ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 165, 665-671 (1974)

Properties of Purified Kidney Microsomal NADPH-Cytochrome c

Reductase’

LUCY LIOU FAN AND BETTIE SUE SILER MASTERS

Department of Biochemistry, The University of Texas Health Science Center at Dallas, Dallas, Texas 75235

Received August 5, 1974

NADPH-cytochrome c reductase, solubilized by lipase digestion of microsomes prepared from perfused porcine kidney cortex, was purified about 3600-fold to give a turnover number of 1230 nmoles cytochrome c reduced per min per nmole flavin. The kinetic determination of K, and V with respect to NADPH, cytochrome c, and NADH, resulted in values similar to those obtained with purified liver reductase. The kidney microsomal enzyme also exhibited a ping-pong kinetic mechanism for NADPH-mediated cytochrome c reduction.

Spectrofluorometric measurements demonstrated the presence of equimolar amounts of FAD and FMN per mole of reductase. The molecular weight was estimated by Sephadex G-ZOO gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 68,000 and 71,000 g per mole, respectively.

Immunochemical techniques, including Ouchterlony double-diffusion studies and inhibition of catalytic activity by antibody to the liver microsomal NADPH-cytochrome c reductase, established the similarity of the purified liver and kidney reductases.

Hepatic microsomal NADPH-cyto- chrome c reductase (EC 1.6.2.3) has been extensively purified and studied in several laboratories (l-7). The liver reductase is a flavoprotein (1, 2) which is quite specific for NADPH as its physiological reductant, but it will utilize both l-electron and 2-electron acceptors as oxidants (5, 7). Recently, the liver microsomal flavoprotein was shown to contain equimolar quantities of FAD and FMN (8).

Preliminary reports of this work have appeared’ (9, 10). The present report de- scribes a study of the physicochemical and immunochemical properties of the purified kidney reductase. The purpose of this investigation was to determine the similar- ities between the kidney and liver micro-

’ Supported in part by USPHS Grants HL 13619, GM 16488, and Grant No. I-453 from The Robert A. Welch Foundation (B. S. S. M.).

’ A preliminary report of this work was presented at the 9th International Congress of Biochemistry in

Stockholm (1973) and at the Biochemistry/Biophysics Meetings in Minneapolis (1974).

somal flavoproteins. Since the kidney cor- tex microsomes utilize NADH as well as NADPH in the cytochrome P-450-depend- ent w-hydroxylation of fatty acids, it was of interest to determine if, unlike the liver flavoprotein, the kidney enzyme could function with NADH as electron donor. Preliminary experiments (ll), in which NADPH- and NADH-cytochrome c and cytochrome P-450 reductase activities were measured in rat kidney cortex mi- crosomes, showed that antibody to the NADPH-cytochrome c reductase flavo- protein inhibited only the NADPH- mediated activities indicating that elec- trons from NADH were not being shuttled through this flavoprotein.

MATERIALS AND METHODS

Reagents. F.4D. FMN, bovine serum albumin, DEAE-cellulose and cytochrome c (horse heart type VI) were obtained from Sigma Chemical Co. Human serum protein (100% human albumin) was obtained from Dade Division, American Hospital Supply Cor- poration. Pancreatic steapsin (lipase) was purchased from Nutritional Biochemicals Corporation and puri-

665

CopyrIght 0 1974 hy Academic Press. Inc. All rights of reprrlduction in an> form reserved.

666 FAN AND MASTERS

tied according to the procedures described by Masters et al. (12). NADH, NADP’, and NADPH were obtained from P-L Biochemicals, Inc. Sephadex and Blue Dextran were furnished by Pharmacia Fine Chemicals, AB. Ammonium sulfate and monopotas- sium and dipotassium phosphates were purchased from J. T. Baker Chemical Co. All other chemicals were obtained as reagent grade from Mallinckrodt Chemical Co.

