Isolation of the Photoactive Reaction Center Complex that Contains Three Types of Fe-S Centers and a Cytochrome c Subunit from the Green Sulfur Bacterium Chlorobium limicola f. thiosulfatophilum, Strain Larsen

The photoactive reaction center (RC) complex from the greensulfur bacterium Chlorobium limicola f. thiosulfatophilum, strainLarsen, was isolated after solubilization and ammonium sulfatefractionation followed by ion-exchange chromatography. The spectrumof the complex was almost identical with that of the similarRC complex isolated by Feiler et al. [(1992) Biochemistry 31:2608–2614] except for the presence of cytochrome c₅₅₁instead of c₅₅₃ in the latter study. A molecular ratio of BChla to P840 of the isolated RC complex was assayed to be 25–35.SDSPAGE analysis revealed that the isolated complex containedthree major polypeptides with apparent molecular masses of 68,41 and 21 kDa, respectively. The 21-kDa polypeptide was identifiedto be a heme-binding protein by staining the gel for peroxidaseactivity. The cytochrome c₅₅₁ was oxidized by flash light ina biphasic manner with half times of 90 and 390 µs, respectively,that coincided with the reduction half times of P840⁺. Threedistinct iron-sulfur centers assigned to F_A, F_B and F_x, respectively,from their g-values were detected by EPR spectroscopy at cryogenictemperature. These results suggest that the present preparationcontains a minimal functional unit of the RC of this bacterium,and that this complex appears to lie on a evolutionary linebetween RC's of purple bacteria and photosystem I. (Received August 18, 1992; Accepted October 28, 1992)     

Leukotriene B4 omega-hydroxylase in rat liver microsomes: identification as a cytochrome P-450 that catalyzes prostaglandin A1 omega-hydroxylation, and participation of cytochrome b5

The omega-hydroxylation of leukotriene B4 (LTB4) by rat liver microsomes requires NADPH and molecular oxygen, suggesting that the hydroxylation is catalyzed by a cytochrome P-450 (P-450)-linked monooxygenase system. The reaction is inhibited by CO, and the inhibition is reversed by irradiation of light at 450 nm in a light-intensity-dependent manner. The extent of the reversal is strongly dependent on the wavelength of the light used, the 450-nm light is most efficient. The finding provides direct evidence for the identification of the LTB4 omega-hydroxylase as a P-450. The P-450 seems to be also responsible for prostaglandin A1 (PGA1) omega-hydroxylation, but not for lauric acid omega-hydroxylation. The LTB4 omega-hydroxylation is competitively inhibited by PGA1, but not affected by lauric acid. The Ki value for PGA1 of 38 microM agrees with the Km value for PGA1 omega-hydroxylation of 40 microM. LTB4 inhibits the PGA1 omega-hydroxylation by rat liver microsomes in a competitive manner with the Ki of 43 microM, which is consistent with the Km for the LTB4 omega-hydroxylation of 42 microM. An antiserum raised against rabbit pulmonary PG omega-hydroxylase (P-450p-2) inhibits slightly the omega-hydroxylations of LTB4 and PGA1, while it has stronger inhibitory effect on lauric acid omega-hydroxylation. In addition to NADPH-cytochrome P-450 reductase, cytochrome b5 appears to participate in the LTB4 omega-hydroxylating system, since the reaction is inhibited by an antibody raised against the cytochrome b5 as well as one raised against the reductase.     

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)     

Synthesis and Herbicidal Activity of 6-Phenyl-5-Propylamino-2-Pyrazinecarbonitriles and Related Compounds

6-Phenyl- and 5-phenyl-2-pyrazinecarbonitriles with or without a propylamino group at the 3-, 5- or 6-position of the pyrazine ring were prepared together with some related compounds from the corresponding 2,3-pyrazinedicarbonitriles. Their herbicidal activities against barnyardgrass and broadleaf weeds were examined in pot tests. The 6-phenyl-2-pyrazinecarbonitriles were relatively potent compared with the 5-phenyl derivatives. Moreover, the presence of a propylamino group at the 5-position of the 6-phenyl-2-pyrazinecarbonitriles was closely related to an increase in activity.     

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.     

15-Cis-carotenoids found in the reaction center of a green sulfur bacterium Chlorobium tepidum and in the Photosystem I reaction center of a cyanobacterium Synechococcus vulcanus

Carotenoids were extracted, at 4 °C in complete darkness and under nitrogen atmosphere, from the reaction center (RC) of a green-sulfur bacterium and the Photosystem (PS) I RC of a cyanobacterium; each extract was subjected to high-performance liquid chromatography (HPLC) using an apparatus equipped with a two-dimensional diode-array detector in order to spectroscopically identify cis–trans carotenoids while performing HPLC analysis. In the extract from the RC of Chlorobium tepidum, 15-cis and all-trans--carotenes as well as 13-cis-, 15-cis- and all- trans-chlorobactenes (in the order of elution) were identified, whereas in the extract from the PS I RC of Synechococcus vulcanus, 15-cis-, all-trans- and 9-cis--carotenes were found. Thus, the universal presence of 15- cis carotenoids in the 'iron sulfur-type' RCs has been shown in addition to the previous cases of the 'quinone-type' RCs.