Phosphorylation of cytochrome P450: Regulation by cytochrome b5

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 271, No. 2, June, pp. 424-432,1989

Phosphorylation of Cytochrome P450: Regulation by Cytochrome bg

PAUL M. EPSTEIN,* MARIO CURTI,* INGELA JANSSON,* CHI-KUANG HUANG,j- AND JOHN B. SCHENKMAN*s’

Lkprtments of *Pharmacology and fPathoLogy, University of Connecticut

Health Center, Farmington, Connecticut 06032

Received October 17.1988, and in revised form February $1989

Rabbit liver cytochrome P450 LM2 and several forms of rat liver cytochrome P450 are phosphorylated by CAMP-dependent protein kinase (PKA) and by protein kinase C. Under aqueous assay conditions at neutral pH LM2 is phosphorylated only to a maxi- mum extent of about 20 mol% by PKA. We show that detergents or alkaline pH greatly enhance the extent of phosphorylation of the cytochrome P450 substrates of CAMP-dependent protein kinase. In the presence of 0.05% Emulgen, PBRLM5, which appears to be the best cytochrome P450 substrate for CAMP-dependent protein kinase, incorporates phosphate up to about 84 mol% of enzyme. We reported previously (I. Jansson et al. (1987) Arch. Biochem. Biophys. 259,441-448) that cytochrome bS inhibits the phosphorylation of LM2 by CAMP-dependent protein kinase. In this paper, using PBRLM5, we demonstrate, by analysis of initial rates, that the inhibition of phosphorylation by cytochrome b5 is competitive, with a K, = 0.48 PM. We also show that a number of forms of cytochrome P450 can be phosphorylated by protein kinase C, and that the phosphorylation of these forms by protein kinase C is also inhibited by cytochrome bS. These data suggest that the phosphorylation site(s) of cytochromes P450 may be located within or overlap the cytochrome bS binding domain of the enzymes. cc> 1989 Academic Press. Inc.

Protein phosphorylation has come to be recognized as the most common type of post-translational reversible protein mod- ification in eukaryotic organisms (1). Many different types of protein kinases exist, with most of these capable of phosphory- lating many different protein substrates; additionally, many protein substrates can be phosphorylated by several different protein kinases, often with the same amino acid being phosphorylated (1, 2). This al- lows for a great deal of complexity in the regulation of enzyme activity and protein- protein interactions by protein phosphorylation (1). It is also recognized that an im- portant means by which hormones control enzyme activities and cellular proccsscs is through phosphorylation mechanisms, which are brought into play in response to

’ To whom correspondence should be addressed.

the generation of second messengers (3,4). Much attention has therefore focused on the regulation of protein phosphorylation by second messenger-dependent protein kinases.

A number of earlier reports have appeared which indicate that rabbit cytochrome P450 LM2 (5-7) and several different rat cytochromes P450 (7, 8) can be phosphorylated iw vitro by CAMP-dependent protein kinase (PKA).” The site of

‘Abbreviations used: PKA, cyclic AMP-dependent protein kinase; PKC, Ca”+-regulated, phospholipid activated protein kinase; RLMG, rat liver microsomal P450, band 6 (anode to cathode) in the 45- to 60-kDa region of SDS-PAGE gels; SDS-PAGE, sodium dodecyl sulfate-polyacrylamidc gel elcctrophoresis; fRLM4, female-specific P450, band 4; PBRLM5, phenobarbital-induced P450, band 5; LM2, phenobarbital-induced rabbit liver microsomal P450, band 2; P450 CAM, P460 isolated from Pseudomonas putida