Instruments. Flavin spectra were measured with a Cary Model 14R recording spectrophotometer ther- mostated at 25°C. Kinetic determinations were per- formed with a Hitachi 124 recording spectrophotome- ter thermostated at 25°C. Spectrofluorometric analy- ses were performed with an Aminco Bowman spectro- photofluorometer outfitted with a Sargent SRG re- corder.

Purification of NADPH-cytochrome c reductase. Porcine kidney cortex microsomes were prepared and solubilized with lipase according to the procedure of Masters et al. (12). The use of pancreatic steapsin has been consistently used for the solubilization of the liver microsomes for the purification of NADPH-cyto- chrome c reductase. From liver microsomes, this procedure yielded a preparation essentially free of cytochrome b, in the supernatant fraction, and the resulting soluble enzyme was purified to homogeneity for the mechanism studies of Masters et al. (5, 7). It was, therefore, considered desirable to obtain a solu- ble preparation of the kidney cortex microsomal reduc- tase for purposes of comparison with the liver flavo- protein utilizing this solubilization technique. Subse- quent ammonium sulfate fractionation (precipitation between 30 and 70% saturation), Sephadex G-100 superfine, DEAE-cellulose, and Sephadex G-200 su- perfine column chromatography were performed by a modification (13) of the method of Omura and Take- sue (14).

Assay of enzyme actiuity. NADPH-cytochrome c reductase was assayed as previously described (12). One unit of enzyme activity is defined as that amount of enzyme which will catalyze the reduction of cyto- chrome c at an initial rate of 4.76 x 10m5 mmoles per min per ml of reaction mixture, utilizing the cM for reduced minus oxidized cytochrome c of 21 x lo3 M-km-’ (15). Protein was determined by the biuret method (16) using human serum albumin as stan- dard.

Determination of flnuin content. Commercial sam- ples of FAD and FMN were chromatographed on DEAE-cellulose using 0.1 M potassium phosphate (pH 6.8) as eluting buffer. The FAD and FMN solutions gave absorbance ratios (375 nm:45Onm) ot 0.81 and 0.83, respectively, in agreement with Beinert (17). Chromatographic, spectrophotometric, and spectrophotofluorometric procedures were performed in subdued light. The flavin content of this reductase was determined by using the spectrofluorometric method of Faeder and Siegel (18).

Electrophoresis. Polyacrylamide disc gel electro- phoresis was performed at constant current with Hoeffer equipment using the Tris-glycine buffer sys- tem of Davis (19). Electrophoresis in the presence of sodium dodecyl sulfate was performed as described by Weber and Osborn (20).

Immunochemical studies. Double-diffusion agar plates were run by the method of Ouchterlony (21)

TABLE I

PURIFICATION OF NADPH-CYTOCHROME c REDUCTASE FROM PORCINE KIDNEY MICROSOMES

Procedures Enzyme activity Percent units” yield

start RIXOV- erY

1 Lipase solubilization 2300 1500 65 2 (NH,),SO, saturation 1500 1300 57

(30-70s) 3 Sephadex G-100 super- 1300 1300 57

fine column 4 DEAE-cellulose col- 1106 700 36

umn 5 Sephadex G-200 super- 600 500 30

fine column

0 Enzyme activity was assayed as described in Materials and Methods.

FIG. 1. Polyacrylamide disc-gel electrophoresis of purified liver and kidney NADPH-cytochrome c re- ductase. (A, C) Stained with amido black; liver and kidney reductase, respectively. (B, D) Stained for NADPH-neotetrazolium reductase activity; liver and kidney reductase, respectively. All gels con- tain tracking dye and protein (about 30 pg).

