NADPH-CYTOCHROME c REDUCTASE AND ITS ROLE IN MICROSOMAL CYTOCHROME

Transcription

1 DRG METABOLISM AND DisposiTioN Copyright #{174} 1973 by The American Society for Pharmacology and Experimental Therapeutics vol. 1 No. 1 Printed in.s.a. NADPH-CYTOCHROME c REDCTASE AND ITS ROLE IN MICROSOMAL CYTOCHROME P45-DEPENDENT REACTIONS BETTIE SE SILER MASTERS. EDWARD B. NELSON. BRENT A. SCHACTER.2 JEFFREY BARON.3 AND ELIZABETH L. ISAACSON niversity of Texas Southwestern Medical School at Dallas The study of liver microsomal electron transport and drug metabolism activities involving NADPH-cytochrome c reductase has been performed using immunochem ical techniq ues ( I -4). This approach has been particularly valuable in establishing the role of this flavoprotein in the reduction of microsomal cytochrome P-45 and, thus, in the metabolism of drugs (2, 3). The existence of NADPH-cytochrome c reductase activity in a variety of tissues including kidney, spleen, adrenal cortex, heart, and lung has led to a closer examination of the role of this enzymic activity in cytochrome P-45-mediated reactions in these tissues. It has been demonstrated by Masters and Ziegler (5) that the two NADPH-dependent electron transport systems in porcine liver microsomes contain separate and distinct flavoproteins (fig. I): NADPH-cytochrome c reductase and NADPH-dependent mixed function amine oxidase (N-oxidase). This conclusion was based on the differential purification of the two enzymic activities, the induction of the re- Supported by.s. Public Health Service Grant HL from the National Heart and Lung Institute and by Grant from the Robert A. Welch Foundation (B.S.S.M.) and by.s. Public Health Service Grant 1-P1 1 GM An Established Investigator of the American Heart Association. 2 Present address: Department of Internal Medicine. niversity of Manitoba Faculty of Medicine. 7 Bannatyne Avenue. Winnipeg. Manitoba. Canada R3E V9. Present address: Department of Pharmacology. niversity of Iowa College of Medicine. Iowa City. Iowa Send reprint requests to: Dr. Bettie Sue Masters. Department of Biochemistry. niversity of Texas Southwestern Medical School Harry Hines Blvd.. Dallas. Texas ductase (but not N-oxidase) activity by phenobarbital pretreatment, the comparison of the physical properties of the purified enzymes, and, finally, the immunochemical nonidentity of the two enzymes, either purified or microsomal. Focusing on the upper scheme in fig. I in which NADPH-cytochrome c reductase is involved in the transfer of electrons to cytochrome P-45, it can be seen that inhibition of electron transfer from the reductase should result in inhibition of the overall activity catalyzed by such a system if the initial transfer of electrons is rate limiting. It can be seen in fig. 2 that inhibition of NADPHcytochrome c reductase activity in porcine liver microsomes by anti-reductase -y-globulin results in the concomitant inhibition of ethylmorphine demethylation catalyzed by these microsomes, showing that the oxidative metabolism of this drug is absolutely dependent upon electron transfer via NADPH-cytochrome c reductase. A similar experiment was performed using human liver microsomes in which NADPH-cytochrome C reductase, NADPH-cytochrome P-45 reductase, and NADPH-dependent oxidative demethylation of aminopyrine were measured as a function of increasing concentrations of antibody to the reductase. In fig. 3 the concomitant inhibition of these three activities is clearly