THE JOURNAL OF BIOLOGICAL CHEMISTRY
`(0 1986 by The American Society of Biological Chemists, Inc.
`
`Vol ,261, NO. 2, Issue of January 15, pp. 909-921,1986
`Printed in U.S.A.
`
`Human Liver Microsomal Cytochrome P-450 Mephenytoin
`4-Hydroxylase, a Prototype of Genetic Polymorphism
`in Oxidative Drug Metabolism
`PURIFICATION AND CHARACTERIZATION OF TWO SIMILAR FORMS INVOLVED IN THE REACTION*
`
`(Received for publication, April 29,1985)
`
`Tsutomu Shimadag, Kunio S. Misono, and F. Peter Guengerichg
`Fram the ~ e ~ r ~ r n e n t
`~ ~ x i c ~ l a g y , Vanderbilt University School of Medicine,
`
`of E ~ ~ ~ r n i s t ~ and Center in ~
`a
`~
`c
`~
`r
`
`Nashville, Tennessee 37232
`
`similar forms of human P-450 are involved in S-me-
`phenytoin .l-hydroxylation, an activity which shows
`genetic polymorphism.
`
`~
`
`~~
`
`~
`
`~
`
`- -~ -
`
`Two forms of cytochrome P-450 (P-450), designated
`P-450nrp.l and P-450Mp.a, were purified to electropho-
`retic homogeneity from human liver microsomes on the
`basis of mephenytoin 4-hydroxylase activity. Purified
`P-450Mp-1 and P-45oMp.2 contained 12-17 nmol of P-
`450/mg of protein and had apparent monomeric mo-
`Cytochrome P-450 enzymes catalyze the monooxygenation
`lecular weights of 48,000 and 50,000, respectively.
`of a wide variety of chemicals, including drugs, carcinogens,
`P‘45oMP.1 and P-450Mp-2 were found to be very simi-
`pesticides, pollutants, and other xenobiotics as well as endog-
`lar proteins as judged by chromatographic behavior
`enous steroids, fatty acids, and prostaglandins (Conney, 1967;
`on n-octylamino-Sepharose 4B, hydroxylapatite, and
`Sat0 and Omura, 1978; Wislocki et al., 1980). In recent years,
`DEAE- and CM-cellulose columns, spectral properties,
`numerous biochemical studies have led to the purification of
`amino acid composition, peptide mapping, double im-
`many of the isozymes from experimental animals, determi-
`munodiffusion analysis, immunoinhibition, and N-ter-
`minal amino acid sequences. In vitro translation of
`nation of substrate specificities, the development of immu-
`liver RNA yielded polypeptides migrating with P-
`nochemical techniques for quantitative estimation of individ-
`or P-45oMp.2, depending upon which form was
`ual isozymes, determination of primary sequences, and the
`~ ~ O M P - ~
`in each sample, indicating that the two P-450s are
`application of recombinant DNA methods to questions con-
`translated from different mRNAs.
`cerning the regulation of these enzymes (Imai and Sato, 1974;
`When reconsituted with NA~PH-cyt~hrome-P-450
`Haugen and Coon, 1976; Norman et al., 1978; Ryan et al.,
`~-a-dilauroyl-sn-glyceryo-3-phospho-
`reductase and
`1979; Guengerich, 1979; Guengerich et al., 1982b; Fujii-Kuri-
`choline, P‘45o”p-l and P-45oMP-2 gave apparentfy
`yama et al., 1982; Cheng et al., 1984; Yabusaki et al., 1984;
`higher turnover numbers for mephenytoin 4-hydrox-
`Kimura et al., 1984).
`ylation than did the P-450 in the microsomes. The
`Humans, of course, also contain P-450s.’ Individual forms
`addition of purified rat or human cytochrome b5 to the
`have been isolated and purified, and ~iochemical evidence
`reconstituted system caused a significant increase in
`supports the existence of isozymes (Wang et al., 1980, 1983).
`the hydroxylation activity; the maximum stimulation
`However, little is known about the similarity of individual
`was obtained when the molar ratio of cytochrome b6 to
`forms in different people and the substrate specificities of
`b6
`P-450 was 3-fold. Rabbit anti-human c ~ o c ~ o m e
`these P-450s. One of the more interesting aspects of the P-
`inhibited NADH-cytochrome-c reductase and S-me-
`450s in humans relates to the polymo~hic variation in the
`phenytoin 4-hydroxylase activities in human liver mi-
`crosomes. In the presence of cytochrome b5, the K,,,
`activities of certain of these enzymes toward the oxidation of
`certain drugs (Kupfer and Preisig, 1983; Mahgoub et at., 1977;
`value for S-mephenytoin was 1.25 mM with all five
`Scott and Poffenbarger, 1979; Kupfer et al., 1984). In several
`purified cytochrome P-450s preparations, and V,,,
`values were
`0.8-1.25 nmol of
`4-hydroxy product
`cases, this polymorphism has a strong genetic component.