PROPERTIES OF KIDNEY NADPH-CYTOCHROME c REDUCTASE 667

0 40 80 20 40 60 SO 100 --!e &,-I [Cyt 4

FIG. 2. Left portion. Lineweaver-Burk plots of NADPH-cytochrome c reductase activity varying NADPH and cytochrome c concentration. Reaction rates were determined as described by Masters et al. (1). The sample cuvette contained enzyme with a flavin concentration of 1 x 1Om8 M. The cytochrome c concentrations are 0.01 mM (O-a, 0.02 mM (A-A), 0.05 mM ([7--O), and 0.1 mM

(O-O). All reactions in a total volume of 1.1 ml in 0.05 M potassium phosphate buffer (pH 7.71 with lo-’ M EDTA. Blanks contained no NADPH. Right portion. Reciprocal plot of the V value obtained with each cytochrome c concentration from left-hand side of figure versus cytochrome c concentra- tion. From this replot a so-called doubly infinite V and turnover number at the intercept was calculated to be 1245 nmoles cytochrome c reduced x min’ x nmole-’ flavin.

and antibody titrations were performed as described previously (22).

RESULTS

Enzyme purification. The purification procedures are outlined under Materials and Methods. The final yield of purified kidney reductase from the microsomal sus- pension is approximately 30% (Table I). The purified kidney enzyme was not homo- geneous (Fig. l), but when compared with the liver reductase by disc gel electrophore- sis, the striking similarity between the two enzymes is apparent. Note that some of the minor bands of the kidney and liver reduc- tase preparations produce activity bands when tested for NADPH-neotetrazolium red tase activity, indicating that enzy- matically active proteolytic products are formed during the preparation procedures.

Kinetic studies. Steady-state kinetic studies were performed on the partially purified reductase and the following K, values (Figs. 2 and 3) were obtained: K m N .AD P H = 4.6 PM, Kncytoct,rome c’ = lo.0 PM, and KmNADII = 24 mM, which are sim- ilar to those obtained with purified liver mi- crosomal NADPH-cytochrome c reduc-

tase3 (1). Figure 2 (left) presents a series of parallel lines, consistent with a ping-pong- type kinetic mechanism for NADPH- mediated cytochrome c reduction. These data suggest that the kidney cortex flavo- protein can be reduced by NADPH in the absence of electron acceptor generating a reduced enzyme intermediate which can, in the absence of NADP+, be reoxidized by cytochrome c and other electron acceptors (5). A doubly infinite turnover number of 1240 nmoles cytochrome c reduced per min per nmole flavin is obtained (Fig. 2, right). In addition, NADP+ was shown to be a potent competitive inhibitor of the kidney reductase with a K, of 4.4 pM (Fig. 4), similar to the liver flavoprotein.

Molecular weight determination. The molecular weight of kidney NADPH-cyto- chrome c reductase was estimated to be about 68,000 by Sephadex G-200 chroma- tography in that it exhibited a similar elution volume relative to the void volume of Blue Dextran (V, of kidney reductase =

3R. A. Prough and B. S. S. Masters, unpublished observations regarding the K,NADH for liver micro-

somal NADPH-cytochrome c reductase.

668 FAN AND MASTERS

140-- . “0 ; 120..

E loo--

: - F 60.-

D

.A

l / NADH

=*25mM

d 60.-

:::A” l

I I1 I I I I, /,I,

-40 -20 0 20 40 60 60 100 120 140 IW 160 200 220 240 260

i&l M-’

FIG. 3. A double-reciprocal plot of the absorbance changes at 550 nm, associated with the addition of increasing concentrations of NADH to a reaction mixture of partially purified kidney NADPH- cytochrome c reductase containing 5 mg per ml,

I I I I I I 0 40 80 I20 160 200

- mM-’ [N ADPH]

FIG. 4. A Lineweaver-Burk plot exhibiting the competitive inhibition of pig kidney NADPH-cyto- chrome c reductase by NADP’. The concentration of NADP+ was varied as indicated in the figure. The turnover number calculated at infinite concentrations of NADPH was 1180 nmoles cytochrome c reduced x min.’ x nmolee’ flavin.