demonstrated. Since there is no evidence for a concentration lag in the inhibition pattern as the antibody titer is increased, these data demonstrate an absolute dependence upon electron flux through the reductase flavoprotein. For these studies, it was important to establish the species cross-reactivity of the antireductase -y-globulin. As shown in table I, the amount of antibody prepared to porcine liver reductase required to inhibit NADPH-cytochrome c reductase activity 5% is approximately the same in porcine and human liver microsomes. On the other hand, approximately 5 121

2 122 MASTERS ET AL. CYTOCHROME C DRG 2 H2 DRG-OH TPNH -P FIAVOPROTEIN -+ X --P CYTOCHROME P-45 (FAD) 2 H2 TPNH -P FLAVOPROTEINId (FAD) DRG CYTOCHROME C FIG. 1. The NA DPH (TPNH)-dependent electron transport systems of porcine liver microsomes. taming 61.6 mg of microsomal protein and varying amounts of preimmune and immune -y-globulin and assayed for ethylmorphine demethylase and NADPHcytochrome c reductase activities. Formaldehyde formation from ethylmorphine was measured at 38#{176}C and NADPH-cytochrome c reductase activity was determined at 25#{176}C.The data are presented as percentage of control activity to demonstrate the concomitant inhibition of both activities by anti-reductase -y-globulin. NADPH-cytochrome c reductase activity was calcutimes as much antibody is required to inhibit > -I z I z a cytochrome c reduction by rat microsomes, presumably due to a relative lack of immunochemical similarity between the enzymes from pig and rat. Having established the varying degrees of species cross-reactivity of the antibody to reductase, it was decided to test the inhibition of NADPH-cytochrome c reductase activity in other tissues. Fig. 4 shows the NADPH-specific electron transport systems of adrenal cortex mitochondria and microsomes, both of which are directed toward the reduction of a cytochrome P-45 which serves as the terminal oxidase for the hydroxylation of specific steroids by the adrenal gland. It is interesting to note that the adrenocortical mitochondria are specific for the I I fi- and 18-hydroxylation reactions of steroids as reported by Sweat (6) and his colleagues, and mg. OF IMMNE 7 - GLOBLIN IN PREINCBATION FIG. 2. Concomitant inhibition ofporcine liver microsomal activities. Microsomes from phenobarbital-treated pigs were previously incubated with varying amounts of -y-globulin prepared from pooled antisera to purified, lipase-solubilized NADPH-cytochrome c reductase. Aliquots were removed from previously incubated mixtures con- the adrenocortical microsomes catalyze the 17- and 2l-hydroxylations of steroids according to Plager and Samuels (7) and Cooper, Estabrook, and Rosenthal (8). The presence of an iron-sulfur protein, adrenodoxin, as an intermediate electron carrier in the adrenocortical mitochondrial system between the flavoprotein and cytochrome P-45 has been established by the studies of Omura et a!. (9, 1). These studies involved the resolution and reconstitution of the I l9-hydroxylation of deoxycorticosterone in which a requirement for adrenodoxin was demonstrated. lated on the basis of controls without y-globulin (#{149}-#{149})and with preimmune -y-globulin (A-A). Ethylmorphine demethylase was calculated on the basis of controls without -y-globulin (-) and with preimmune y-globulin (E.-i). Control activities were: NADPH-cytochrome c reductase, 147 nmol x min x mg, and HCHO formation, 15.1 nmol x min x mg.

