the omega- and (omega-1)-hydroxylation of fatty acids such as caprate, laurate, myristate, and palmitate, although P-450 k-1 exhibits a higher specific activity with all fatty acids tested. In addition, P-450 k-1 is capable of hydroxylating prostaglandin (PG) A1 and A2 at the omega-position, whereas P-450 k-2 has no activity toward PGs. These activities are all stimulated by addition of cytochrome b5. The two P-450s give different peptide map patterns when partially digested with Staphylococcus aureus V8 protease or papain.     

Effects of cholesterol and adrenodoxin binding on the heme moiety of cytochrome P-450scc: a resonance Raman study

The effects of cholesterol and adrenodoxin binding on resonance Raman spectra of cytochrome P-450scc in both oxidized and CO-reduced states were examined. Upon cholesterol binding, oxidized cytochrome P-450scc showed a significant shift of spin equilibrium from low-spin to high-spin state. Addition of adrenodoxin caused a complete conversion of cholesterol-bound oxidized cytochrome P-450scc to a pure high-spin state that was considered to be in the hexacoordinated state judged by the v10 mode at 1620 cm-1 and v3 mode around 1485 cm-1. Cholesterol in substrate binding site may oppose a linear and perpendicular binding of carbon monoxide to the reduced heme iron, leading to the distorted Fe-C-O linkage. This is based on the following observations: (1) an increase of the Fe-CO stretching frequency to 483 from 477 cm-1 upon addition of cholesterol; (2) an enhanced photodissociability of bound carbon monoxide of CO complex of cytochrome P-450scc in the presence of cholesterol. As another aspect of the effect of cholesterol on the CO complex form of cytochrome P-450scc, the enhanced stability of the native form ("P-450" form) was observed. There was no additional effect of reduced adrenodoxin on the Raman spectra of the CO-reduced form of cytochrome P-450scc.     

Replacing the carboxy-terminal 28 residues of rabbit liver P-450 (laurate (omega-1)-hydroxylase) with those of P-450 (testosterone 16 alpha-hydroxylase) produces a new stereospecific hydroxylase activity

cDNA for chimeric P-450 consisting of the amino-terminal 462 residues of P-450 (laurate (omega-1)-hydroxylase) and the remaining 28 residues of P-450 (testosterone 16 alpha-hydroxylase) was constructed and expressed in yeast cells. The resulting chimera could catalyze laurate (omega-1)-hydroxylation and benzphetamine N-demethylation at much higher rates than the parental P-450s, but exhibited the same specificity towards fatty acid substrates as the wild-type laurate hydroxylase. When testosterone was examined as a substrate, the 16 beta-hydroxylated product, which cannot be formed by either of the parental P-450s, was detected, suggesting that the laurate hydroxylase contains a structure that is capable of binding testosterone at a proper orientation so that it can be hydroxylated at the 16 beta position.     

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)     

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.     

Surface enhanced resonance Raman study of phenobarbital-induced rabbit liver cytochrome P-450 LM2

Surface enhanced resonance Raman (SERR) spectroscopy has been used to study the vibrational spectra of the heme of purified rabbit liver cytochrome P-450 LM2 which was adsorbed on colloidal silver suspensions or on a silver electrode. Bases on a comparison with the resonance Raman (RR) spectra of the 'solute' species the high sensitivity of the SERR technique is demonstrated. Two different features were chosen in order to determine the structural and functional integrity of the adsorbed P-450. Both, substrate-induced spin state changes on the oxidized P-450 and the effect of the thiolate ligand on the oxidation state marker band v4 in the reduced P-450 could be observed in the SERR spectra of the adsorbed as well as in the RR spectra of the dissolved enzyme. These findings indicate that the protein structure near the substrate binding site and the coordination by thiolate are not affected by the interaction with the metal surface. Both structural elements are crucial for the function of P-450. Thus the elementary processes of the enzymatic action of P-450 can be investigated by this highly sensitive version of RR spectroscopy.     