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424

PHOSPHORYLATION OF CYTOCHROME P450 425

phosphorylation of LM2 has been reported to be serine at position 128 (6). The functional significance of this phosphorylation is, at present, not known. LM2 and several other forms of cytochrome P450 have been shown to undergo complex formation with cytochrome b5, an action resulting in changes in both the spin state of the cytochrome P450 and in the affinity of the cytochrome P450 for substrate (9,10). One such complex has been trapped by covalent binding and shown to exhibit enhanced substrate turnover in the reconstituted monooxygenase system (11); such stimulation is seen by cytochrome bs addition to free cytochrome P450 (10). Since phosphorylation of LM2 has been shown to be inhibitory of monooxygenase activity (‘7, 12) and of complex formation between cytochromes P450 and bs (7), and since the effects of cytochrome bs are stimulatory (g-11), the possibility exists that the two phenomena are opposite controls of monooxygenase activity. In this paper we show that cytochrome b, competitively inhibits the phosphorylation of cytochrome P450 by PKA.3 This leads us to hypothesize that the phosphorylation site on cytochrome P450 is in the binding domain for cytochrome bg, and that phosphorylation may act to regulate cytochrome P450 interaction with cytochrome bg.

EXPERIMENTAL PROCEDURES

Puri$cation. of microsomal enzymes. The purifica- tions of individual forms of rat liver microsomal cytochrome P450 were as reported earlier: RLM2 (13).

RLM3 and RLM5 (14), RLM5a and fRLM4 (15), RLM5b and RLM6 (16), and PBRLM5 (17) and rabbit LM2 (10). Cytochrome b:, was purified as described (9).

Rat liver cytochrome bS and rabbit cytochrome b, were purified for use with the individual rat P450 and LM2, respectively.

grown on camphor; Hepes, 4-(2-hydroxyethyl)-l-pi- perazineethanesulfonic acid.

’ A preliminary account of this investigation was previously presented as an abstract [I. Jansson, P. Ep-

stein, M. Curti, and J. B. Schenkman (1988) in Bio- chemistry and Biophysics of Cytochrome P450

(Schuster, I., Ed.), Taylor & Francis, London] for the 6th International Conference on Biochemistry and Biophysics of Cytochrome P450, Vienna, July, 1988.

Phosphorylation of proteins. The method of phos-

phorylation of cytochrome P450 by PKA was modified from that of Pyerin et al. (5) as described previously

(7). Except where otherwise noted, reaction mixtures (0.2 ml) contained 50 mM Hepes buffer, pH 7.4,10 mM

MgC&, 12 pM ATP containing 1 &i [T-~*P]ATP (New

England Nuclear), 4 pg protein (27 U/pg) of the catalytic subunit of PKA (Sigma), and 25-100 pmol of cy- toehrome P450 substrate, as indicated. For phosphor-

ylation by PKC, reaction mixtures (0.2 ml) contained 10 mM Hepes buffer, pH 7.4, 1 mM Mg&, 12 +M ATP containing 1 &i [y-a2P]ATP, 0.1 mg/ml phosphatidyl-

serine (Sigma, from bovine brain), 0.5 mM CaCI,, amounts of cytochrome P450 substrates as indicated, and 1 pg of PKC. PKC was purified from calf brain by

DEAE-cellulose and phenyl-Sepharose chromatogra- phy (18) followed by protamine-agarose chromatog-

raphy (19). For both PKA and PKC, reaction mixtures were incubated at 37°C for various times and stopped by the addition of 1 ml of cold 10% trichloro-

acetic acid-3% sodium pyrophosphate; samples were left on ice for 10 min and precipitates collected on Whatman glass microfiber filters and processed as de-

scribed previously (7). Assays. Spectra were recorded on a Shimadzu UV-

3000 spectrophotometer for quantification of hemoproteins as described earlier (10,14). All radioactive quantitation was as reported previously (7).

RESULTS

Conditions for Phosphorylation

In our earlier report (7) we noted that the extent of phosphorylation of LM2 did not exceed 21% with the catalytic subunit of CAMP-dependent protein kinase (PKA). In the current study attempts were made to optimize conditions of phosphorylation. To obviate interference due to aggregation of cytochrome P450 during phosphorylation, Emulgen 911, a nonionic detergent, was added to the reaction mixture. Using a level of Emulgen 911 of 0.05% it was pos- sible to increase the extent of phosphorylation (Fig. 1A) from 20 to 37%. Raising the concentration of Emulgen 911 above 0.1% resulted in a subsequent decline in phosphorylation. Triton X-100, another nonionic detergent, was almost as effective as Emulgen 911 in enhancing the extent of LM2 phosphorylation (Fig. lB), with maxi- mal effects seen at 0.01% Triton X-100. Concentrations of Triton X-100 above 0.1% were less effective.

