Identification and characterization of the herpes simplex virus type 2 gene encoding the essential capsid protein ICP32/VP19c.

We describe the characterization of the herpes simplex virus type 2 (HSV-2) gene encoding infected cell protein 32 (ICP32) and virion protein 19c (VP19c). We also demonstrate that the HSV-1 UL38/ORF.553 open reading frame (ORF), which has been shown to specify a viral protein essential for capsid formation (B. Pertuiset, M. Boccara, J. Cebrian, N. Berthelot, S. Chousterman, F. Puvian-Dutilleul, J. Sisman, and P. Sheldrick, J. Virol. 63: 2169-2179, 1989), must encode the cognate HSV type 1 (HSV-1) ICP32/VP19c protein. The region of the HSV-2 genome deduced to contain the gene specifying ICP32/VP19c was isolated and subcloned, and the nucleotide sequence of 2,158 base pairs of HSV-2 DNA mapping immediately upstream of the gene encoding the large subunit of the viral ribonucleotide reductase was determined. This region of the HSV-2 genome contains a large ORF capable of encoding two related 50,538- and 49,472-molecular-weight polypeptides. Direct evidence that this ORF encodes HSV-2 ICP32/VP19c was provided by immunoblotting experiments that utilized antisera directed against synthetic oligopeptides corresponding to internal portions of the predicted polypeptides encoded by the HSV-2 ORF or antisera directed against a TrpE/HSV-2 ORF fusion protein. The type-common immunoreactivity of the two antisera and comparison of the primary amino acid sequences of the predicted products of the HSV-2 ORF and the equivalent genomic region of HSV-1 provided evidence that the HSV-1 UL38 ORF encodes the HSV-1 ICP32/VP19c. Analysis of the expression of the HSV-1 and HSV-2 ICP32/VP19c cognate proteins indicated that there may be differences in their modes of synthesis. Comparison of the predicted structure of the HSV-2 ICP32/VP19c protein with the structures of related proteins encoded by other herpes viruses suggested that the internal capsid architecture of the herpes family of viruses varies substantially.

Effects of gossypol on DNA replication in mammalian cells

Gossypol, a binaphthalene compound, possesses male infertility effects. However, its mechanism of action and effects on somatic cells are not yet understood. The purpose of this study was to examine the effects of gossypol on mammalian cell growth and DNA replication, using tissue culture cells (HeLa) as an in vivo model.^ Gossypol inhibited DNA synthesis in HeLa cells at low doses, without affecting RNA or protein synthesis. This caused cells to accumulate in S phase without affecting cells in other phases of the cell cycle. The inhibition of DNA synthesis was both dose- and time-dependent. This irreversible block was associated with a decrease in HeLa plating efficiency. Gossypol did bind to DNA but did not measurably affect its ability to serve as a template for DNA polymerase $\alpha$, the major replicative enzyme. Only in the absence of serum could gossypol induce single-strand DNA breaks in HeLa cells; no DNA-DNA or DNA-protein crosslinks were formed.^ Gossypol exhibited dose-dependent inhibition of a number of eukaryotic and prokaryotic replicative DNA polymerases both in vitro and in vivo. This inhibition was kinetically non-competitive with respect to the DNA template and dNTP substrates. Both a filter binding assay and polyacrylamide gel electrophoresis were used to study gossypol binding to DNA polymerase. Inhibition resulted from drug binding to two adjacent amino acid residues on the enzyme. Binding was found to be irreversible and mediated through either non-covalent interactions or by Schiff's base formation between the aldehyde groups of gossypol and the $\varepsilon$-NH$\sb2$ groups of amino acid residues on the polymerase. Structure-function studies using eleven gossypol derivatives revealed that both aldehyde and hydroxyl groups function independently to effect inhibition of DNA polymerase and DNA replication. The activities of DNA polymerase $\beta$ and ribonucleotide reductase were also inhibited by increasing gossypol concentrations.^ These studies demonstrate that the gossypol-mediated inhibition of DNA replication is due in part to inhibition of key replicative enzymes, such as DNA polymerase $\alpha$. The study of DNA polymerase may serve as a model for the interaction of enzymes with gossypol, a drug which may prove useful as a chemotherapeutic agent. ^

B cells in ADA deficiency

Deficiency of the enzyme adenosine deaminase (ADA) results in severe lymphopenia in humans. Mice with an inactivating mutation in the ADA gene also exhibit profound lymphopenia, as well as pulmonary insufficiency and ribcage abnormalities. In fact, the mouse model has a phenotype that is remarkably similar to that of the human disease, making the mice valuable tools for unraveling the mechanism of lymphocyte destruction in absence of this housekeeping gene. T cell deficiency in ADA deficiency has been extensively studied by others, revealing a block in early thymocyte development. In contrast, our studies revealed that early B cell development in the bone marrow is normal. ADA-deficient mice, however, exhibit profound defects in germinal center formation, preventing antigen-dependent B cell maturation in the spleen. ADA-deficient spleen B cells display significant defects in proliferation and activation signaling, and produce more IgM than their normal counterparts, suggesting that extrafollicular plasmablasts are overrepresented. B cells from ADA-deficient mouse spleens undergo apoptosis more readily than those from normal mouse spleens. Levels of ADA's substrates, adenosine and 2′-deoxyadenosine, are elevated in both bone marrow and spleen in ADA-deficient mice. S ′-adenosyihomoeysteine hydrolase (SAH hydrolase) activity is significantly inhibited in both locales, as well. dATP levels, though, are only elevated in spleen, where B cell development is impaired, and not in bone marrow, where B cell ontogeny is normal. This finding points to dATP as the causative agent of lymphocyte death in ADA deficiency. ADA deficiency results in inhibition of the enzyme ribonucleotide reductase, thereby depleting nucleoside pools needed for DNA repair. Another mouse model that lacks a functional gene encoding a protein involved in DNA repair and/or cell cycle checkpoint regulation, p53-binding protein 1, exhibits blocks in T and B cell development that are similar to those seen in ADA-deficient mice. Unraveling the mechanisms of lymphocyte destruction in ADA deficiency may further understanding of lymphocyte biology, facilitate better chemotherapeutic treatment for lymphoproliferative diseases, and improve gene and enzyme therapy regimens attempted for ADA deficiency. ^

