296 Chem.-Biol. Interactions Elsevier Publishing Company, Amsterdam

Printed in The Netherlands

Immunochemical studies utilizing ant ibody t o NADPH-cy tochrome c reductase as a specific inhibitor of microsomal electron transport

BETTIE SUE S. MASTERS, EDWARD B. NELSON, DANIEL M. ZIEGLER, JEFFREY BARON, P. PRITHVI RAJ and ELIZABETH L. ISAACSON Department o f Biochemistry, The University of Texas Southwestern Medical School at Dallas, Texas 75235 (U.S.A.)

The study of microsomal electron transport has been greatly aided in recent years by the introduction of specific inhibitors in the form of antibodies to electron transport components of liver microsomes by Omura I . Although Omura reported the inhibition of NADPH-cytochrome c reductase activity and the NADPH-dependent aniline hydroxylation in rat liver microsomes, inhibition of rabbit liver microsomal hydroxylation and cytochrome c reduction by antibodies to trypsin-solubilized rat liver reductase was not demonstrable. The present report will demonstrate the production of antibodies to hpase- solubilized porcine liver NADPH-cytochrome c reductase which concomitantly inhibit the NADPH-dependent demethylation of Type I substrates, such as ethylmorphine and amino- pyrine, and NADPH-cytochrome c reductase in porcine liver microsomes. A 7-globulin fraction prepared from pre-immune sera from the same rabbits, subsequently injected with antigen, or from non-immune sera from other uninjected rabbits had no inhibitory effect on any of these activities.

The concomitant inhibition of these activities by antibody to lipase-solubilized NADPH-cytochrome c reductase, which itself cannot reconstitute drug metabolism* or co-hydroxylation 2 in a resolved system, indicates that the solubilized reductase still possesses antigenic determinants in common with the native, intact flavoprotein reductase. Furthermore, the reductase solubilized from porcine liver shares common antigenic determinants with reductases from both higher and lower mammals in the phylogenetic series, since the antibody inhibits similar activities in rat, rabbit and human liver microsomes. Samples obtained from autopsy specimens of human liver were studied with respect to inhibition by anti-reductase "y-globulin. As with porcine and rat liver, the NADPH-cytochrome c reductase activity of human liver microsomes is inhibited concomitantly with NADPH-cytochrome P-450 and NADPH-dependent aminopyrine demethylase activities (Fig. 1).

The specificity of the anti-reductase 7-globulin was also demonstrated in studies designed to differentiate the cytochrome P-450-dependent oxidative demethylation pathway from the flavoprotein-dependent N-oxidation pathway for mixed-function oxidation of various drugs. Immunochemical differences between the 2 purified NADPH- specific flavoproteins were demonstrated by lack of inhibition or precipitation of the NADPH-dependent mixed-function amine oxidase by the anti-reductase 7-globulin. Furthermore, the anti-N-oxidase V-globulin, which precipitates but does not inhibit the enzymatic activity of the purified N-oxidase, did not result in a precipitin reaction with

~rUnpublished observations of B.S.S. Masters.

Chem.-Biol. Interactions, 3 (1971) 296-299

ANTIBODY AS INHIBITOR OF MICROSOMAL ELECTRON TRANSPORT

1

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297

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75.

25-

3.0 9.0 15.0 immune- 7'- globulin (rag)

Fig 1. The concomitant inhibition of aminopyrine demethylation, NADPH-cytochtome c reductase and NADPH-cytochrome P-450 reductase by antibody to porcine NADPH-cytochtome c teductase. The samples were prepared by mixing antibody in the amount designated in the figure with microsomes. Aliquots were then withdrawn from preincubation mixtures containing 20 rag microsomal protein for each of the various assays: e, aminopyrine demethylation; A, NADPH-cytochrome c reductase; =, NADPH-cytochrome P-450 reductase. The dashed line indicates non-immune garnma globulin. Control activities were: NADPH-cytochrome c reductase, 33 nmoles, rain -1 •mg -1 ; NADPH- l cytochrome P-450 reductase, 1.1 nmoles, rain -~ • mg -1 ; and HCHO formation, 2.0 nmoles, rain- •mg -1

purified porcine liver NADPH-cytochrome c reductase (Table I). Treatment o f the N-oxidase with lipase (used to solubilize the reductase from microsomes) or sodium dodecyl sulfate failed to elicit either cytochrome c reductase activity or immunochemical interaction with anti-reductase 7-globulin. The physical properties of the 2 flavoproteins are seen to differ markedly (Table I) and the 2 enzymatic activities are differentially purified. In addition, phenobarbital treatment has been shown to increase NADPH-cy tochrome c reductase and N,N-dimethylaniline and aminopyrine demethylase activities, but not the formation ofN, N-dimethylaniline-N-oxJde in porcine liver microsomes.

Chem.-BioL Interactions, 3 (1971)296-299

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TABLE I

A COMPARISON OF THE PROPERTIES OF NADPH-CYTOCHROME c REDUCTASE AND NADPH-DEPENDENT MIXED-FUNCTION AMINE OXIDASE FROM PORCINE LIVER MICROSOMES

Flavoprotein Molecular weight FAD content NADPH-cytochrome N3V-Dimethyl-n. Precipitin (moles FAD/ c r e d u c t a s e octylamtne-dependant reaction with mole enzyme) (nmole/mtn/mg) NADPH oxidation

(nmole/min/mg) Anti. Ant~ reductase oxidase

bO ~O OO

NADPH-cytochrome c reductase Equil. 68 800 2 38 100

sed.

Sephadex G-200 65 800

NADPH-dependent Equil. 474 000 mixe&function sed. +_23 000 amine oxidase

0 + 0

6 1 1400 0 +

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,.-]

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ANTIBODY AS INHIBITOR OF MICROSOMAL ELECTRON TRANSPORT 299

This work was supported in part by Grant No. 1-453 from the Robert A. Welch Foundation, by USPHS Grant No. 13619 from the Heart and Lung Institute and by Grant No. 68750 from the American Heart Association (BSSM), by USPHS Grant No. 12360 from the Institute of General Medical Sciences (DMZ) and USPHS Grant No. 1P11GM 16488.

REFERENCES

1 T. Omura, in J.R. Gillette, A.H. Conney, G.J. Cosmides, R.W. Estabrook, J.R. Fouts and G.J. Mannering (Eds.), Mierosomes and Drug Oxidations, Academic Press, New York, 1969, pp. 160-162.

2 A.Y.H. Lu, K.W. Junk and M.J. Coon, I. BioL Chem., 244 (1969) 3714.

Che~-BioL Interactions, 3 (1971) 296-299