Since protein structure may be influ- enced by pH, we examined the effect of pH

426 EPSTEIN ET AL.

0 0 01 01 I 0 0 01 0 I I I% oatargsnt I% Detergent

FIG. 1. Influence of the nonionic detergents, Emulgen 911 (A) and Triton X-100 (B) on the extent

of phosphorylation of cytochrome P450 LMZ. Reaction mixtures (0.2 ml) contained 50 HIM Hepes, pH 7.4,10 mM MgC&, 12 pM ATP containing 1 nCi [y-32P]ATP, 4 pg (protein) of the catalytic subunit of PKA, 100 pmol (500 nM) LMZ, and detergents as indicated. Incubations were for 20 min at 37°C.

Results represent the means + the range of duplicate determinations,

on the extent of phosphorylation of LM2 (Fig. 2). As the pH was raised, from 6.3 to 8 in the presence of detergent, the extent of phosphorylation more than doubled. Be- tween pH 8 and pH 9 no apparent further increase was seen, but above pH 9 the extent of phosphorylation again increased, to about 64%. The extent of LM2 phosphorylation was independent of the hemoprotein concentration (Fig. 3) at both pH 7.4 and pH 10.0, with a greater degree of phosphorylation occurring at pH 10 at each substrate level. Thus, the difference seen is not due to differences in affinity for LM2 at the different pH values.

In our prior paper (7) we examined the extent of phosphorylation of a number of forms of rat liver microsomal cytochromes P450 at pH 7.4 in the absence of detergents. Of these, only the major phenobarbital-in- ducible rat form, PBRLM5 (17), was phosphorylated to any appreciable extent, 12%) compared with a 20% phosphorylation of LM2; the partial phosphorylation of this latter enzyme in vitro was shown not to be due to prior phosphorylation in vivo (7). Using conditions optimal for phosphorylation but minimally deleterious for the cytochrome P450, i.e., pH 8 in medium containing 0.05% Emulgen 911, the extents of

FIG. 2. Effect of pH on the extent of phosphoryla-

tion of LM2 by PKA. Conditions were as in Fig. 1 except that the reaction mixtures contained 50 pmol (250 nM) LM2,0.05% Emulgen 911, and 50 mM potas- sium phosphate (A) or 50 mM Tris-HCl (0) buffer. In- cubation was for 20 min at 37°C. Results represent the means of duplicate determinations.

01 , I , , 125 250 375 500

J

LM 2 l”M)

FIG. 3. Lack of effect of LM2 concentration on the extent of phosphorylation of LM2 by PKA. Conditions

were as in Fig. 2 using 50 mM Hepes buffer, pH 7.4 (o), or 50 mM Tris-HC1 buffer, pH 10 (0). Results represent the means k the range of duplicate determinations.


FIG. 4. Comparison of the extent of phosphorylation of different cytochromes P450 by PKA. Condi- tions were as in Fig. 1 except that reaction mixtures

contained 50 mM Tris-HCl, pH 8.0, 0.05Y0 Emulgen 911,25 pmol(125 nM) of each form of P450, and incubation was for 45 min at 37°C.

phosphorylation of a number of forms of rat cytochrome P450 were compared with that of LM2 (Fig. 4). Under these conditions, LM2 phosphorylation reached 38% while that of PBRLM5 was 84%. Other forms of rat cytochrome P450 that were appreciably phosphorylated include RLM5 (30%) and RLM6 (24%). Only small amounts of phosphorylation of RLM2 (1.3% ) or fRLM4 (0.7% ) were obtained.