Characterization of rabbit liver cytochrome P-450 (laurate omega-1 hydroxylase) synthesized in transformed yeast cells

Three cDNAs for chimeras between cytochrome P-450s (pHP3 and pHP2-1) were constructed and inserted between the alcohol dehydrogenase promoter and terminator regions of the yeast expression vector pAAH5 to form expression plasmids, pAH3P2, pAH3E2, and pAH3A2. pAH3P2 contained the entire coding sequence of cytochrome P-450 (pHP2-1) except for the 3rd, the 8th, the 36th, and the 42nd residues of the total of 490 amino acids. Nucleotide sequences of pAH3P2 were replaced with those of cytochrome P-450 (pHP3) in the region coding for the NH2-terminal 210 and 262 amino acid residues to yield pAH3E2 and pAH3A2, respectively. The three expression plasmids were introduced into Saccharomyces cerevisiae AH22 cells and cytochrome P-450 s (3P2, 3E2, and 3A2) were purified from the microsomal fractions of the transformed yeast cells. In the oxidized state either of the cytochromes exhibited a low- and high-spin mixed-type spectrum of cytochrome P-450. The reduced CO complex of the cytochromes showed a Soret absorption maximum at 450 nm. When laurate or caprate was added to ferric cytochrome P-450 s (3P2 and 3E2), the spectrum was converted to that of the typical high-spin type, indicating the binding of the fatty acids to the substrate site of the cytochromes. On the other hand, the addition of the fatty acids to ferric cytochrome P-450 (3A2) induced no spectral change. Only chemicals having a carboxyl group caused such spectral conversion of cytochrome P-450 (3P2) among dodecyl compounds examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unusual CO bonding geometry in abnormal subunits of hemoglobin M Boston and hemoglobin M Saskatoon

To clarify the role of the proximal histidine (F8-His), distal His (E7-His), and E11 valine (E11-Val) in ligand binding of hemoglobin (Hb), we have investigated the resonance Raman (RR) spectra of the carbon monoxide adduct of Hbs M (COHb M) in which one of these residues was genetically replaced by another amino acid in either the alpha or beta subunit. In the fully reduced state, all Hbs M gave v3 at approximately 1472 cm-1 and vFe-His at 214-218 cm-1, indicating that they have a pentacoordinate heme and the heme iron is bound to either E7-His or F8-His. The porphyrin skeletal vibrations of the COHb M were essentially unaltered by replacements of E7- or F8-His with tyrosine (Tyr) and of E11-Val by glutamic acid (Glu). The vCO, vFe-CO, and delta Fe-C-O frequencies of COHb M Iwate (alpha F8-His----Tyr), COHb M Hyde Park (beta F8-His----Tyr), and COHb M Milwaukee (beta E11-Val----Glu) were nearly identical with those of COHb A. In contrast, the RR spectra of COHb M Boston (alpha E7-His----Tyr) and COHb M Saskatoon (beta E7-His----Tyr) gave two new Raman bands derived from the abnormal subunits, vFe-CO at 490 cm-1 and vCO at 1972 cm-1, in addition to those from the normal subunits at 505 cm-1 (vFe-CO) and 1952 cm-1 (vCO). The CO adduct of the abnormal subunits exhibited apparently no photodissociation upon illumination of CW laser with a stationary cell under which the normal subunit exhibited complete photodissociation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple forms of cytochrome P-450 in rabbit colon microsomes

Three cytochrome P-450 preparations, designated as cytochrome P-450ca, cytochrome P-450cb, and cytochrome P-448c fraction, were separated and purified about 23-, 50-, and 29-fold, respectively, from the cholate extracts of rabbit colon mucosa microsomes. Their specific contents were 1.2, 2.6, and 1.5 nmol of cytochrome P-450 per mg of protein, respectively. Cytochrome P-450ca and cytochrome P-450cb migrated as heme-containing polypeptide bands with molecular weights of about 53,000 and 57,000, respectively, on SDS-polyacrylamide gel electrophoresis. The CO-reduced difference spectra of cytochrome P-450ca, cytochrome P-450cb, and cytochrome P-448c fraction showed maxima at 451, 450, and 449 nm, respectively. Cytochrome P-450ca efficiently catalyzed the omega-hydroxylation of prostaglandin A1 (PGA1) and the omega- and (omega-1)-hydroxylation of caprate, laurate, and myristate in the reconstituted system containing cytochrome P-450ca, NADPH-cytochrome P-450 reductase, cytochrome b5, and phosphatidylcholine. In contrast, cytochrome P-450cb and cytochrome P-448c fraction had no detectable activity toward PGA1 and fatty acids. Both catalyzed aminopyrine and benzphetamine N-demethylation. Cytochrome P-448c fraction also hydroxylated benzo(a)pyrene, and phosphatidylinositol or phosphatidylserine exhibited a stimulatory effect on this activity. The results show that rabbit colon microsomes contain catalytically different cytochrome P-450, one of which is specialized for the omega-oxidation prostaglandins, the others being involved in the metabolism of exogenous compounds such as drugs and polycyclic hydrocarbons.     