`formed per min/nmol of P-450. P-45OMP is a relatively
`Debrisoquine 4-hydroxylation has probably been the most
`selective P-450 form that metabolizes substituted hy-
`dantoins well. Reactions catalyzed by purified P-
`
`4 5 0 ~ ~ - I and P - 4 5 0 ~ p - ~ preparations and inhibited by
`anti-P-45OMP in human liver microsomes include S-
`mephenytoin 4-hydroxylation, S-nirvanol 4-hydrox-
`ylation, S-mephenytoin ~-demethylation, and diphen-
`ylhydantoin 4-hydroxylation. Thus, at least two very
`
`The abbreviations used are: P-450, liver microsomal cytochrome
`P-450; NaDodS04, sodium dodecyl sulfate; IgG, immunoglobulin G
`fraction (of sera); PTH, phenylthiohydantoin; P-45O~p.l and P-
`
`450~p.~, the respective 48,000- and 50,000-dalton forms of the human
`S-mephenytoin 4-hydroxylase discussed in this report; P-45008, the
`human liver debrisoquine 4-hydroxylase purified elsewhere (Distler-
`ath et d., 1985); P-450 PA, the human liver phenacetin 0-deet.hylase
`purified elsewhere (Distlerath et al., 1985). Human liver samples are
`denoted “HL” with a code number for each individual person. The
`
`properties of the rat P-450s designated P-450P~~.E and P-450UT., have
`been described elsewhere (Guengerich et ~ l . , 1982a; Waxman et d.,
`1985). Other preparations which appear to correspond to P’450pCN.E
`(p-450PB/p~N.E) include those designated ‘‘P-45Opc~” (Elshourbaa
`and Guzelian, 1980) and “PB-2a” (Waxman et
`a[., 1985). Other
`
`preparations which appear to correspond to P-450”~.1 include “female-
`specific P-450” (Kamataki et at., 1983), ”P-45Oi” (Ryan et al., 1984)
`and “P-450 2d” (Waxman et al., 1985).
`
`* This research was supported in part by Grants CA 30907 and ES
`00267 from the National Institutes of Health. The costs of puhlication
`of this article were defrayed in part hy the payment of page charges.
`This article must therefore
`be hereby marked “advertisement” in
`accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
`$ Present address: Osaka Prefectural Institute of Public Health,
`Nakamichi, Higashinari-ku, Osaka 537, Japan.
`5 Burroughs Wellcome Scholar in Toxicology (1983-1988). To
`whom correspondence should he addressed.
`
`909
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`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`

`
`Human Mephenytoin P-450s
`910
`studied of the polymorphisms (Mahgoub et al., 1977); other
`examples include phenacetin 0-deethylation and mepheny-
`toin 4-hydroxylation. We have previously purified and char-
`acterized the human liver P-450s involved in debrisoquine 4-
`hydroxylation and phenacetin 0-deethylation (Distlerath et
`al., 1985, Distlerath and Guengerich, 1984).
`S-Mephenytoin (5-phenyl-5-ethyl-N-methylhydantoin) is
`an anticonvulsive drug which is metabolized by N-demethyl-
`ation and 4-hydroxylation (of the aromatic ring) (Kupfer and
`Preisig, 1984; Troupin et al., 1976; Kupfer et al., 1981). About
`5-10% of Caucasian people display a “slow” metabolism phe-
`notype in vivo (Kupfer and Preisig, 1983, 1984; Troupin et al.,
`1976; Kupfer et al., 1981; Wedlund et al., 1984). In order to
`better understand
`the biochemical basis for this polymor-
`phism and develop knowledge that could be applied clinically,
`we sought to purify this enzyme from human liver and char-
`acterize it. In this article, we report the purification of the
`enzyme from five different liver samples and the biochemical
`comparison of the different preparations. We also report here
`a study of the specificity of this enzyme toward a series of
`hydantoin derivatives as well as other typical P-450 substrates
`using techniques of enzyme reconstitution and immunochem-
`ical inhibition; we compare the steady-state kinetic parame-
`ters of two M,variants of the enzyme; and we explore the role
`of cytochrome b6 in the reaction using reconstitution and
`immunochemical inhibition techniques.
`
`ANTI-P-45O(mg/flmOl P-450)
`FIG. 2. Inhibition of mephenytoin 4-hydroxylase activity
`by anti-P-450s in human liver microsomes. Human liver micro-
`somes (sample HL 38) containing 20 pmol of P-450 were preincubated
`at 23 “C for 10 min with various amounts of anti-P-4508 (O), anti-P-
`4503 (0), or preimmune antibody (H) IgG fractions. S-Mephenytoin
`4-hydroxylase activity of the liver microsomes was measured as
`described under “Experimental Procedures.” The activity without
`IgG was 0.18 nmol of 4-hydroxymephenytoin formed per min/nmol
`of P-450.