Kinetics of Phosphorylntim

The kinetics of phosphorylation of PB- RLM5 and the influence of cytochrome b5 on these kinetics were examined (Fig. 5). Decreasing the amount of PKA in the assay (Figs. 5A-5C) resulted in decreases in the rates of phosphorylation. Cytochrome bs was found earlier (7) neither to undergo phosphorylation nor to inhibit PKA phosphorylation of histone; it inhibited LM2 phosphorylation by PKA (7) and also inhibited the phosphorylation of PBRLM5 (Figs. 5A-5C). The extent of this inhibition was greater at lower PKA levels. A plot of the ratio of inhibited to noninhibited activity as a function of PKA concentration indicated that the inhibition could be over- come by increasing the PKA concentration (Fig. 5D). A Dixon plot of the activity at two different PKA concentrations as a

function of cytochrome b5 concentration showed the inhibition to be competitive, with a K% of 0.48 @M cytochrome b5 (Fig. 6). This is very close to the KD of cytochrome b, for P450 (0.54 PM for LM2) determined

I 2 3 4 Time (mln)

0.5 IO Protein Kinase (ug)

FIG. 5. PKA dependence of the rate of PBRLM5

phosphorylation and influence of PKA concentration on inhibition of phosphorylation by cytochrome hr,. PKA phosphorylation assays were run at 3’7°C from 1 to 4 min in 50 1nM Hepes, pH 7.4, in the absence of

detergent with 50 pmol of PBRLM5 as substrate. The PBRLM5 was preincubated for 30 min at room temperature in the absence (@) or presence (A) of 400

pmol(2 pM) of rat cytochrome bs, prior to initiation of the phosphorylation reactions. Panels A-C represent different amounts of PKA added to the reaction as

follows: (A) 1 /~g of PKA; (B) 0.5 rg of PKA; (C) 0.25 pg of PKA. (D) Initial rates were determined for each concentration of PKA (A-C) in the presence and absence of cytochrome b5 and plotted in D with the ordi-

nate as percentage activity in the presence vs the absence of cytochrome br,.

428 EPSTEIN ET AL.

Cytochrome b5 (phi)

FIG. 6. Kinetics of cytochrome b, inhibition of PKA-catalyzed phosphorylation of PBRLM5: Dixon plot of reciprocal initial rates versus cytochrome b5 concentration. Conditions were as in Fig. 5. Initial rates (V) were calculated from the slopes of reactions run from 0 to 4 min at 37°C using 0.25 pg PKA, various amounts of cytochrome & as indicated, and PBRLM5 as substrate at 50 pmol(0.25 pM) (e) or 100 pmol (0.5 FM) (A). Since the lines on the Dixon plot intersect above, and not on, the abscissa, it rules out noncompetitive inhibition. This type of plot does not in itself distinguish between competitive and linear mixed-type inhibition. However, when the slope of the lines in the Dixon plot are plotted vs l/[P450], a straight line is obtained which transects the origin (inset); this clearly rules out linear mixed-type inhibition and indicates that inhibition by cytochrome bs is competitive (20).

by spectral analysis (9). As seen by the inset (Fig. 6), the inhibition is not of the mixed type as slopes extrapolate through zero (20).

Phosphorylation by PKC

A number of different regulatory protein kinases exist (2). One of these, a Ca2+- regulated, phospholipid activated enzyme, PKC, has been reported (8) to phosphorylate several forms of rat cytochrome P450. In agreement with that report, PKC has been found to phosphorylate a number of different forms of rat P450, plus rabbit LM2 (Fig. 7). While the extents of phosphorylation of the different enzymes var- ied, the levels of 32P incorporated in all in- stances, except for fRLM4, were considerably diminished relative to phosphorylation by PKA; with fRLM4 the level increased from 0.7 to 4.3%. The relative extents of phosphorylation of the different forms by PKC also differed from those by PKA.

With PKC the greatest degree of phosphorylation was seen using RLM5 as substrate, followed by RLM6 and PBRLM5; only little phosphorylation was obtained with LM2 (Fig. 7). In contrast, with PKA

FIG. 7. Comparison of the extent of phosphorylation of different cytochromes P450 by PKC. Assay conditions were as described under Experimental Procedures using 50 pmol (0.25 PM) of each P450 as substrate. Incubation was for 60 min at 37°C.