3 NADPH-CYTOCHROME c REDCTASE IN VARIOS TISSES TABLE Specificity of antibody to porcine liver NA DPHcytochrome c reductase NADP1-cytochrome c reductase activities in the presence of anti-reductase y-globulin were determined as described previously (3). I Liver Microsome. from Mg y-globuiin/nit Reductaae Activity Producing SO Inhib,tion Pig Human Rat Rabbit C C.) from sonicated mitochondria, called S2 (I I), containing flavoprotein and adrenodoxin, and was found to inhibit the NADPH-dependent reduction of cytochrome c but not 2,6-dichlorophenolindophenol (12). Table 2 shows the results of an experiment in which sonicated mitochondna were titrated with anti-adrenodoxin -y-globulin; NA DPH-cytochrome c reductase, NADPH-cytochrome P-45 reductase, and I I $-hydroxylase activities were measu red. The data show that all three activities are inhibited to approximately the same extent, which mdicates a requirement for adrenodoxin in the catalysis of these three reactions by adrenocortical 3 5#{149} minima - globulin (aiga) FIG. 3. The concomitant inhibition ofhuman microsomal aminopyrine demethylation, NA DPH-cytochrome c reductase, and NA DPH-cytochrome P-45 eductase by antibody to porcine NA DPH-cytochrome c reductase. The samples were prepared by mixing a known amount of -y-globulin with 2 mg of microsomal protein in.5 M potassium phosphate buffer, l M EDTA, ph 7.7, at 2#{176}C.Aliquots were then withdrawn from this mixture and added to the various assays as described in the text: S-S. aminopyrine demethylation; A-A, NADPH-cytochrome c reductase; #{149}---i, NADPH-cytochrome P-45 reductase. The dashed line indicates preimmune -y-globulin. Control activities were: NADPH-cytochrome c reductase, 33 nmol x min x mg : NADPH-cytochrome P-45 reductase, 1.1 nmol x min x mg ; and HCHO formation, 2. nmol x min x mg. In order to differentiate between the electron transport systems of adrenocortical mitochondria and microsomes, antibody to homogeneous preparations of adrenodoxin was prepared. This antibody was added to a soluble fraction prepared 15- mitochondria. When the antibody to adrenodoxin was tested in adrenocortical microsomes for its effect on the 2 1 -hydroxylation of I 7a-hydroxyprogesterone, it was found to have no effect (table 3). On the other hand, if the flavoprotein of adrenocortical microsomes was similar to the NADPHcytochrome c reductase of liver microsomes, the antibody to this ezyme might be expected to inhibit both cytochrome c reduction and 21-hydroxylation in this tissue. In experiments performed in collaboration with Dr. George T. Bryan of the niversity of Texas at Galveston, anti-reductase y-globulin was shown to inhibit the formation of I l-deoxycorticosterone from progesterone (fig. 5) from 7.8% at the highest level of antibody tested. Similar results were obtained when the formation of I l-deoxycortisol from I 7 a-hydroxyprogesterone was measured (fig. 5). The antibody used in this experiment was elicited in goats and purified to the 7-globulin fraction. The lack of concomitant inhibition of the NADPH-cytochrome c reductase activity can be explained by contamination with adrenocortical mitochondria in which the anti-reductase 7-globulin has no effect (3). Cytochrome

4 124 MASTERS ET AL f- L.LY.L!Ak2 ADRENAL MICROSOMES CYTOC ROME C SBSTRATE H SSSTRATE-OH TPNH - Fl.AVOPROT(IN +X -P CYTOCHROM( P-45 IFADI ADRENAL cortical MITOCHONDRI CYTOCHROME c STEROID STEROID-OH TPNH -..- FLAVOPROTEIN -r NON-HEME IRON PROTEIN --PCYTOCHROME P-45O IFADI rs ADRENXINI 2 H2O FIG. 4. The cytochrome P45-dependent electron transport systems of liver and adrenal cortical microsomes and adrenal cortical mitochondria. TABLE 2 Comparison of the inhibitory efftcts of the antibody to adrenodoxin on NA DPH-dependent activities in bovine adrenocortical mitochondria Bovine adrenocortical mitochondria were frozen and thawed five times prior to the determination of enzymatic activities. The determination of 1 19-hydroxy1ase activity in bovine adrenocortical mitochondria was made using I l-deoxycorticosterone as substrate by measuring