Int. J. Biochem. Vol. 18, No. 8, pp. 731-741, 1986 0020-711X/86 $3.00+0.00 Printed in Great Britain Pergamon Journals Ltd KINETIC AND STRUCTURAL PROPERTIES OF BIOCATALYTICALLY ACTIVE SHEEP LUNG MICROSOMAL NADPH-CYTOCHROME c REDUCTASE MESUDE Y. |~CAN and EMEL ARiN¢~ Department of Biological Sciences,Middle East Technical University, Ankara Turkey [Tel. 237100/3105] (Received 16 December 1985) Abstract--1. NADPH-cytochrome c reductase was purified to electrophoretic homogeneity from detergent solubilized sheep lung microsomes. 2. The specific activity of the purified enzyme ranged from 56 to 67 #mol cytochrome c reduced/min/mg protein and the yield was 48-52% of the initial activity in lung microsomes. 3. The reductase had Mr of 78,000 and contained I mol each of FAD and FMN. 4. K~, values obtained in 0.3 M phosphate buffer, pH 7.8 at 37°C for NADPH and cytochrome c were 1 I. 1 _ 0.70/~M and 20.0 _+ 2.15 # M. Lung reductase was inhibited by its substrate, cytochrome c when its concentration was above 160/~M. 5. The lung reductase exhibited a ping-pong type kinetic mechanism for NADPH mediated cytochrome c reduction. 6. Purified lung reductase was biocatalytically active in supporting benzo(a)pyrene hydroxylation reaction when coupled with lung cytochrome P-450 and lipid. INTRODUCTION Many environmental pollutants enter into the body via inhalation and may result in carcinogenic activity in lung tissue (Kushner et al., 1957; Saffiotti et al., 1968). It has been recognized that lung, similar to liver, contains microsomal mixed-function oxidase system which can oxidatively metabolize a large number of foreign compounds, including carcinogens (Uehleke, 1968; Oppelt et al., 1970; Matsubara and Tochino, 1971; Bend et al., 1972; Abe and Watanabe, 1982; Armq and I~can, 1983; |~can et al., 1984; Arln~, 1985). This system consists of cytochrome P-450, NADPH-cytochrome c reductase (EC 1.6.2.4.) and phospholipid (Ann~ and Philpot, 1976; Guengerich, 1977b; Wolf et al., 1978; Serabjit-Singh et al., 1979; Arm~, 1980). NADPH-cytochrome c reductase func- tions in the transfer of reducing equivalents from NADPH to cytochrome P-450 during catalysis. Although liver NADPH-cytochrome c reductase was first purified from pigs in 1950 by Horecker, the purification of lung reductase was accomplished in the 1970s. In 1975, Buege and Aust purified the enzyme from bromelain-solubilized rat lung micro- somes. However, catalytic activities of this lung re- ductase preparation in hydroxylation reactions were not reported. The preparation of biocatalytically active detergent solubilized and partially purified rabbit lung reductase was first described by Arm~ and Philpot (1976). Since then, considerable progress has been made towards characterizing the rabbit lung reductase. Guengerich (1977b, 1978) and Serabjit- Singh et al (1979) purified rabbit lung microsomal enzyme by the method of Yasukochi and Masters (1976) and described the structural, biocatalytic and immunological properties of the enzyme in some detail. However, any information on the kinetic mech- anism for the reduction of cytochrome c by the purified lung reductase has not been reported before. In this study, some structural and kinetic properties of electrophoretically homogeneous and bio- catalytically active sheep lung NADPH-cytochrome c reductase are characterized. MATERIALS AND METHODS Chemicals 2',5'-ADP Sepharose 4B, Sephadex G-25 were purchased from Pharmacia Fine Chemicals. Emulgen 913 was pro- vided by Kao-Atlas Co. Ltd. Porapak Q was obtained from Waters Associates. DEAE-Cellulose (DE-52) was purchased from Whatman Biochemicals Ltd. Hydroxylapatite was obtained from Bio-Rad Laboratories. The following chem- icals were purchased from Sigma Chemical Company: NADP ÷, NADPH, E-amino-n-caproic acid (~-ECA) phenylmethylsulfonyl fluoride (PMSF), 3-Methylcholan- trene (3-MC), 2'AMP~ benzo(a)pyrene, rabbit muscle phos- phorylase a, bovine liver catalase, bovine serum albumin, hog stomach muscle pepsin, horse heart cytochrome c (Type VI). FAD and FMN were obtained from Calbiochem. (G-3H)-Benzo(a)pyrene (5 mCi/ml toluene) was purchased from the Radiochemical Centre Ltd. All other chemicals were of the highest grade commercially available. Preparation of sheep lung microsomes The lungs from well bled Akkaraman sheep, about (~12 months old, were obtained from slaughter house (Et ve Balik Kurumu-Ankara) immediately after killing. Lung microsomes were prepared as described by Armq and I~can (1983) except that, 0.25 mM E-ACA and 0.25 mM PMSF were added to the homogenization medium and the washed microsomal pellet was suspended in 25% glycerol, contain- ing 1 mM EDTA, 0.25 mM E-ACA. The homogenized microsomal suspension containing approximately 20-25 mg protein per ml was gassed with nitrogen in small plastic bottles and stored at -20°C until used. 731 B.C. 18/8--D