An anomaly in the resonance Raman spectra of cytochrome P-450cam in the ferrous high-spin state.

Resonance Raman spectra of cytochrome P-450cam (P-450cam) and its enzymatically inactive form (P-420) in various oxidation and spin states were measured for the first time. The Raman spectrum of reduced P-450cam was unusual in the sense that the "oxidation-state marker" appeared at an unexpectedly lower frequency (1346 cm-1) in comparison with those of other reduced hemoproteins (approximately 1355-approximately 1365 cm-1), whereas that of oxidized P-450cam was located at a normal frequency. This anomaly in the Raman spectrum of reduced P-450cam can be explained by assuming electron delocalization from the fifth ligand, presumably a thiolate anion, to the antibonding pi orbital of the porphyrin ring. The corresponding Raman line of reduced P-420 appeared at a normal frequency (1360 cm-1), suggesting a status change or replacement of the fifth ligand upon conversion from P-450cam to P-420. The Raman spectrum of reduced P-450cam-metyrapone complex was very similar to that of ferrous cytochrome b5.     

Fatty acid hydroxylation in rat kidney cortex microsomes

Rat kidney microsomes have been found to catalyze the hydroxylation of medium-chained fatty acids to the omega- and (omego-1)-hydroxy derivatives. This reaction, which requires NADPH and molecular oxygen, is a function of monooxygenase system present in the kidney microsomes, containing NADPH-cytochrome c reductase and cytochrome P-450K. NADH is about half as effective as an electron donor as NADPH and there is an additive effect in the presence of both nucleotides. Cytochrome P-450K absorbs light maximally at 452-3 nm, when it is reduced and bound to carbon monoxide. The extinction coefficient of this complex is 91 mM(-1) cm(-1). Electrons from NADPH are transferred to cytochrome P-450K via the NADPH-cytochrome c reductase. The reduction rate of cytochrome P-450K is stimulated by added fatty acids and the reduction kinetics reveal the presence of endogenous substrates bound to cytochrome P-450K. Both cytochrome P-450K concentration and fatty acid hydroxylation activity in kidney microsomes are increased by starvation. On the other hand, phenobarbital treatment of the rats has no effect on either the hemoprotein or the overall hydroxylation reaction and 3,4-benzpyrene administration induces a new species of cytochrome P-450K not involved in fatty acid hydroxylation. Cytochrome P-450K shows, in contrast to liver P-450, high substrate specificity. The only substances forming enzyme-substrate complexes with cytochrome P-450K are the medium-chained fatty acids and certain derivatives of these acids. The chemical requirements for substrate binding include a carbon chain of medium length and at the end of the chain a carbonyl group and a free electron pair on a neighbouring atom. The distance between the binding site for the carbonyl group and the active oxygen is suggested to be in the order of 16 A. This distance fixes the ratio of omega- and (omega-1)-hydroxylated products formed from a certain fatty acid by the single species of cytochrome P-450K involved. The membrane microenvironment seems also to be of importance for the substrate specificity of cytochrome P-450K, since removal of the cytochrome from the membrane lowers its binding specificity to some extent. A comparison between the liver and kidney cytochrome P-450 systems suggests that the kidney cytochrome P-450K system is specialized for fatty acid hydroxylation.     

Purification and characterization of two forms of cytochrome P-450 with omega-hydroxylase activities toward prostaglandin A and fatty acids from rabbit liver microsomes