the rate of formation of corticosterone by the fluorometric method of Mattingly (13). p-giobuiin NADPH.Cytochrome Reductase c NADPH-Cytochrome P.45 Reductase Ii $-HydroxyIa Amount % control Amount 5 control Amount 5 control mg/mg mi:oclso.sdrioj protein nmol/ mg/mm nmol/mg/ini:ial ti sec nmol/ mg/mm 2:1 (immune) 2:l(preimmune) TABLE 3 Effect ofantibody to adrenodoxin on the C-21 hydroxylation of! 7 a-hydroxypmgesterone in bovine adrenocortical microsomes The C-21 hydroxylation of 17a-hydroxyprogesterone in adrenocortical microsomes was determined by measuring the rate of formation of 1 1-deoxycortisol employing the blue tetrazolium method of Elliott eta!. (14). 7-Globulin mg/mg microsomal protein 1 (immune) l(preimmune) I Deoxycortisol nmol/mg mmcrosomoi protein/mm % Control P-45 reductase activity was not measured in these experiments but has previously been shown to be inhibited in bovine adrenocortical microsomes by antibody to the porcine liver reduc These data show that adrenocortical mitochondria contain an iron-sulfur protein, adrenodoxin, which is required for the reduction of cytochrome c and cytochrome P-45, and for the I I $-hydroxylation of steroids. There is no spectral evidence for the existence of an ironsulfur protein in liver microsomes (15, 16) and, indeed, anti-adrenodoxin 7-globulin has no effect on any of the electron transport activities catalyzed by either liver or adrenal cortex microsomes. In addition, the NADPH-cytochrome c reductase activities from both liver and adrenocortical microsomes are immuno.. chemically similar, so that antibody to the liver reductase inhibits all activities catalyzed by the enzyme in adrenal cortex microsomes. It has thus been shown that NADPH-cytochrome C reductase catalyzes the reduction of cytochrome P.45 in the microsomal fractions of both liver and adrenal cortex (3) and is required tase (3). in the overall electron transport sequence for

5 NADPH-CYTOCHROME c REDCTASE IN VARIOS TISSES 125 D TPP4HCytochrome #{231} Reductase I7,2l-Dihydroxyprogestgrone Deoxycorticosterone 8 C I 5 6 C 4 O\\ i-globulin 1mg microsomes FIG. 5. The inhibition of 21-hydroxylation in bovine adrenocortical microsomes by antibody to NA DPH-cytochrome c reductase. Assays were performed at 29#{176}Cin a water bath shaker. Incubation mixtures consisted of an NADPHgenerating system containing NADP, isocitrate, isocitrate dehydrogenase, and microsomes (.9 mg of protein per ml) in.5 M Tris-.3 M MgCl2 buffer, ph 7.4, in a total volume of 1 ml. The microsomes had been preincubated with the various ratios of 7-globulin indicated in the figure. The substrates, either tritiated progesterone or tritiated I 7-hydroxyprogesterone, were labeled in the 7a-position and purified chromatographically before use. The final concentrations were 76 sm progesterone and 56 sm 17-hydroxyprogesterone. Samples were removed at, 15, and 3 mm, added immediately to methylene chloride, extracted twice, and developed on treated silica gel thin layer chromatography plates in a benzene-acetone (3: 1) system. Spots were compared with known markers and eluted and counted in a liquid scintillation spectrometer. the hydroxylation of drugs in liver microsomes and steroids in adrenal cortical microsomes. To complement studies on the spleen microsomal electron transport system which has been implicated in the catabolism of heme by Tenhunen et a!. (17, 18), Schacter et a!. (19) extended this experimental approach. The relationship between microsomal heme oxygenase and the NADPH-cytochrome P-45 electron transport sequence is schematically depicted in fig. I of Dr. Schmid s paper in this Symposium.4 It can be seen that if the mixed function oxidation of 4 R. Schmid. this Symposium. p mg y-globulin/mg microsomol protein FIG. 6. Concomitant inhibition of rat spleen microsomal NA DPH-cytochrome c reductase and heme oxygenase by antibody to the reductase. Heme oxygenase activity was assayed as previously described (19). Control activities were:.42 nmol of bilirubin x min x mg for heme oxygenase and 18.6 nmol x min x mg for NADPH-cytochrome