`
`TABLE I
`Purification of P - 4 5 0 ~ p . ~ from human liver microsomes
`
`P-450
`
`EXPERIMENTAL PROCEDURES’
`
`RESULTS
`Identity of P-4508 and P-45o~p.l in Human Liver Micro-
`somes-We
`first purified P-45oMp from liver sample HL 31
`according to the general method described under “Experimen-
`tal Procedures,” assaying catalytic activity at each step after
`removal of detergent and reconstitution. In
`this case, the
`hydroxylapatite column became severely compressed, and
`overall recovery of P-450 from the solubilized microsomes
`was less than 0.5%. However, the purified preparation gave a
`single protein band on NaDodSO4-polyacry1amide gel electro-
`phoresis and had S-mephenytoin 4-hydroxylase activity (be-
`cause of the presence of residual Emulgen 913 in the prepa-
`ration, we could not determine the exact turnover number).
`We examined the immunochemical cross-reactivity of the
`purified enzyme with other isozymes of human liver P-450
`which had already been purified in this laboratory (Wang et
`al., 1983; Distlerath et al., 1985). Purified P-45oMp reacted
`strongly with antibody raised to P-4508 (Wang et al., 1983) in
`analysis with immunochemical blotting but did not react with
`antibodies raised against human P-4502, P-4503, P-4504, P-
`4505, P-450DB, or P-45OPA (data not shown). To compare the
`contents of P-4508 and P-450Mp.l in several human liver mi-
`crosomes, antibodies raised in rabbits to each antigen were
`used with immunochemical blotting analysis. The content of
`P-45oMp.1 in 12 different human liver microsomal preparations
`was found to correlate well with the amounts of P-4508 in
`these preparations ( r = 0.956), suggesting that the two P-450s
`are identical or very similar (data not shown). Fig. 2 shows
`* Portions of this paper (including “Experimental Procedures” and
`Fig. 1) are presented in miniprint at the end of this paper. Miniprint
`is easily read with the aid of a standard magnifying glass. Full size
`photocopies are available from the Journal of Biological Chemistry,
`9650 Rockville Pike, Bethesda, MD 20814. Request Document No.
`85M-1428, cite the authors, and include a check or money order for
`$3.20 per set of photocopies. Full size photocopies are also included
`in the microfilm edition of the Journal that is available from Waverly
`Press.
`
`Protein
`
`Total Specific
`content
`
`nmol
`mg
`1830
`2400
`Microsomes”
`1800
`2230
`Solubilized supernatant
`609
`Octylamino-Sepharose 4B 135
`160
`19
`Hydroxylapatite
`9.8 134
`DE52
`68
`CM52
`4.8
`Sample HL 36 was used.
`
`nmol’mg
`protein
`0.76
`0.81
`4.5
`8.6
`13.7
`14.3
`
`Purifi- Yield
`cation
`
`__
`
`-fold
`1
`1.1
`5.9
`11
`18
`19
`
`%
`100
`98
`33
`9
`7
`4
`
`that rabbit anti-P-4508 completely inhibited S-mephenytoin
`4-hydroxylase activity in human liver microsomes. IgG frac-
`tions from rabbits immunized with the other human P-450s
`(see above) did not inhibit catalytic activity; in Fig. 2, only
`the lack of effect of anti-P-4503 is shown for example.
`Purification of P-450Mp.1 and P-450Mp.2-Chromatography
`was used to purify the P-450 responsible for the 4-hydroxyl-
`ation of S-mephenytoin from human livers, and enzyme frac-
`tions were monitored by the use of NaDodSO4-polyacry1amide
`gel electrophoresis and immunochemical detection of resolved
`proteins (transferred to nitrocellulose sheets) with rabbit anti-
`P-450Mp1 as described above (Table I). Hydrophobic affinity
`chromatography of cholate-solubilized microsomes on a n-
`octylamino-Sepharose 4B column yielded the majority of P-
`
`4 5 0 ~ p . ~ in fractions eluted with the buffer containing 0.06%
`(w/v) Emulgen 913 (Guengerich and Martin, 1980; Wang et
`al., 1983; Distlerath et al., 1985). The pooled fractions were
`purified further by chromatography on a hydroxylapatite col-
`umn. Most of the P-450Mp.1 was routinely found in the hem-
`oprotein peak eluted with 300 mM potassium phosphate buffer
`(after extensive washing with 40, 90, and 180 mM buffers).
`Since fractions from this peak contained two major polypep-
`tides and minor polypeptide Contaminants as shown in Fig.
