Catalytic properties of rabbit kidney fatty acid omega-hydroxylase cytochrome P-450ka2 (CYP4A7).

We have examined in detail the substrate specificity of a rabbit kidney fatty acid omega-hydroxylase, designated cytochrome P-450ka2 (CYP4A7). The hydroxylation products were identified as omega- and (omega - 1)-hydroxy fatty acids mainly using gas chromatography-electron impact mass spectrometry. [1] Straight-chain saturated fatty acids ranging from 10 to 19 carbons were effectively hydroxylated at the omega- and (omega - 1)-position. The ratios of omega- to (omega - 1)-hydroxylation activity decreased with increasing the carbon chain length of fatty acids. [2] Both isomyristate and anteisomyristate, and isopalmitate were hydroxylated several fold more rapidly than myristate and palmitate, respectively, with iso-branched chain fatty acids being hydroxylated at the omega-position solely. [3] Both palmitoleate and palmitoelaidate, and both oleate and elaidate were hydroxylated much more rapidly than palmitate and stearate, respectively. [4] Linoleate, gamma-linolenate, and arachidonate were also excellent substrates for this enzyme. [5] Prostaglandin (PG) A1 and PGA2 were efficiently hydroxylated at the omega-position solely, with PGE1 and PGE2 being much less active. [6] Arachidonic acid not only showed a Km value significantly lower than those for lauric acid, gamma-linolenic acid and PGA1, but also it is a potent competitor for lauric acid and PGA1, showing a very high affinity for the enzyme. It is possible that arachidonic acid is the physiological substrate for kidney P-450ka2.

Some properties of the fatty alcohol oxidation system and reconstitution of microsomal oxidation activity in intestinal mucosa.

This paper describes the metabolism of fatty alcohols by microsomal and cytosolic fractions from intestinal mucosa. Microsomes of rabbit intestinal mucosa had a high activity of [1-14C]dodecanol oxidation as did those of liver. The intestinal cytosolic fraction also exhibited oxidation activity to a lesser extent than the microsomes did. The reaction product was determined as lauric acid using thin-layer chromatography. Laurylaldehyde was detected as another product, when semicarbazide was added to the incubation system. Cyclodextrins exhibited a stimulation effect similarly to bovine serum albumin on the microsomal activity. We have compared the stimulatory effects of dimethyl-beta-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin and alpha-cyclodextrin, which decrease in that order. Effects of NAD+ and dodecanol concentrations, pH and pyrazole on microsomal activity were compared with those on cytosolic activity. Dodecanol oxidation activity was solubilized and reconstituted with a fatty alcohol dehydrogenase and a fatty aldehyde dehydrogenase separated from the intestinal microsomes. These findings indicate that both the dehydrogenases participate in microsomal oxidation of fatty alcohols to fatty acids with fatty aldehydes as intermediates in the reaction.

Purification of aldehyde dehydrogenase reconstitutively active in fatty alcohol oxidation from rabbit intestinal microsomes.

This work presents the purification and further characterization of the aldehyde dehydrogenase reconstitutively active in fatty alcohol oxidation, from rabbit intestinal microsomes. Microsomal aldehyde dehydrogenase was solubilized with cholate and purified by using chromatography on 6-amino-n-hexyl-Sepharose and 5'-AMP-Sepharose. The purified enzyme migrated as a single polypeptide band with molecular weight of 60,000 on SDS-polyacrylamide gel. By gel filtration in the presence of detergent, its apparent molecular weight was estimated to be 370,000. In the detergent-free solution, in contrast, it had a much higher molecular weight, indicating its association in forming large aggregates. The pH optimum was 9.0 when pyrophosphate buffer was used. The enzyme was active toward various aliphatic aldehydes with more than three carbons. The Km value for substrate seemed to decrease with increase in the chain length. The microsomal aldehyde dehydrogenase was not affected by disulfiram and MgCl2, which were, in contrast, highly inhibitory towards the activity of the cytosolic aldehyde dehydrogenase separated from intestinal mucosa.

Purification and NH2-terminal amino acid sequences of human and rat kidney fatty acid omega-hydroxylases.