c reductase in microsomes from phenobarbitaltreated rats. NADPH-cytochrome c reductase; O-O, heme oxygenase , activity in the presence of preimmune 7-globulin. heme is dependent upon reducing equivalents from the reductase to cytochrome P-45, then the inhibition of this flavoprotein by antibody would result in inhibition of heme oxygenase activity. This inhibition is shown in Fig. 6, in which NADPH-cytochrome c reductase and heme oxygenase activities in spleen microsomes are titrated with anti-reductase 7-globulin which results in concomitant inhibition of both activities as the titer is increased. In addition, microsomes were prepared from the livers of untreated rats and rats pretreated with methemalbumin, which induces heme oxygenase activity. These data (Fig. 7) show that even under conditions in which the heme oxygenase activity is increased 2-fold, there is an absolute dependence upon reducing equivalents from NADPH-cytochrome c reductase. These data, however, do not bear upon the introduction of the second and third atoms of oxygen into the biliverdin molecule, but the oxidation of the a-methene bridge to carbon monoxide has now been shown to be a mixed function oxidation requiring NADPHcytochrome c reductase and cytochrome P-45. In collaboration with Dr. Sten Orrenius of the

6 126 MASTERS ET AL. Heme Oxygersase NADPH-Cytochrome c Reductase 8.7 ZZ > 4 -J l2 l8 24 mg y -globulin /mg microsomol protein FIG. 7. Concomitant inhibition of liver microsomal z ITPNH TPNH NA DPH-cytochrome c reductase and heme oxygenase activities from untreated rats and rats pretreated with methemalbumin by antibody to the reductase. Methemalbumin treatment consisted of four injections of methemalbumin ip (2 Mmol/ 1 g) given at 12-hr intervals. ntreated rats received equal volumes of a 2% (w/v) solution of human serum albumin ip. Heme oxygenase activity was assayed as previously described (19). Control activities were.27 and.41 nmol of bilirubin produced per mg of protein per mm for heme oxygenase in untreated and methemalbumintreated rats, respectively, and 15 and 11 nmol per mg of microsomal protein per mm for NADPH-cytochrome c reductase in untreated and methemalbumm-treated rats, respectively. Left, titration of microsomal heme oxygenase from untreated (O-O) and methemalbumin-treated (#{149}-#{149}) rats with antireductase 7-globulin. Right, titration curve of microsomal NADPH-cytochrome c reductase from untreated (-) and methemalbumin-treated (A-A) rats. In both parts ofthe figure - _ - indicates activity in presence of preimmune 7-globulin. Karolinska Institute in Stockholm, Sweden, we have begun a series of experiments on the role of NADPH-cytochrome c reductase in the cytochrome P-45-dependent,- and -l-hydroxylation of lauric acid by kidney cortex microsomes. Since the data of Ellin et a!. (2) showed that NADH can support the hydroxylation of laurate to at least 5% of the rate with NADPH, it was of interest to determine the role of NADPH-cytochrome c reductase in the reduction of cytochrome P-45 by both pyridine nucleotides. Our studies show that anti-reductase 7-globulin inhibits cytochrome P-45 reduction in kidney microsomes approximately 7-8% in various experiments when NADPH is the electron donor (Fig. 8). With NADH as electron donor, however, there is little or no inhibition of cytochrome c or cytochrome Cytochrome P-45 ReductIon - Cytochrome #{163} Reduction 2 3 P-45 reduction by the antibody. In these cxmg y-globlin / mg MICROSOMES FIG. 8. The titration of kidney microsomal electron transport activities with anti-reductase 7-globulin. Rat kidney microsomes were preincubated with varying amounts of anti-reductase 7-globulin or preimmune 7-globulin and aliquots were removed for assays of NADPH-cytochrome c and NADPH-cytochrome P-45 reductase activities, performed as previously described (3). Control activities were: NADH-cytochrome c reductase, nmol x mint x mg. NADPH-cytochrome c reductase, 43.8 nmol x min x mg : NADPH-cytochrome P-45 reductase, 7.6 nmol x min x mg : and NADH-cytochrome P-45, 1.2 nmol x min x