`3, further purification with Whatman DE52 and CM52 ion-
`exchange column chromatography was carried out. P-450~p.~
`was eluted in the void fraction of DE52 columns under these
`conditions. This fraction was applied to a CM52 column,
`
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`
`911
`
`Human Mephenytoin P-450s
`blotting analysis, we detected two polypeptides in some
`human liver microsomal preparations that reacted with anti-
`
`P-45oMp.1; the low M,polypeptide is designated P-450MMp.1 and
`the higher hf, protein is termed P"i5oMp.2. Of particular in-
`terest is the observation that one of the humans (sample HL
`32) contained essentially no P-450Mp-1 but only P-45oMp.2. We
`purified this protein using the same chromatographic proce-
`dures described above. Using immunoblotting, we found that
`both proteins have the same chromatographic properties on
`hydroxylapatite and DEAE- and CM-cellulose and react with
`anti-P-450~p.~ with almost equal intensity. As shown in Fig.
`5, P"t5oMP.2 from sample HL 32 was homogeneous on Na-
`DodS04-polyacrylamide gel electrophoresis and had a mini-
`mum M, of about 50,000. When the P-45oMp.2 was mixed with
`P-450Mp.1, the two P-450s could be clearly separated by
`NaDodS04-polyacrylamide gel electrophoresis.
`of Cytochromes P-450Mp.1 and
`Spectral Properties
`P-4~50Mp.~-Fig. 6 shows the spectral properties of P-45oMp.1
`(from sample HL 38) and P-45oMp.2 (from sample HL 32).
`Both P-450s were found to have similar spectral properties,
`CY band at 567-
`i.e. in the oxidized states, both proteins had an
`568 nm, a p band at 534-536 nm, and a Soret peak at 416 nm,
`and the wavelength maximum of the reduced carbon monox-
`ide complex was found to be 449.5 nm in both cases. The
`ferric proteins appear to be mainly low-spin. The two prepa-
`rations (and others) contained relatively little cytochrome P-
`420.
`amino acid compositions
`Amino Acid Compositions-The
`
`of two preparations of P-45o~p.~ (from samples HL 36 and
`HL 38) and one preparation of P-45oMp.Z (from sample HL
`32) are presented in Table 11. These compositions are based
`only on 24-h hydrolysis and do not include cysteine and
`tryptophan values; the compositions are calculated on the
`basis of apparent M, values of 48,000 and 50,000 for P-
`450Mp-1 and P"t50Mp.2, respectively.
`The amino acid compositions of these preparations were
`found to be very similar except that the methionine content
`in the P-45oMp.2 preparation was somewhat less than that in
`P-45oMp.1 preparations.
`The difference indices (Metzger et al., 1968) for the three
`preparations were calculated and compared with other human
`
`P-450s, including P - 4 5 0 ~ ~ and P - 4 5 0 ~ ~ . The difference index
`for two P-450Mp.1 preparations (samples HL 36 and HL 38)
`was only 2.7, indicating similar amino acid composition. The
`indices between P'450Mp-1 and P-45oMp.2 were calculated to be
`2.7 and 3.7, suggesting that the two proteins are also very
`
` 2 3 4 5 M
`M I
`FIG. 3. NaDodS0,-polyacrylamide gel electrophoresis of
`purified P-450nm.l fractions of human liver microsomes. Elec-
`trophoresis was carried out with solubilized microsomes (lane I ) , n-
`octylamino-Sepharose 4B eluate (lane 2), hydroxylapatite eluate (lane
`3), DE52 eluate (lane 4 ) , and CM52 eluate (lane 5, 2 pg of protein).
`All samples were derived from sample HL 36 microsomes. The
`molecular mass standards (lanes M) used were bovine serum albumin
`(accepted 68 kDa), Escherichia coli L-glutamate dehydrogenase (53
`kDa), and equine liver alcohol dehydrogenase (43 kDa). The anode
`was at the bottom of the figure. The gel was stained with a silver
`method (Wray et al., 1981).
`
`68kDa-
`
`53kDa-
`
`43kDo-
`
`A
`
`E
`
`""
`
`1 2 3 4 5
`1 2 3 4 5
`FIG. 4. NaDodS0,-polyacrylamide gel electrophoresis and
`immunoblotting of human liver microsomes. Ten pg of
`liver
`microsomes from samples HL 70 (lane I ) , HL 72 (lane 21, HL 31
`(lane 3), HL 32 (lane 4 ) , and HL 33 (lane 5) was electrophoresed. The
`anode was at the bottom of the figure. After electrophoresis, the gel
`was stained with a silver method (Wray et al., 1981) (A) or the
`proteins were transferred to nitrocellulose and treated with anti-P-
`45o~p.1(1:100 dilution of antisera) (23).