Two forms of cytochrome P-450 (P-450), designated as P-450LPGA omega 1 and P-450LPGA omega 2, have been purified to specific contents of 17.9 and 11.1 nmol P-450/mg protein, respectively, from liver microsomes of rabbits treated with di(2-ethylhexyl)phthalate (DEHP), a peroxisomal proliferator. The purified P-450LPGA omega 1 and P-450LPGA omega 2 were found to have apparent molecular weights of 52,500 and 53,000, respectively. They showed absorption maxima at 451 and 450 nm in the carbon monoxide-difference spectra for their reduced forms, respectively. The two P-450s both efficiently catalyzed the omega-hydroxylation of prostaglandins A1 (PGA1) and A2 (PGA2), as well as the omega- and (omega-1)-hydroxylation of fatty acids such as laurate, myristate, and palmitate. In a reconstituted system, various metal ions such as Na+ and Mg2+ stimulated these reactions. The P-450s exhibited no detectable activity toward several xenobiotics tested. The two P-450s showed different peptide map patterns following limited proteolysis with Staphylococcus aureus V8 protease or papain. The NH2-terminal amino acid sequences (ALNPTRLPGSLSGLLQVAGL and ALSLTRLPGSFSGFLQAxGLLGLLL) of P-450LPGA omega 1 and P-450LPGA omega 2 were identical at 18/20 and 19/24 positions with that of the lung prostaglandin omega-hydroxylase from pregnant rabbits, respectively. An antibody against P-450LPGA omega 2 recognized a 52,000-53,000 molecular weight protein(s) in rabbit liver microsomes. The intensity of the immunoblot was significantly increased in liver microsomes from rabbits treated with DEHP, but not with phenobarbital or 3-methylcholanthrene.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Effects of cholesterol analogues and inhibitors on the heme moiety of cytochrome P-450scc: a resonance Raman study

Interactions of cholesterol analogues and inhibitors with the heme moiety of cytochrome P-450scc were examined by resonance Raman spectroscopy. The Raman spectra of ferric cytochrome P-450scc complexed with inhibitors such as cyanide, phenyl isocyanide, aminoglutethimide, and metyrapone were characteristic of low-spin state and were very similar. However, the effect of exchange of the sixth ligand from the oxygen atom (ferric low-spin state) to the nitrogen atom upon aminoglutethimide and metyrapone binding was seen as down-frequency shifts of the v3 band from 1503 to 1501 and 1502 cm-1, respectively, while cyanide and phenyl isocyanide binding caused an up-frequency shift of the v3 band to 1505 cm-1. The effects of cholesterol analogues [22(R)-hydroxycholesterol, 22(S)-hydroxycholesterol, 22-ketocholesterol, 20(S)-hydroxycholesterol, and 25-hydroxycholesterol] on a Fe2+-CO stretching frequency of cytochrome P-450scc in ferrous CO form were examined. The 22(R)-hydroxycholesterol complex could not give a clear Fe2+-CO stretching Raman band due to a strong photodissociability. 22(S)-Hydroxycholesterol and 25-hydroxycholesterol complexes gave the Raman bands at 487 and 483 cm-1, respectively, whereas 20(S)-hydroxycholesterol and 22-ketocholesterol complexes gave Fe2+-CO stretching frequencies (478 cm-1) almost identical with that without substrate (477 cm-1). These findings suggest the existence of the following physiologically important natures of the cytochrome P-450scc active site: (1) there is a strong steric interaction between heme-bound carbon monoxide and the 22(R)-hydroxyl group or the 22(R)-hydrogen of the steroid side chain and (2) the hydroxylation at the 20S position may cause a conformational change of the side-chain group relative to the heme.     

Fatty acid omega and (omega-1)-Hydroxylation in rabbit intestinal mucosa microsomes.

The microsomes from rabbit intestinal mucosa which had been washed quickly and thoroughly with phenylmethylsulfonyl fluoride were found to catalyze the hydroxylation of fatty acids in the presence of NADPH and molecular oxygen. Myristic and palmitic acids were converted to the corresponding omega-and (omega-1)-hydroxy fatty acids, whereas lauric acid was converted only to 12-hydroxylauric acid, and capric acid, to 9-and 10-hydroxycapric acids together with an unknown polar acid.Among these fatty acids, both myristic and lauric acids appeared to be the most efficient substrates. The inhibition of the hydroxylation by SKF 525-A and carbon monoxide suggested that the activity depended upon cytochrome P-450. The specific activity of the fatty acid hydroxylation was almost constant along the small intestine, while the aminopyrine N-demethylation activity and the cytochrome P-450 content were highest at the proximal end of the intestine and progressively declined toward the caudal end. The cytochrome P-450 was solubilized from the intestinal microsomes and purified by 6-amino-n-hexyl Sepharose 4B chromatography. The partially purified cytochrome P-450 was active in fatty acid hydroxylation in combination with intestinal NADPH-cytochrome c reductase and phosphatidylcholine.     

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.