A cytochrome P-450 (P-450), designated P-450HK omega, has been isolated and purified from human kidney microsomes to a specific content of 13 nmoles of P-450/mg of protein. P-450HK omega showed an apparent molecular weight of 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Absolute spectra of the oxidized form indicated that this P-450 was largely in the low-spin state and partly in the high-spin state. It catalyzed the omega- and (omega-1)-hydroxylation of fatty acids such as laurate, myristate, and palmitate, with no activity toward prostaglandin A1, benzphetamine, 7-ethoxycoumarin, or 7-ethoxyresorufin. The first 35 NH2-terminal amino acid sequence of P-450HK omega had about 70% homology with those of rabbit kidney fatty acid omega-hydroxylases of the P-450 IVA gene subfamily, P-450ka-1, P-450ka-2, and P-450kd, except for four undetermined residues. Moreover, Western blot and immuno-inhibition studies showed that P-450HK omega reacted with an antibody against the rabbit kidney fatty acid omega-hydroxylase. The results suggest that P-450HK omega is a member of the same P-450 gene family (IVA subfamily) as the rabbit enzymes. In addition, the terminal sequence of P-450HK omega also showed 54% homology with that of P-450k-2, a fatty acid omega-hydroxylase from rat kidney microsomes. To our knowledge, this is the first time that a P-450 specific for fatty acid omega-hydroxylase activity has been isolated to homogeneity from human tissues.

Purification and characterization of two new cytochrome P-450 related to CYP2C subfamily from rabbit small intestine microsomes.

Two forms of cytochrome P-450, designated P-450id and P-450ie, were purified to specific contents of 14.3 and 15.0 nmol of P-450/mg of protein, respectively, from small intestine mucosa microsomes of rabbits. P-450id and P-450ie showed apparent molecular weights of 50 and 49 kDa, respectively, on SDS-PAGE. Both P-450s catalyzed N-demethylation of nitrosodimethylamine. The NH2-terminal amino acid sequence (first 19 residues) of P-450id exhibited 74-90% identity with those of six members of the rabbit P-450 2C subfamily, except for P-450 2C3. Similarly, the NH2-terminal sequence (first 22 residues) of P-450ie showed 73-86% identity with those of the same members of the rabbit P-450 2C subfamily. The peptide mapping patterns of the two P-450s were quite different from each other. In addition, P-450id did not cross-react with the guinea-pig antibodies against P-450ie. The results indicate that rabbit small intestine mucosa contain two new distinct forms of P-450s, both of which may be classified into the 2C subfamily.

Omega- and (omega-1)-hydroxylation of arachidonic acid, lauric acid and prostaglandin A1 by multiple forms of cytochrome P-450 purified from rat hepatic microsomes.

The metabolism of arachidonic acid, lauric acid and prostaglandin A1 by rat hepatic microsomes and multiple forms of cytochrome P-450 purified from rat hepatic microsomes was studied. Arachidonic acid was hydroxylated by hepatic microsomes of male rats by omega- and (omega-1)-hydroxylation. Phenobarbital treatment of rats decreased the hydroxylation activity slightly, but 3-methylcholanthrene treatment increased the hydroxylation activity 2-fold. However, lauric acid and prostaglandin A1 omega- and omega-1)-hydroxylation activities decreased after treatment with phenobarbital and 3-methylcholanthrene. Arachidonic acid and lauric acid were metabolized with similar ratios of omega- and (omega-1)-hydroxylation, but prostaglandin A1 was efficiently metabolized at the omega-position by hepatic microsomes of untreated male rats. In a reconstituted system with purified cytochromes P-450, P450 UT-1, UT-2 (P-450h), MC-1 (P-450d) and MC-5 (P-450c) effectively hydroxylated arachidonic acid at both the omega- and (omega-1)-position. P450 UT-8 hydroxylated arachidonic acid only at the omega-position. P450 DM (P-450j) hydroxylated arachidonic acid at the (omega-1)-position efficiently. Lauric acid was also hydroxylated by P450 UT-1, UT-2, PB-1, PB-2, MC-1, IF-3 (P-450a) and DM, at the (omega - 1)-position only. Only P450 UT-8 could hydroxylate laruic acid at the omega-position. Prostaglandin A1 was efficiently and specifically metabolized by P450 UT-8 with omega-hydroxylation. P450 UT-2 and PB-1 could hydroxylate prostaglandin A1 by (omega-1)-hydroxylation, but with low activity.