FIG. 8. Inhibition of PKC-catalyzed phosphoryla-

tion of RLM6 by cytochrome b5. Assay conditions were as described under Experimental Procedures using 50 pmol(0.25 HIM) of RLM6 and various amounts

of cytochrome b:, as indicated. RLMS was preincu- hated with and without cytochrome b, for 30 min at room temperature prior to the addition of the assay

reagents necessary for PKC phosphorylation. Phos- phorylation reactions were then carried out for 30 min at 37°C.

the highest activity was observed with PB- RLM5 as the substrate, RLM5 and LM2 had similar extents of phosphorylation but less than PBRLM5.

Injke?sce oj’Cytochrome bj on PKC rrctivity

As with PKA, cytochrome b5 was inhibitory of the phosphorylation of P450 by PKC (Fig. 8). The extent of RLM6 phosphorylation, after preincubation of the hemoprotein with varying amounts of cytochrome bsr declined with increasing cytochrome b5 concentration. At a 1:l ratio of cytochrome b5 to P450 inhibition reached 30%) while at a 4:l ratio inhibition of phosphorylation was 65% (Fig. 8). Similar results were obtained with RLM5 (data not shown).

DISCUSSION

Cytochrome b, is a component of a number of different electron transfer pathways (21). In the reconstituted P450 monoxygen- ase system it has been shown to either stimulate or have no influence on the turnover of substrates (10,22-24). Two reports have also appeared (23,24) which indicated that with rabbit cytochrome P450, cyto-

chrome bS was inhibitory of turnover of some substrates. The inhibition in at least one of these studies (23) was subsequently attributed to the order of addition of the hemoproteins to the assay (25).

The stimulation of substrate turnover that occurs when cytochrome b5 is added to cytochrome P450 in the reconstituted system is the result of formation of a binary complex, a heterodimer held together by complementary charge pairing plus hydro- phobic interactions of the hemoproteins (9). The stimulation was suggested (26) to be due to the ability of the complex to ac- cept two electrons from NADPH-cytochrome P450 reductase, thereby shorten- ing the lag that might occur for reductase to rebind to the cytochrome P450 molecule for input of the second electron. Using a covalently attached functional cytochrome P450-b, heterodimer (11) it was shown that the affinity of the complex for reductase was the same as that of the uncom- plexed cytochrome P450 (27). If interaction between cytochrome b5 and cytochrome P450 is prevented, as by phosphorylation, the expected effect would be that of a loss of the stimulatory activity. In fact, such a loss was observed (7), but m addition phosphorylation of LM2 also resulted in a decline in the activity even in the absence of cytochrome b5 (7,12).

At least a dozen different constitutive forms of cytochrome P450 are found in liver microsomes. A number of potential regulatory mechanisms may exist for these forms. For example, since the concentration of NADPH-cytochrome P450 reductase is about 10% the molar concentration of total cytochrome P450, changes in affinity for binding to one form or another could serve as a control. Similarly, an increase in the affinity of cytochrome P450 for cytochrome b5 when substrate binds (9) could preferentially serve to stimulate the turnover of substrate by a specific form of cytochrome P450. Stimula- tion of substrate turnover by specific forms of cytochrome P450 by cytochrome b5 appears to depend both upon substrate and form of cytochrome P450 (10, 22). These would be examples of positive controlling influences. Recent observations that cyto-

430 EPSTEIN ET AL

chromes P450 are phosphorylatable (6-8, 28) suggest that phosphorylation, too, may serve as a control, although a negative one (7,121.

In the present study, cytochrome b5 is seen to competitively inhibit the rate of cytochrome P450 phosphorylation. In our prior study (7) we also observed phosphorylation of cytochrome P450 to diminish its interaction with cytochrome b5. In this manner, phosphorylation may serve as a mechanism to prevent enhancement of the monooxygenase activity, The site of PKA phosphorylation of LM2 has been reported to be residue 128, a serine molecule (6). This residue is contained in the PKA substrate recognition sequence, Arg-Arg- Phe-Ser-Leu (29), and is also found in PB- RLM5 (30) with serine as residue 128, and in RLM6 (31), where the serine is residue 129, and in a similar sequence, Arg-Arg- Phe-Ser-Ile, in RLM5 (32), with serine as residue 127. From the observation that cytochrome b5 inhibits the phosphorylation of LM2 (7), PBRLM5, RLM5, and RLMG, it is suggested that the kinase substrate recognition sequences lie within or overlap the site to which cytochrome b5 binds. Studies are currently underway to identify the cytochrome b5 binding site using the covalently complexed cytochrome b5-cytochrome P450 LM2 heterodimer (11).