`
`68kDo
`
`53kDo
`
`concentration of
`where P'45oMp.1 was eluted with a NaCl
`about 60 mM. The purified P-45oMp.1 thus obtained was ho-
`mogeneous as judged by NaDodS04-polyacrylamide gel
`electrophoresis, as shown in Fig. 3, and contained 12-17
`nmol of P-450/mg of protein in all cases. The minimum M,of
`I. 2 3 4 5 6 M
`M
`P-450Mp.1 was determined to be about 48,000. The fold puri-
`electrophoresis of
`FIG. 5. NaDodS04-polyacrylamide gel
`fication of P-45oMp.1 was greater than 18 (based on total
`purified human P-45OMP preparations used. P-45oMp.l from
`microsomal P-450), and the apparent recovery from sample
`samples HL 36 (lane 3), HL 37 (lane 4 ) , HL 38 (lane 5), and HL 91
`36 liver microsomes was about 4% (Table I).
`(lane 6 ) and P-45oMp.z from samples HL 32 (lane I ) and HL 33 (lane
`Fig. 4 shows both NaDodS04-polyacrylamide gel electro-
`2) are shown. Molecular mass standards (lanes M) were also used.
`Dhoresis and immunochemical staining (with anti-P-450~~.,)
`Conditions for electrophoresis were as described in the legend to Fig.
`. .. ._
`of five human microsomal preparations. With immuno-
`J.
`
`43 kDo
`
`I .
`
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`
`912
`
`WAVELENGTH (nm)
`FIG. 6. Spectral properties of P - 4 5 0 ~ ~ - , and P"t50Mp-Z.
`Spectra of P-450 (in 100 mM potassium phosphate buffer (pH 7.7)
`containing 20% (v/v) glycerol, 1 mM EDTA, 0.4% (w/v) sodium
`cholate, and 0.4% (w/v) Emulgen 913) were obtained with a Cary 219
`spectrophotometer in the automatic base-line correction mode. The
`absolute spectra of P-450Mp.l (from sample HL 38) and P-45oMp.2
`(from sample HL 32) are shown in A and B, respectively (Fe3+, Fez+,
`and Fez+-CO). The insets ( B and D) show Fez+-CO versus Fez+
`difference spectra of each purified P-450. Reduction was done by the
`addition of solid Na2S2O4.
`
`TABLE I1
`Amino acid compositions of P-45o~p.1 and P-45Ohlp-2
`Number of residues"
`
`mytoin P-450s
`Human Mephc
`tion by proteases (Staphylococcus aureus V8 protease, a-
`chymotrypsin, or papain) followed by NaDodSOr-polyacryl-
`amide gel electrophoresis was also used to compare P-45oMP.l
`and P-450Mp.2. Fig. 7 shows that limited proteolysis of these
`two forms of P-450 gave very similar peptide maps, but some
`slightly different cleavage products were obtained. Particu-
`larly in the case of digestion by papain, some differences in
`polypeptides were noted. The same conclusion was reached
`from a comparison of the tryptic peptides using high perform-
`ance liquid chromatography. The tryptic peptides obtained
`from P-45OMp.1 (samples HL 36 and HL 38) and P-450Mp.2
`(sample HL 32) were very similar (data not shown).
`Double Immunodiffusion Analysis-Rabbit antisera against
`P-45oMp.1 and P-45oMp.2 were prepared separately. Double
`immunodiffusion analysis was performed to examine the im-
`munochemical relationship of p-45oMp.l and P-45oMP.2. As
`shown in Fig. 8, anti-P-450Mp.l (P-45oMp.l from sample
`HL 38) formed a single precipitin line with both P-45oMP.l
`from samples HL 36, HL 37, HL 38, and HL 91, and anti-P-
`
`4 5 o ~ p - ~ (P-45oMp.2 from sample HL 32) gave the almost same
`results obtained with anti-P-450Mp.l. The same results were
`obtained with either antiserum and with detergent-solubilized
`human liver microsomes. Both antisera formed single precip-
`itin lines (and no spurs) with the solubilized microsomes from
`samples HL 38 and HL 32 as well as with purified P-45oMP.l
`(sample HL 38) and P-45oMp.z (sample HL 32), consonant
`with high immunochemical similarity. No spurs were observed
`al., 1983; see
`when the anti-P-4508 preparation (Wang et
`below) was compared with anti-P-450Mp.l or anti-P-450Mp.z.