Separation of two constitutive forms of cytochrome P-450 active in aminopyrine N-demethylation from rabbit intestinal mucosa microsomes.

Two constitutive forms of cytochrome P-450, designated P-450ib and P-450ic, were purified from intestinal mucosa microsomes of untreated rabbits. P-450ib and P-450ic have minimal molecular weights of 56 000 and 49 000, respectively, as determined by calibrated sodium dodecyl sulphate polyacrylamide gel electrophoresis. The CO-reduced difference spectral maximum of cytochrome P-450ib is at 450 nm and P-450ic is at 451 nm. Both the cytochromes preferentially demethylate aminopyrine, benzphetamine and N,N-dimethylaniline in the presence of NADPH-cytochrome P-450 reductase. Cytochrome P-450ib has absorption maxima at 417, 535 and 573 nm in the oxidized form, indicating that this cytochrome is in a low-spin state. Ouchterlony double-diffusion studies show that cytochrome P-450ib does not cross-react with antisera against liver cytochrome P-450LM2 purified from phenobarbital-treated rabbits, but P-450ic cross-reacts with spur formation. Unlike cytochrome P-450ib, P-450ic is very similar, if not identical, to liver cytochrome P-450LM2 on the basis of its molecular weight, spectral properties, catalytic activities and immunochemical properties.

Cloning and expression of a novel form of leukotriene B4 omega-hydroxylase from human liver.

We have isolated and sequenced a cDNA for human liver LTB4 omega-hydroxylase. The cDNA encoded a protein of 520 amino acids with a molecular weight of 59,853 Da. The cDNA-deduced amino acid sequence showed 87.3% homology to that of human polymorphonuclear leukocytes (PMN) LTB4 omega-hydroxylase (CYP4F3). Northern blot analysis revealed that the mRNA hybridized to the specific cDNA fragment is expressed in human liver, but not in human PMN. The microsomes from yeast cells transfected with the cDNA catalyzed the omega-hydroxylation of LTB4 with a Km of 44.8 microM. These results clearly show that a new form of the CYP4F LTB4 omega-hydroxylase exists in human liver.

Expression and induction of cytochrome P450s in rabbit parotid glands.

Earlier, we isolated and purified five different P450 isoforms from rabbit kidney cortex microsomes, three of which are members of the CYP4A subfamily (CYP4A5, CYP4A6, and CYP4A7), with the others being CYP2B4 and CYP1A1. In contrast, P450s in parotid glands were unknown. The fact that the parotid glands bear a marked morphological and functional resemblance to kidney tissue prompted us to investigate P450s in these glands. The present study was undertaken to determine which P450 isoforms are expressed in this tissue. Microsomes from parotid glands of untreated rabbits were found to contain 42.3 pmol of P450/mg protein and to catalyze the omega-hydroxylation of laurate. Administration of di(2-ethylhexyl) phthalate (DEHP) resulted in a 7-fold increase of laurate omega-hydroxylation. This enzyme activity was greatly inhibited by pretreatment with antibodies against CYP4A5. Furthermore, parotid gland CYP4A5, CYP4A6, and CYP4A7 mRNAs were identified by RT-PCR. Moreover, the CYP4A enzymes were demonstrated immunohistochemically to be localized exclusively in the ducts of these glands. In addition to the CYP4A enzymes, immunoblot analysis revealed that CYP2B4 is constitutively present, and that CYP1A1 is induced in these glands by treatment with 3-methylcholanthrene. Taken together, we can conclude that the P450 isoforms expressed in rabbit kidney cortex and parotid glands are identical in composition.

Characterization of human liver leukotriene B(4) omega-hydroxylase P450 (CYP4F2).