10 ml) as bovine serum albumin (V, of BSA = 10 ml) (Fig. 5). SDS polyacrylamide disc gel electrophoresis (Fig. 6) indicated that the kidney and liver flavoproteins migrate

similarly and a molecular weight of 71,000 was calculated from these data. (Note the similarity to human serum albumin).

Flavin content. The kidney microsomal reductase contains equimolar quantities of FAD and FMN (Table II). These results are in agreement with those published by Iyanagi and Mason (8) and confirmed by this laboratory for the liver reductase (23).

Immunochemical studies. In order to test for subtle differences in the liver and kidney reductases which were not obvious in the physicochemical studies, and to demonstrate immunochemical similarities between the two enzymes, double-diffusion experiments were performed (Fig. 7). It is apparent that the liver and kidney reduc- tases are immunochemically similar, since the major precipitin bands are confluent. Adsorbing-out experiments (not shown) re- sulted in complete disappearance of the liver reductase-antibody precipitin line when increasing amounts of kidney reduc- tase were added to the antibody-conta+ing wells, further indicating immunochemical similarity. Antibody titration was em- ployed, also, as shown in Fig. 8. Although the purified kidney reductase is inhibited to a greater extent than is the activity in the kidney microsomes by antibody to the liver reductase, both respond to the anti- body. Therefore, the antibody to liver

PROPERTIES OF KIDNEY NADPH-CYTOCHROME c REDUCTASE 669

NADPH-cytochrome c reductase is a po- tent inhibitor of the kidney enzyme, puri- fied or microsomal.

DISCUSSION

Kidney microsomal NADPH-cyto- chrome c reductase is difficult to purify due to its instability during the prepara- tion procedures and to the small amount of reductase in kidney cortex microsomes, approximately one-fifth that contained in the liver microsomes. From the over-all yield presented in this paper, it is apparent that sufficient quantities of material for

characterization and immunochemical studies can be obtained only after the purification procedure has been repeated several times. However, owing to the rela- tive stability of the purified enzyme this task has not been beyond the scope of possibility.

‘In Fig. 1, the electrophoretic patterns of kidney microsomal reductase revealed more bands than a similar preparation of liver enzyme. The occurrence in kidney cortex of easily released lysosomal cathep- sins or soluble cathepsins probably con- tributes to the further proteolysis of the

I 1 I I

(InI) -77 - 4.5 -1.3 0 1.9 5.1 IO.0 II.5 I47 16.1

ELUTION VOLUME RELATIVE TO VOID VOLUME

‘Ilx_ Afir n .A 2.5 75 11

(ml) -7.1 -3.9 0 2.5 5.7 100 12.1 15.3 ELUTION VOLUME RELATIVE TO VOID VOLUME

Fro. 5. Chromatography of NADPH-cytochrome c reductase on Sephadex G-2OO. Gel filtration of Blue Dextran (0.1 mg) and BSA (1 mg) are shown in the upper figure, while Blue Dextran (0.2 mg) and kidney microsomal reductase (about 500 enzyme units) are shown in the lower figure with the same column (r = 0.8, cm, 1 = 23 cm). The proteins were eluted with 0.05 M phosphate buffer. (pH 7.7) containing 0.1 mM EDTA.

50

1

. PHOSPHORYLASE (I

\ *A

48 t

HSP

GDH. *Mw of Pig Liver NADPH Cyt E

&+u of Ptg Kldney NADPH Cyl G

461

\

Reductase = 71,000

Reductose = 71,000 ALDOLASE.

PEPSIN.

0 02 04 06 OS IO

MOBILITY

FIG. 6. Estimation of the molecular weight of NADPH-cytochrome c reductase by SDS polyacryl- amide disc-gel electrophoresis. Gels and samples were prepared as indicated by Weber (20). The marker proteins were phosphorylase a, human serum protein, glutamic acid dehydrogenase, aldolase, pepsin, myo- globin, and cytochrome c. All proteins were run on duplicate gels, separately, and then on one gel con- taining all proteins together, except human serum protein. The mark (*) and (A) indicated the mobility of liver and kidney reductase, respectively.