`In Vitro Translation of P-45o~p.l and P-450Mp.2-None of
`the preceding studies really distinguished P-45oMp.1 from P-
`~ ~ O M P . ~ , with the exception of NaDodS04-polyacrylamide gel
`electrophoresis. The possibility existed that P-45oMP.l and P-
`450Mp.2 differ only in post-t,ranslational modifications or are
`artifacts arising from post-mortem changes or storage. We
`isolated RNA from four human liver samples and translated
`proteins in
`the presence of [35S]methionine and a rabbit
`reticulocyte lysate system. Anti-P-450~p-~ was used to precip-
`itate products of interest using a solid-phase method; the
`recovered material was electrophoresed, and a fluorograph is
`
`Amino acid
`
`P-45O~p.2,
`P-45O~p.1
`sample HL 32
`SamDle HL 36
`SamDle HL 38
`Ala
`27
`26
`28
`16
`19
`17
`Arg
`Asx
`40
`40
`43
`Glx
`55
`54
`58
`57
`61
`53
`GlY
`His
`14
`13
`12
`Ile
`19
`21
`21
`Leu
`33
`36
`37
`22
`24
`25
`LYS
`Met
`7
`4
`7
`Phe
`19
`19
`18
`Pro
`22
`19
`21
`Ser
`56
`55
`58
`Thr
`21
`24
`21
`12
`11
`11
`TY r
`Val
`26
`26
`27
`(Total)b
`(463)
`(445)
`(447)
`' Values presented are means of duplicate determinations.
`Not including cysteine or tryptophan.
`
`similar. P-45oMp.l and P-45oMp.2 had different amino acid
`compositions than P-450D~ and P-45OPA, for the indices be-
`tween them were calculated to be greater than 6. When we
`compared P-45oMp.1 and P-45oMp.2 to the reported composi-
`tions of rat P-450s (Haniu et al., 1984), the difference indices
`were all in the range of 5.5-12.
`Peptide Mapping of P - ~ ~ O M P - ~
`and P-450~p-~-Partial diges-
`
`"-
`
`1 2 3 1 2 3
`
`1 2 3 1 2 3
`+
`D
`C
`B
`A
`FIG. 7. Comparison of proteolytic digests of purified P-
`450Mp.l and P-450hm.z using NaDodS04-polyacrylamide gel
`electrophoresis. The P-450s were undigested in A or were digested
`with S. aureus V8 protease (B), a-chymotrypsin (C), or papain (D)
`as described under "Experimental Procedures." Lane 1 contained P-
`450Mp.1 (from sample HL 38), lane 2 contained P-450~p.1 (from sample
`
`HL 36), and lane 3 contained P-45o~p.~ (from sample HL 32). The
`gel was stained with silver (Wray et al., 1981).
`
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`

`
`B
`
`913
`
`Human Mephenytoin P-450s
`shows comparisons with reported sequences of rat and rabbit
`P-450s (Haniu et al., 1984; Black and Coon, 1985). The most
`homologous proteins were rat P-45OUT.*, P-450uT.I, P-45OPB.
`C, and P-450f. These rat proteins have 10-15 of the first 20
`residues in common with P-45oMp. Rat P-450, showed 9 of
`the first 15 residues. The other rat P-450s seem to be less
`related, and little homology to the rabbit proteins was noted,
`except for P - ~ ~ O L M . ~
`and P-4501,"~b (7 and 8 of the first 20
`residues, respectively, were identical).
`Reconstitution of Mephenytoin 4-Hydroxylase Activity by
`Purified P-450Mp-l and P-450Mp.2 and the Effects of Rat
`and Human Cytochrome b5-S-Mephenytoin
`4-hydroxylase
`activity was reconstituted in a
`system containing purified
`P"k5oMP.l or P-45oMp.2, NADPH-cytochrome-P-450 reduc-
`tase, and ~-a-dilauroyl-sn-glyceryo-3-phosphocholine. The
`activity was dependent on the incubation time (Fig. 1lA) and
`on the presence of NADPH-cytochrome-P-450 reductase (Fig.
`11B) and purified P-450Mp.1 (in this case, the preparation was
`used from liver microsomal sample HL 37) (Fig. llD). ~ - a -
`Dilauroyl-sn-glyceryo-3-phosphocholine activated the hy-
`droxylase activity about 2-fold with the optimal effect at a
`concentration of 40-50 pg/ml (Fig. 11C).
`Addition of rat cytochrome b5 to the complete reconstituted
`system caused further stimulation of S-mephenytoin 4-hy-
`droxylase activity; a 3-fold increase in the activity was ob-
`tained with a molar ratio of cytochrome b, to P-45oMp.l of 2-
`3 (Fig. 12A). Similar results were obtained when the rat
`cytochrome b5 was replaced by human cytochrome b5 which
`had been treated extensively to remove any residual detergent
`(Fig. 12B).
`Table IV compares the S-mephenytoin 4-hydroxylase activ-
`ities of the reconstituted monooxygenase system containing
`four different preparations of P-45oMp.l and one preparation
`of P-45oMp.Z with the activities of the respective human liver
`microsomes. The effects of cytochrome b5 on the activities of
`the five P-450 preparations are also included in Table IV in
`some instances. In the presence of cytochrome b5, the activi-
`ties of the reconstituted systems were higher than the respec-
`tive human liver microsomes. (Liver microsomes from sample
`HL 91 contained higher activities than the
`reconstituted
`system devoid of cytochrome b5.) A maximal purification
`factor of 6 (based on turnover numbers derived from total P-
`450 concentrations) was observed when the reconstituted
`system containing P-450Mp.1 from sample HL 36 liver micro-
`somes was used. P-45oMp.2 seemed to have only slightly lower
`mephenytoin hydroxylase activity than P"i5oMP.1. CytO-
`chrome b5 stimulated the S-mephenytoin 4-hydroxylase activ-
`ity in all of the reconstituted systems containing P-45oMP-1 Or
`P'45oMp-2.