We previously reported the cloning of a human liver leukotriene B(4) (LTB(4)) omega-hydroxylase P450 designated CYP 4F2 [Kikuta et al. (1994) FEBS Lett. 348, 70-74]. However, the properties of CYP 4F2 remain poorly defined. The preparation solubilized using n-octyl-beta-D-glucopyranoside from microsomes of CYP 4F2-expressing yeast cells catalyzes v- hydroxylation of LTB(4), 6-trans-LTB(4), lipoxin A(4), 8-hydroxyeicosatetraenoate, 12-hydroxyeicosatetraenoate, and 12-hydroxystearate in the presence of rabbit liver NADPH-P450 reductase. In addition, the enzyme shows ethoxycoumarin O-deethylase and p-nitroanisole O-demethylase activities. The enzyme was purified to apparent electrophoretic homogeneity from yeast cells by sequential chromatography of solubilized microsomes through amino-n-hexyl-Sepharose 4B, DEAE-HPLC, and hydroxylapatite HPLC columns. The final preparation showed a specific content of 11.1 nmol of P450/mg of protein, with an apparent molecular mass of 56.3 kDa. CYP 4F2 was distinguished from the closely homologous CYP 4F3 (human neutrophil LTB(4) omega-hydroxylase) by its much higher K(m) for LTB(4), inability to omega-hydroxylate lipoxin B(4), and extreme instability.

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.

Reconstruction of aryl hydrocarbon hydroxylase from rabbit intestinal mucosa microsomes.

Benzo(a)pyene hydroxylation activity was solubilized from rabbit intestinal mucosa microsomes and reconstituted with a cytochrome P-450 preparation obtained by fractionation with 6-amino-n-hexyl Sepharose 4B, hydroxylapatite and CM-Sephadex C-50, and partially purified NADPH-cytochrome c reductase. Phosphatidylserine was required for the maximal activity, while phosphatidylcholine had no stimulatory effect. The carbon monoxide difference spectrum of the cytochrome P-450 fraction showed a maximum peak at 450 nm. Although another cytochrome P-450 fraction was active for hexadecane hydroxylation, this fraction had little activity. The results indicate that more than one cytochrome P-450 exists in the intestinal mucosa microsomes.

Superoxide dismutase from Mycobacterium tuberculosis.

1. A superoxide dismutase [EC 1.15.1.1] was purified about 275-fold with a yield of 34% from Mycobacterium tuberculosis, strain H37Ra (attenuated strain), grown on a Sauton medium for two months. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis, and by analytical ultracentrifugation and sedimentation equilibrium studies. 2. The molecular weight of the enzyme was estimated to be approximately 88,000 by sedimentation equilibrium analysis. Since the molecular weight of the subunit was 21,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme appears to be composed of four subunits of equal size. 3. Electron spin resonance (ESR) spectra showed that the enzyme contained ferric iron, and metal analysis showed that the enzyme contained ferric iron, and metal analysis showed that approximately 3.7 atoms of iron were present per mole of the enzyme, indicating the occurrence of 1 atom of iron per subunit. 4. The amino acid composition was apparently similar to those of the iron-containing superoxide dismutases from Escherichia coli, luminous bacteria, Pseudomonas ovalis, and blue-green alga. 5. Antibodies against the enzyme were raised in rabbits and immunological studies were performed. The enzyme from M. tuberculosis, strain H37Rv (virulent strain), was found to have antigenic structures identical with those of the H37Ra enzyme. On the other hand, the manganese-containing superoxide dismutases from other species of mycobacteria, i.e., Mycobacterium species, strain Takeo, M. phlei and M. lepraemurium, showed only partial immunological identity with the H37Ra enzyme. 6. During the growth of M. tuberculosis, strain H37Ra, the enzyme was found to be secreted into the culture medium.

Multiple forms of cytochrome P-450 in kidney cortex microsomes of rabbits treated with 3-methylcholanthrene.

Cytochrome P-450 was purified from kidney cortex microsomes of rabbits treated with 3-methylcholanthrene. 6-Amino-n-hexyl-Sepharose 4B column chromatography of the cholate-solubilized microsomes yielded two cytochrome P-450 fractions, one of which was eluted from the column with 20 mM potassium phosphate buffer in the presence of 0.4% cholate and 0.08% Emulgen 913. This fraction was partially purified to a specific content of 4.49 nmol of cytochrome P-450/mg of protein. This P-450 fraction catalyzed myristate omega- and (omega-1)-hydroxylation with a turnover rate of 5.0 nmol/nmol of cytochrome P-450 in a reconstituted system containing NADPH-cytochrome c reductase, cytochrome b5 and phosphatidylethanolamine. It had no benzo(a)pyrene hydroxylation activity. The other cytochrome P-450 fraction, which was eluted from the column with 0.1 M potassium phosphate buffer in the presence of 0.4% cholate and 0.08% Emulgen 913, was purified to a specific content of 12.0 nmol of cytochrome P-450/mg of protein. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the final preparation gave a major polypeptide band with a molecular weight of 58,000. This cytochrome P-450 showed a maximal peak at 448 nm in the carbon monoxide difference spectrum of its reduced form. Its absolute spectrum of the oxidized form had low-spin characteristics. It catalyzed benzo(a)pyrene hydroxylation with a turnover rate of 3.63 nmol/nmol of cytochrome P-450 in a reconstituted system containing NADPH-cytochrome c reductase and phosphatidylcholine, whereas little myristate hydroxylation activity was detected. The results demonstrate the occurrence of multiple forms of cytochrome P-450 in rabbit kidney cortex microsomes.