TABLE II

SPECTROPHOTOFLUOROMETRIC DETERMINATION OF THE FLAVIN CONTENT OF KIDNEY NADPH-CYTOCHROME c

REDUCTASE

670 FAN AND MASTERS

an extent which has permitted a compari- son of its kinetic, physical, and immuno- chemical properties with the liver micro- somal reductase purified to homogeneity in our laboratory (7), we must, therefore, conclude that only subtle differences exist

FIG. 7. The immunochemical similarity of NADPH-cytochrome c reductase from pig liver and kidney microsomes determined with y-globulin pre- pared against pig liver microsomal reductase. Anti- body was applied in the center well while the two enzymes were applied in alternate outer wells. Ap- proximately 10 ~1 of each enzyme preparation (3 mg mll’ protein) were introduced into the agar wells.

Porcine kidney Relative Concentration % NADPH- fluorescence Cm) Flavin

cytochrome e intensity reductase pH 2.611.1”

ID 1.23 FAD 23.4 56 FMN 18.2 44

II’ 1.24 FAD 16.6 50 FMN 16.4 50

D Fluorescence measurements were performed as described in Materials and Methods. The relative fluorescence intensity ratio (pH 2.6ipH 7.7) of the FMN standard and the FAD standard gave 0.76 and 4.49, respectively.

b Enzyme containing 42.6 nM flavin was used in the spectrofluorometric determinations.

c Enzyme containing 36.9 nM flavin was used in the spectrofluorometric determinations.

kidney preparation and its consequent instability.

FIG. 8. The concomitant inhibition of kidney mi- crosomal and purified NADPH-cytochrome c reduc- tase by antibody to porcine liver reductase. The incubations with antibody were performed as de- scribed previously (22). Control activity for cyto- chrome c reduction by NADPH were, purified NADPH-cytochrome c reductase, (1200 nmole x min’ x nmole-’ flavin); microsomes (9.0 nmoles x min’ x mg-‘). Purified kidney reductase (0-O); kidney microsomes (m-m). The corresponding Since we have purified the kidney micro-

somal NADPH-cytochrome c reductase to dashed lines indicate preimmune y-globulin.

PROPERTIES OF KIDNEY NADPH-CYTOCHROME c REDUCTASE 671

which are qualitatively indicated by the immunochemical studies shown in Fig. 7. Since the role of this enzyme in kidney, as well as in liver, is ultimately to reduce cytochrome P-450, which itself serves as the terminal oxidase and the site of sub- strate specificity, it is not surprising that the kidney and liver reductases possess similar physicochemical properties.

At the outset of this work it was our intention to obtain pure kidney reductase which would allow a comparison with puri- fied liver reductase. Ongoing studies in this laboratory are leading to further investiga- tion of the roles of NADPH- and NADH- specific enzymes in kidney microsomal electron transport activities.

Recent studies by Sasame et al. (24), have suggested a role for cytochrome b, in NADH- and NADPH-mediated w- and (w-1)-hydroxylation of laurate in both liver and kidney cortex. This report (24) also confirmed our observations (11) that NADH-mediated activities were not inhib- ited by anti-NADPH-cytochrome c reduc- tase. The present paper strengthens the evidence that electrons from NADH are not transferred by NADPH-cytochrome c reductase in either liver or kidney mi- crosomes. The interaction of cytochrome 6, with the terminal oxidase for o-hydroxyla- tion activities in liver and kidney, deter- mined by Kupfer and Orrenius (25) and Jakobsson et al. (26) to be cytochrome P-450, will be the subject of further investi- gation.