`Fig. 13 shows the kinetic analysis of mephenytoin 4-hy-
`droxylase activity of reconstituted systems containing five
`different preparations of purified P-45oMp in the presence of
`cytochrome b5. The K,,, value was estimated to be about 1.25
`mM in all of the systems containing the different preparations
`of P - 4 5 0 ~ ~ . The VmaX value varied from 0.83 to 1.25 nmol of
`4-hydroxymephenytoin formed per min/nmol of P-450.
`Inhibition of Mephenytoin 4-Hydroxylase Activity of Human
`Liver Microsomes by Anti-human Cytochrome bs-In order to
`better address the possible role of cytochrome b5 in 4-hydrox-
`ylation of mephenytoin, the effect of anti-human cytochrome
`b5 on the activity was examined. The antibody raised to
`purified human liver cytochrome b5 recognized only a single
`polypeptide in three different human liver microsomal prep-
`arations as judged by immunoblotting analysis (e.g. Fig. 1).
`Since in the NADH-dependent electron transfer system cy-
`tochrome c accepts an electron via cytochrome b5 (Noshiro
`
`FIG. 8. Comparison of P - 4 5 0 ~ p - ~ and P - 4 5 O ~ p . z by double
`immunodiffusion analysis. Anti-38 and anti-32 represent the rab-
`bit antisera raised to P - 4 5 0 ~ p . ~ (from sample HL 38) and P"t5oMp.2
`
`(from sample HL 32), respectively. In A, four preparations of purified
`P-45oMp.l (from samples HL 36, HL 37, HL 38, and HL 91) and one
`(from sample HL 32) were included.
`preparation of purified P - 4 5 o ~ p . ~
`In B, the solubilized microsomes from samples HL 38 and HL 32
`were applied as well as the purified preparations of P-45oMp.1 (sample
`HL 38) and P"kjoMp.2 (sample HL 32).
`
`presented in Fig. 9. RNA isolated from an individual contain-
`ing primarily only P-45oMp.2 (sample HL 32) produced only a
`labeled polypeptide with the same apparent monomeric M, as
`P-45oMp.2 (50,000), and RNA isolated from an individual con-
`taining only P-45oMp.l (sample HL 93) produced only a labeled
`polypeptide with the sample apparent M, as P-45oMP-1
`(48,000). (Both samples HL 32 and HL 93 also yielded traces
`of a lower M, 3 5 S - p r o d ~ ~ t which is not thought to be related
`to P"i5oMp.) Liver samples HL 91 and HL 92 each contained
`
`both P'450Mp.1 and P-450~p.~ (Fig. 9); the RNA samples pre-
`pared from these two particular samples yielded only low
`levels of total translation products (Fig. 9A shows very weak
`bands in the position of P-45oMp.1). The apparent translation
`frequency was estimated to be in the range of 0.05% in all
`four cases.
`and P-
`N-terminal Amino Acid Sequences of P-45oMp-l
`450Mp.2-The sequences determined using automated Edman
`degradation are shown in Fig. 10, and yield data are given in
`Table 111. The sequences were identical at all residues which
`could be defined with certainty. The yields of the PTH
`derivatives of tyrosine and tryptophan at positions 10 and 13
`were rather low in P"i50Mp.lr and positions 10 and 13 in P-
`450Mp.2 were not identified. Cysteine products could not be
`found, even after prior performate oxidation or reduction and
`carboxymethylation with i~do['~C]acetic acid. Fig. 10 also
`
`Vanda Exhibit 2006 - Page 5
`
`

`
`Y P - 2 \
`
`U P - 1 r
`
`1 UP-2
`7 UP-1
`
`I
`
`"I
`
`J
`
`c
`
`i'
`- i
`/
`
`o l ,
`
`Vanda Exhibit 2006 - Page 6
`
`

`
`Human Mephenytoin P-450s
`
`915
`
`P T H
`derivative
`
`Yield"
`
`P-450
`
`Met
`ASP
`
`Leu
`
`Val