Some properties of a hexadecane hydroxylation system in rabbit intestinal mucosa microsomes.

Among many different tissues of rabbit, hexadecane hydroxylation activity was found in only small intestine and liver microsomes. Both activities for hexadecane and decane hydroxylation in intestinal microsomes were significantly higher than those in liver microsomes. In contrast, the hydroxylation activities of p-xylene, benzo(a)pyrene, and decanol, and the demethylation activity of aminopyrine in the former were much lower than those in the latter. The intestinal microsomes converted [1-14C]hexadecane to cetyl alcohol, but not further to palmitic acid. Hexadecane hydroxylation activity was found to be markedly stimulated by non-ionic detergents such as Triton X-100, Nonident P-40, and Emulgen 913. Phosphatidylcholine and phosphatidylethanolamine stimulated the activity to a lesser extent. The hydroxylation was inhibited by various aliphatic hydrocarbons with carbon numbers larger than 10, but not by aromatic and polycyclic hydrocarbons. Hexadecane hydroxylation activity was solubilized from the intestinal microsomes and reconstituted with a partially purified cytochrome P-450 fraction, and intestinal NADPH-cytochrome c reductase, or spinach ferredoxin and ferredoxin-NADP reductase. The chromatography of the crude cytochrome P-450 preparation on hydroxylapatite separated at least two cytochrome P-450 fractions; one preferentially hydroxylating hexadecane, and the other preferentially hydroxylating myristic acid. The results suggest that rabbit intestinal mucosa microsomes had a cytochrome P-450 species specialized for hexadecane hydroxylation.

Effect of cytochrome b5 on fatty acid omega- and (omega-1)-hydroxylation catalyzed by partially purified cytochrome P-450 from rabbit kidney cortex microsomes.

Cytochrome P-450 was solubilized from kidney cortex microsomes of rabbits treated with 3-methylcholanthrene and partially purified by chromatography on 6-amino-n-hexyl Sepharose 4B and heparin-Sepharose CL-6B columns. Fatty acid omega- and (omega-1)-hydroxylation activity was reconstituted from the partially purified cytochrome P-450 and NADPH-cytochrome c reductase, with phosphatidylethanolamine or phosphatidylcholine. The activity was further stimulated by addition of detergent-solubilized cytochrome b5 from rabbit liver microsomes. Trypsin-solubilized or boiled detergent-solubilized cytochrome b5 had no effect. Among fatty acids tested, caprate, laurate, myristate, and palmitate were the most effective substrates. When caprate and laurate were used as the substrates, the products were the corresponding omega- and (omega-1)-hydroxy fatty acids. The ratio of these products was altered by addition of cytochrome b5. On the other hand, when myristate and palmitate were the substrates, small amounts of unknown polar fatty acids were also formed besides omega- and (omega-1)-hydroxy fatty acids, and the ratio of these products was not affected by addition of cytochrome b5. Benzo(a)pyrene hydroxylation activity was also reconstituted from the same cytochrome P-450 preparation, NADPH-cytochrome c reductase, and phosphatidylserine. However, cytochrome b5 showed only a slight stimulation. The possibility that different cytochrome P-450 species are involved in fatty acid and benzo(a)pyrene hydroxylations is discussed.

Effect of peroxisomal proliferators on microsomal prostaglandin A omega-hydroxylase.