mg periments, some inhibition of both NADPHand NADH- supported activities was obtained with nonimmune y-globulin, so that the slight inhibition obtained with immune 7-globulin was considered negligible. These data clearly show, nevertheless, that NADH is not reducing cytochrome P-45 through the NADPH-cytochrome c reductase flavoprotein. The data of Ellin el a!. (2) would further suggest that separate systems are involved, since the t-hydroxylation and cytochrome P45 reductase activities from the two pyridine nucleotides are additive. In summary, studies with antibody to porcine liver microsomal NADPH-cytochrome c reductase have shown: I) that this enzymic activity is responsible for cytochrome P-45 reduction in the microsomes of liver, adrenal cortex, spleen, and kidney; and 2) that the microsomal electron transport system of which this flavopro-

7 DISCSSION 127 tein is an integral part is responsible for the specific mixed function oxidation reactions catalyzed by these various tissues. References I. T. Omura, in Microsomes and Drug Oxidations (J. R. Gillette, A. H. Conney, G. J. Cosmides, R. W. Estabrook, J. R. Fouts, and G. J. Mannering, eds.) pp Academic Press, New York, B. S. S. Masters, E. B. Nelson, D. M. Ziegler, J. Baron, P. P. Raj, and E. L. lsaacson, Chem.- Biol. Interactions 3, 296 (1971). 3. B. S. S. Masters, J. Baron, W. E. Taylor, E. L. Isaacson, and J. J. LoSpalluto. J. Biol. Chem. 246, 4143 (1971). 4. R. I. Glazer, J. B. Schenkman, and A. C. Sartorelli, Mo!. Pharmacol. 7, 683 (1971). 5. B. S. S. Masters and D. M. Ziegler. Arch. Biochem. Biophys. 145, 358 (1971). 6. M. L. Sweat, J. Amer. Chem. Soc. 73, 456 (1951). 7. J. E. Plager and L. T. Samuels. J. Biol. C/tern. 211,21 (l954). 8. D. Y. Cooper, R. W. Estabrook, and. Rosenthal, J. Biol. Chem. 238, 132 (1963). 9. T. Omura, R. Sato, D. Y. Cooper,. Rosenthal, and R. W. Estabrook. Fed. Proc. 24, 1181 (1965). 1. T. Omura, E. Sanders, D. Y. Cooper,. Rosenthal, and R. W. Estabrook, in Non-Heme Iron Proteins (A. San Pietro, ed), p. 41. Charles F. Kettering Laboratory, Yellow Springs, Ohio, I I. T. Omura. E. Sanders, R. W. Estabrook, D. Y. Cooper, and. Rosenthal, Arch. Biochem. Biophys. 117, 66 (l966). 12. J. Baron, W. E. Taylor, and B. S. S. Masters, Arch. Biochem. Biophys. 15, 15 (1972). 13. C. Mattingly, J. Clin. Pathol. (London) 15, 374 (1962). 14. F. H. Elliott, M. K. Birmingham. A. V. Schally, and E. Schonbaum, Endocrinology 55, 721 (1954). IS. Y. Miyake, H. S. Mason, and W. Landgraf, J. Biol. Chem. 242, 393 (1967). 16. Y. Ichikawa and T. Yamano, Biochim. Biophys. Acta 2, (197). 17. R. Tenhunen, H. S. Marver, and R. Schmid, J. Biol. C/tern. 244, 6388 (1969). 18. R. Tenhunen, H. S. Marver, N. R. Pimstone, W. F. Trager. D. Y. Cooper, and R. Schmid, Biochemistry 11, 1716(1972). 19. B. A. Schacter, Edward B. Nelson, H. S. Marver, and B. S. S. Masters, J. Biol. Chern. 247, 361 ( l972). 2. A. Ellin, S. V. Jakobsson, J. B. Schenkman, and S. Orrenius, Arch. Biochem. Biophys. 15, 64 (1972). Discussion Dr. Coon,. Michigan: How does rate of heme oxygenation compare with that of drug hydroxylation? Dr. Masters: It occurs at a very low rate. The rates of spleen microsomal heme oxygenase activity that we observed in untreated animals, that is, animals which were not pretreated with methemalbumin, were about.23 nmol/min/mg of protein. With methemalbumin treatment, the activity increased approximately 4-fold to about.95 nmol/min/mg. Other workers have obtamed similar values with various pretreatments, such as red cell antibodies, zymosan, etc., but these never exceed.5-1. nmol/min/mg in spleen. Dr. Coon: This rate is approximately /o- /o of the rate obtained in liver microsomal reactions utilizing optimal substrates. Dr. Masters: That is correct. In addition, the NADPH-cytochrome c reductase activity as determined in spleen microsomes from untreated rats is 4-7% of that obtained with rat liver microsomal preparations: but the rate of electron flux in the spleen microsomal system is more than sufficient to account for the heme oxygenase measured as the rate of