`Leu
`
`Leu
`
`Leu
`Leu
`Leu
`Leu
`Ser
`Leu
`Trp
`Arg
`Gln
`Ser
`
`1.79
`0.59
`0.08
`1.74
`1.54
`1.31
`1.04
`
`0.89
`
`0.73
`
`0.71
`
`0.80
`0.87
`
`0.40
`0.10
`0.13
`0.07
`
`(4.7)
`
`0.05
`
`G ~ Y
`
`~~
`
`TABLE I11
`
`Yie'd"
`nmol
`2.28
`1.83
`
`2.21
`
`1.10
`1.75
`
`1.31
`0.20
`1.36
`
`0.15
`1 .oo
`0.82
`1.18
`1.17
`1.15
`
`0.96
`0.19
`
`0.16
`
`
`
`
`
`nmol
`
`Ser
`
`Val
`
`
`
`Val
`
`Ser
`
`
`
`
`
`
`
`Cycle
`number
`
`PTH
`derivative
`
`
`
`
`
`
`
`Met
`ASP
`Ser
`Leu
`Val
`Val
`Leu
`Val
`Leu
`Tyr
`
`Leu Leu
`Ser
`TrP
`Leu
`Leu
`Leu
`Leu
`Ser
`Leu
`Trp
`Arg
`Gln
`Ser
`Ser
`GlY
`
`1
`2
`
`3
`4
`
`5 1.26
`6
`7
`1.45
`8
`9
`10
`11
`
`12
`1.3
`14
`15
`16
`17
`18
`19
`20
`21
`22
`23
`24
`25
`26
`27
`28
`29
`"Sequence analysis was done with 3.0 nmol of P-450~p.~ (from
`(from sample HL 32)
`sample HL 38) or 2.2 nmol of P"k5oMp.2
`(amounts based upon spectral determinations). PTH-serine was not
`quantified in most cases due to low and variable recovery. Identifi-
`cation of PTH-serine was confirmed by identification of the dehydro-
`serine derivative.
`
`TABLE IV
`S-Mephenytooin 4hydroxylase activity of purified P - 4 5 o M P . l and
`P-450MP.z
`S-Mephenytoin-$-hydroxylase activity"
`Purified P - $ 5 0 ~ ~
`
`Liver
`microsomes
`
`--__
`With
`Without
`cytochrome bg -__
`cytochrome b8
`nnol4-hydroxymephenytoinformedjminjnmol
`P-450
`f 0.016 0.267
`f 0.014
`(12)
`(5)
`0.027 f 0.011 0.159 f 0.037 0.387 f 0.015
`(6)
`(14)
`0.047 f 0.008 0.141 f 0.016 0.374
`f 0.035
`(8)
`(3)
`f 0.012 0.413
`k 0.026
`
`f 0.010 0.116
`
`
`
`0.088 f 0.012 0.140
`
`P-45oMp.z 0.022
`(sample HL 32)
`P-450ue.l
`(sample HL 36)
`P-45O~p.l
`(sample HL 37)
`P-45O~p.1
`
`
`(sample HL 38) (1.6)
`0.322 f 0.017 0.241 f 0.009 0.532 2 0.035
`P-45O~p.l
`(sample HL 91)
`(1.7)
`(0.7)
`-__
`' P-450MP.l from microsomes of liver samples HL 36, HL 37, HL
`38, and HL 91 and P-45oMp.Z from microsomes of liver sample HL 32
`were used. Values represent the mean of duplicate experiments F
`S.D. (range). Parentheses indicate the fold purification of the activity
`of purified P-450s as compared with respective microsomal activities.
`All assays used a substrate concentration of 0.5 mM.
`
`I
`
`I
`
`1
`
`I
`
`5-MEPHFNYTOIN ( I / [rnM])
`FIG. 13. Kinetic analysis of mephenytoin 4-hydroxylase
`
`activity by purified P-4501~p.x and P-450~p.~. P-450~p.~ from
`samples HL 36 (VI, HL 37 (O), HL 38 (A), and HL 91 (0) and P-
`450~p.~from sample HL 32 (0) were used (see Fig. 11 for optimization
`of conditions).
`
`"
`
`8
`6
`4
`2
`0
`RAT CYTOCHROME b5
`lmoior ratio of bg to P-45oMp-l
`FIG. 12. Stimulation of P-4tiO~~-l-supported S-mepheny-
`toin 4-hydroxylase activity by purified cytochrome ba. The
`
`standard reaction mixture ( P - 4 5 0 ~ ~ - 1 from sample HL 37) was used
`as described in the legend to Fig. 11. A, rat liver cytochrome bs; 3,
`human liver cytochrome bg.
`
` -
`
`4
`3
`2
`1
`0
`HUMAN CYTOCHROME bs
`
`
`
`benzo(a)pyrene, benzphetamine, and p-nitroanisole (Guen-
`gerich et al., 1982a; Ryan et al., 1982).
`Table V shows results where the activities of microsomes
`and monooxygenase systems from five different individuals
`are compared. In some assays, the effect of cytochrome bs was
`also measured. The S-mephenytoin N-demethylase activity
`of purified P-450~1p.2 was higher in the reconstituted systems
`than in liver microsomes; P-450~p.~ preparations from four
`sa