Earlier, we reported the isolation of a cytochrome P-450 highly active in prostaglandin A (PGA) omega-hydroxylation (PGA omega-hydroxylase) from rabbit kidney cortex, small intestine, and colon microsomes. In the present studies, the effects of peroxisomal proliferating agents on the PGA omega-hydroxylase have been examined. Administration of clofibrate or di(2-ethylhexyl)phthalate (DEHP) resulted in a significant increase in the PGA1 omega-hydroxylase activity of kidney cortex, liver, and small intestine microsomes. Similar findings were also obtained for laurate hydroxylase activity in kidney and liver microsomes. Kidney PGA omega-hydroxylase (designated cytochrome P-450ka) was isolated and highly purified from clofibrate- or DEHP-treated rabbits, with a yield 3 times higher than that from untreated, or phenobarbital- or 3-methylcholanthrene-treated rabbits. Cytochrome P-450ka from clofibrate- or DEHP-treated rabbits exhibited the same properties as those from untreated rabbits. Guinea pig antiserum against cytochrome P-450ka strongly inhibited the omega-hydroxylation of PGA1 by kidney cortex microsomes from clofibrate-treated rabbits. The PGA1 omega-hydroxylase activity of clofibrate-treated liver microsomes was also inhibited by this antiserum, suggesting that a PGA omega-hydroxylase immunochemically related to cytochrome P-450ka exists in liver microsomes.

Occurrence of cytochrome P-450 with prostaglandin omega-hydroxylase activity in rabbit placental microsomes.

The microsomes of placenta and uterus from pregnant rabbits have been found to catalyze the omega-hydroxylation of PGE1, PGE2, PGF2 alpha, and PGA1 as well as the omega- and (omega-1)-hydroxylation of palmitate and myristate in the presence of NADPH. These activities were greatly inhibited by carbon monoxide, indicating the involvement of cytochrome P-450. The apparent Km for PGE1 was 2.38 microM and 2.1 microM with the placental and uterus microsomes, respectively. Cytochrome P-450 has been solubilized with 1% cholate from the placental microsomes, and partially purified by chromatography on 6-amino-n-hexyl Sepharose 4B, DEAE-Sephadex A-50 and hydroxylapatite columns. The partially purified cytochrome P-450 efficiently catalyzed the omega-hydroxylation of various prostaglandins such as PGE1, PGE2, PGF2 alpha, PGD2, and PGA1 in a reconstituted system containing NADPH-cytochrome P-450 reductase, cytochrome b5, and phosphatidylcholine. The reconstituted system also hydroxylated palmitate and myristate at the omega- and (omega-1)-position, but could not hydroxylate laurate. These catalytic properties resemble those of a new form of cytochrome P-450 highly purified from the lung microsomes of progesterone-treated rabbits (Yamamoto, S., Kusunose, E., Ogita, K., Kaku, M., Ichihara, K., and Kusunose, M. (1984) J. Biochem. 96, 593-603). This type of cytochrome P-450, viz., cytochrome P-450 with high prostaglandin omega-hydroxylase activity may play a role in the regulation of prostaglandin levels in pregnancy.

Accumulation and degradation in the endoplasmic reticulum of a truncated ER-60 devoid of C-terminal amino acid residues.

The accumulation and degradation in the endoplasmic reticulum (ER) of a truncated ER-60 protease, from which the C-terminal 89 amino acid residues have been deleted (K 417 ochre), was examined. K 417 ochre overexpressed in COS-1 cells is not secreted into the medium, but accumulates as insoluble aggregates in non-ionic detergent without degradation in unusual clump membrane structures. K 417 ochre, stably expressed, forms soluble aggregates in non-ionic detergent and is distributed in the reticular structures of ER. Under these conditions, K 417 ochre is not secreted into the medium but is degraded with a half-life time of more than 8 h. Since K 417 ochre/C all S, in which all the Cys residues of K 417 ochre are replaced by Ser, also forms aggregates, an inter-disulfide bond appears unnecessary for aggregation. In both types of aggregates, Ig heavy chain binding protein, calnexin, glucose regulated protein 94, calreticulin, ERp72, and protein disulfide isomerase are scarcely found. Since degradation of the stably expressed K 417 ochre was not inhibited by lactacystin, leupeptin, NH(4)Cl, or cytocharasin B, but was inhibited by N-acetyl-leucyl-leucyl-norleucinal, the self-aggregated abnormal protein in the lumen of ER is assumed to be degraded by an unknown protease system other than proteasome, lysosome or autophagy.

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)