While the substrate recognition sequence of PKC is still being elucidated (33), it exhibits a tendency to phosphorylate the same proteins as PKA, although with different relative activities. Since cytochrome b5 also blocks PKC phosphorylation of RLM5 and RLMG, it is tempting to speculate that the phosphorylation site is the same residue which is phosphorylated by PKA. It should be noted, however, that PKC is considerably less effective than PKA in phosphorylation of all of the P450 forms tested except for fRLM4, a female- specific steroid 15-hydroxylase, and even with this form activity was low.

Not all forms of cytochrome P450 con- tain the PKA substrate recognition sequence. From the lack of phosphorylation of RLM2 and RLM3 in this study, it was suspected that they lack the PKA and PKC substrate recognition sequences. In a

manuscript received from Dr. Frank Gon- zalez showing the deduced amino acid sequence of RLMB, the PKA substrate recognition sequence is shown to be present in this enzyme, but with threonine instead of serine at position 130. A recent study (34) indicates that fRLM4 lacks the PKA substrate recognition site. It is also lacking in microsomal P45Oc (35), P450d (36), and P45Op (37), as well as in the mitochondrial P450scc (38). However, P450scc does undergo considerable phosphorylation by PKC (39). Further, considerable homology with P450 CAM exists around residue 291 of P45Oscc. The prokaryotic form, P450 CAM also contains the PKA substrate recognition sequence Arg-Arg-Phe-Ser-Leu, with serine as residue 293 (40). Recent studies by Sligar and associates (41) indicate that P450 CAM, like mammalian P450 (lo), binds cytochrome b5 and undergoes a low to high spin shift in the spin equilib- rium. It will be of interest to determine whether P450 CAM also is phosphorylatable by PKA. However, from the crystal structure of the P450 CAM (42), it would appear the PKA substrate recognition sequence is located at the end of a-helix K, with the serine extending into the substrate binding pocket. Given this location, accessibility to PKA might not be expected.

Although the current study seeks to re- late phosphorylation of cytochrome P450 to modulation of cytochrome b5 stimulation of monooxygenase activity, other pos- sible roles for phosphorylation may also exist. For example, Taniguchi and CO-

workers (43) have reported that phosphorylation of phenobarbital-induced rat liver microsomes resulted in a considerable con- version of cytochrome P450 to P420. More recent studies from Ingelman-Sundberg’s laboratory (44) demonstrated an enhanced rate of degradation of RLM6 (alcohol induced) by stimulation of PKA in primary hepatocyte cultures. In contrast during 3- methylcholanthrene induction of some P450 forms PKA levels rose in liver (45). Further, phenobarbital inhibited brain PKC (46), and phenobarbital induction of P450 was inhibited by TPA, a PKC activa- tor (47). From the number of different


reported effects of phosphorylation of cy- 18. WALTON, G. M., BERTICS, P. J., HULISON, L. G.,

tochrome P450 it is suspected that phos- VEDVICK, T. S., AND GILL, G. N. (1987) Anal.

phorylation may influence the P450 mono- Biochem. 161,425-437.

oxygenase system in a complex manner. 19. WOOTEN, M. W., VANDENPLAS, M., AND NEL, A. E.

Studies such as those reported herein (1987) Eur. J Biochem. 164,461-46’7.

should aid in elucidating the mechanism of 20. SEGAL, I. H., Ed. (1976) Biochemical Calculations,

these controls. 2nd ed., Wiley, New York.

21. SCHENKMAN, J. B., JANSSON, I., AND ROBIE-SIJH,

ACKNOWLEDGMENTS

This work was supported in part by Grants

GM26114, DE08034, and AI 20943 from the United

States Public Health Service. Chi-Kuang Huang is an

Established Investigator of the American Heart As-

sociation.

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Phosphorylation of cytochrome P450: Regulation by cytochrome b5