ACKNOWLEDGMENTS

The authors thank Mm. Elizabeth Isaacson for purifying the liver NADPH-cytochrome c reductase and preparing antibody to this reductase. Also, thanks are due to Dr. Russell A. Prough for his helpful suggestions and Dr. Rene Frenkel for his generous gifts of SDS marker proteins.

REFERENCES

1. WILLIAMS, C. H., JR., AND KAMIN, H. (1962) J. Biol. Chem. 237, 587-595.

2. PHILLIPS, A. H., AND LANGDON, R. G. (1962) J. Biol. Chem. 237, 265222660.

3. JICK, H., AND SCHUSTER, L. (1966) J. Biol. Chem. 241, 5366-5369.

4. OMURA, T., SIEKEVITZ, P., AND PALADE, G. E. (1967) J. Biol. Chem. 242, 2389-2396.

5. MASTERS, B. S. S., KAMIN, H., GIBSON, Q. H., AND WILLIAMS, C. H., JR. (1965) J. Biol. Chem. 240, 921-931.

6. KAMIN, H., MASTERS, B. S. S., GIBSON, Q. H., AND WILLIAMS, C. H., JR. (1965) Fed. Proc. 24, 11641171.

7. MASTERS, B. S. S., BILIMORIA, M. H., KAMIN, H., AND GIBSON, Q. H. (1965) J. Viol. Chem. 240, 4081-4088.

8. IYANAGI, T., AND MASON, H. S. (1973) Biochemis- try 12, 2297-2308.

9. MASTERS, B. S. S., ISAACSON, E. L., PROUGH, R. A., AND MASON, J. I. 9th International Congress of Biochemistry, Stockholm (1973) Abstnn is. 7d2. p. 341.

10. FAN, L. L., AND MASTERS, B. S. S. (1974) Fed. PFOC. 33, 1397.

11. MASTERS, B. S. S., NELSON, E. B., SCHACTER, B. A., BARON, J., AND ISAACSON, E. L. (1973) Drug Metab. Disposition 1, 121-128.

12. MASTERS, B. S. S., WILLIAMS, C. H., JR., AND KAMIN, H. (1967) in Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., eds.), Vol. X, pp. 5655573, Academic Press, New York.

13. PROUGH, R. A., ANDMASTERS, B. S. S. (1973) Ann. N. Y. Acad. Sci. 212, 89-93.

14. OMURA, T., AND TAKESUE, S. (1970) J. Biochem. (Tokyo) 67, 249-257.

15. MASSEY, V. (1959) Biochim. Biophys. Acta 34, 255-256.

16. GORNALL, A. G., BARDAWIU, E. J., AND DAVID, M. M. (1949) J. Biol. Chem. 177, 751-766.

17. BEINERT, H. (1960) in The Enzymes (Lardy, H., and Myrblck, K., eds.), 2nd ed., Vol. 2, pp. 339-416, Academic Press, New York.

18. FAF.DER, E. J., AND SEIGEL, L. M. (1973) Anal. Biochem. 53, 332-336.

19. DAVIS, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404-427.

20. WEBER, K., AND OSBOHN, M. (1969) J. Biol. Chem. 244,4406-4412.

21. OUCHTERLONY, 0. (1958) Progr. Allergy 5, l-78. 22. MASTERS, B. S. S., BARON, J., TAYLOR, W. E.,

ISAACSON, E. L., AND LOSPALLUTO, J. J. (1971) J. Viol. Chem. 246,4143-4150.

23. PROUGH, R. A., MASTERS, B. S. S., AND KAMIN, H. (1974) Fed. PFOC. 33, 1397.

24. SASAME, H. A., THORGEIRSSON, S. S., MITCHELL, J. R., AND GILLETTE, J. R. (1974) Life Sci. 14, 35-46.

25. KUPFER, D., AND ORRENIUS, S. (1970) EUF. J. Biochem. 14, 317-322.

26. JAKOBSSON, S., THOR, H., AND ORRENIUS, S. (1970) Biochem. Biophys. Rex. Commun. 1079&108d.