bilirubin formation in the presence of NADPH. In other words, it is not rate limiting, if we assume that cytochrome P-45 reduction proceeds in the same manner as in liver, kidney, and adrenal cortex. Dr. Conney, Hoffrnann-La Roche: Increasing the concentration of NADPH-cytochrome c reductase antibody decreases hydroxylase activity progressively as the concentration of antibody is increased. However, after 5-75% of the hydroxylase activity is inhibited, the antibody loses its effectiveness to inhibit the hydroxylation reaction. Why is a portion of the hydroxylase activity resistant to inhibition by NADPHcytochrome c reductase antibody? Do these results suggest the presence of two different reductases? Dr. Masters: There are two possible explanations for this, Dr. Conney: I) When we do a titration with purified enzyme, of course, we get essentially l% inhibition (it is inhibited to about 98%, but there may be a 2% residual activity left). In liver microsomes we have obtained 89% inhibition. One must remember that we are extending the use of an antibody prepared against porcine liver reductase to other tissues and other species, thus getting further away from identity as far as antigenic determinants are concerned, so we have to invoke that as a possible explanation for the failure to achieve complete inhibition. 2) The second possibility that occurs to me is that some antibodies which are not inhibitory may be present in the preparation and these may bind to the reductase, preventing it from being inhibited by inhibitory anti-

8 128 DISCSSION bodies. This would be a blocking antibody-type phenomenon. Dr. Coon: I have a general question. There is a good deal of uncertainty about the involvement of cytochrome b5 as an essential or even as an alternate electron donor in hydroxylation reactions. What is your information on this from the immunochemical point of view, or, perhaps, from other laboratories which have made similar studies? It is my recollection that the cytochome b5 antibody does not show an inhibitory effect on hydroxylation. Dr. Masters: This is correct. The studies by Omura certainly indicated that there was no effect of anticytochrome b5 serum on liver microsomal hydroxylation reactions. I believe there will be a report later in this meeting, if my information is correct, about an antibody prepared to cytochrome ba in Gillette s laboratory. We have not been very successful in making good inhibitory preparation to cytochrome b5. Only recently, we have succeeded in obtaining inhibitory preparations of anti-cytochrome b5 serum from rabbits by using cytochrome b5 antigen coupled to human serum protein to increase its immunogenicity. Dr. Maines,. Minnesota: Did you measure the heme oxygenase activity of the spleen? If so, did you measure the cytochrome P45 content of this organ? Dr Masters: Yes, spleen heme oxygenase activity has been measured as I mentioned earlier in reply to Dr. Coon s question. With regard to cytochrome P-45 levels, I wonder if Dr. Schacter would like to comment on these data. Dr. Schacter,. Manitoba: We have been able to determine that small amounts of cytochrome P-45 are present in rat spleen microsomes by measuring the difference spectrum obtained when the contents in the sample cuvet are treated with carbon monoxide and sodium dithionite while the contents in the reference cuvet are treated only with carbon monoxide. This minimizes the problem of spectral distortion by carboxyhemoglobin. The levels of cytochrome P-45 measured are very low, around.2-.3 nmol/mg of microsomal protein. Of interest is the fact that rat spleen cytochrome P-45 is not induced by phenobarbital nor by 3-methylcholanthrene pretreatment of these animals in doses which will induce cytochrome P-45 in rat liver. On the other hand, we have found that benzopyrene and pregnenolone-16 cx-carbonitrile will induce spleen cytochrome P-45. The effect has been more consistent with PCN and levels of cytochrome P-45 as high as.16 nmol/mg of microsomal protein have been obtained which is about 7- or 8-fold above our control range.