Metabolism and actions of 2 -chloro-2$\sp\prime$-fluoro-arabinosyladenine

2-Chloro-9-(2-deoxy-2-fluoro-$\beta $-D-arabinofuranosyl)adenine(Cl-F-ara-A) is a new deoxyadenosine analogue which is resistant to phosphorolytic cleavage and deamination, and exhibits therapeutic activity for both leukemia and solid tumors in experimental systems. To characterize its mechanism of cytotoxicity, the present study investigated the cellular pharmacology and the biochemical and molecular mechanisms of action of Cl-F-ara-A, from entrance of the drug into the cell, chemical changes to active metabolites, targeting on different cellular enzymes, to final programmed cell death response to the drug treatment.^ Cl-F-ara-A exhibited potent inhibitory action on DNA synthesis in a concentration-dependent and irreversible manner. The mono-, di-, and triphosphates of Cl-F-ara-A accumulated in cells, and their elimination was non-linear with a prolonged terminal phase, which resulted in prolonged dNTP depression. Ribonucleotide reductase activity was inversely correlated with the cellular Cl-F-ara-ATP level, and the inhibition of the reductase was saturated at higher cellular Cl-F-ara-ATP concentrations. The sustained inhibition of ribonucleotide reductase and the consequent depletion of deoxynucleotide triphosphate pools result in a cellular Cl-F-ara-ATP to dATP ratio which favors analogue incorporation into DNA.^ Incubation of CCRF-CEM cells with Cl-F-ara-A resulted in the incorporation of Cl-F-ara-AMP into DNA. A much lesser amount was associated with RNA, suggesting that Cl-F-ara-A is a more DNA-directed compound. The site of Cl-F-ara-AMP in DNA was related to the ratio of the cellular concentrations of the analogue triphosphate and the natural substrate dATP. Clonogenicity assays showed a strong inverse correlation between cell survival and Cl-F-ara-AMP incorporation into DNA, suggesting that the incorporation of Cl-F-ara-A monophosphate into DNA is critical for the cytotoxicity of Cl-F-ara-A.^ Cl-F-ara-ATP competed with dATP for incorporation into the A-site of the extending DNA strand catalyzed by both DNA polymerase $\alpha$ and $\varepsilon$. The incorporation of Cl-F-ara-AMP into DNA resulted in termination of DNA strand elongation, with the most pronounced effect being observed at Cl-F-ara-ATP:dATP ratio $>$1. The presence of Cl-F-ara-AMP at the 3$\sp\prime$-terminus of DNA also resulted in an increased incidence of nucleotide misincorporation in the following nucleotide position. The DNA termination and the nucleotide misincorporation induced by the incorporation of Cl-F-ara-AMP into DNA may contribute to the cytotoxicity of Cl-F-ara-A. ^

Studies on the interaction of NADPH cytochrome P-450 reductase and cytochromes P-450

Chemical modification of cytochrome P-450 reductase was used to determine the involvement of charged amino acids in the interaction between the reductase and two forms of cytochrome P-450. Acetylation of 11 lysine residues of the reductase with acetic anhydride yielded a 20-40% decrease in the K$\sb{\rm m}$ of the reductase for cytochrome P-450b or cytochrome P-450c. Modification of carboxyl groups on the reductase with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and methylamine, glycine methyl ester, or taurine as nucleophiles inhibited the interaction with the cytochromes P-450. We were able to modify 4.0, 7.9, and 5.9 carboxyl groups using methylamine, glycine methyl ester, and taurine, respectively. The apparent K$\sb{\rm m}$ for cytochrome P-450c or cytochrome P-450b was increased 1.3 to 5.2 fold. There were varied effects on the V$\sb{\rm max}$. There was no significant change in the conformation of the reductase upon chemical modification. These results strongly suggest that electrostatic interactions as well as steric constraints play a role in the binding and electron transfer step(s) between the reductase and cytochrome P-450. Cytochrome P-450 protected 0.8 moles of carboxyl residues on the reductase from being modified with EDC. These protected amino acids on the reductase are presumably involved in binding to cytochrome P-450. The specific peptide containing these amino acids has been identified. ^

The roles of electrostatic charge pairing in the interaction between cytochrome P-450 and NADPH-cytochrome P-450 reductase

The cytochrome P450 enzyme catalysis requires two electrons transferred from NADPH-cytochrome P450 reductase (reductase) to P450. Electrostatic charge-pairing has been proposed to be one of the major forces in the interaction between P450 and reductase. In order to obtain further insight into the molecular basis for the protein interaction, I used two methods, chemical modification and specific anti-peptide antibodies, to study the involvement and importance of charged amino acid residues. Acetylation of lysine residues of P450c and P450b by acetic anhydride dramatically inhibited the reductase-supported P450c-dependent ethoxycoumarin hydroxylation activity, but P450 activity supported by cumene hydroperoxide is relatively unchanged. The modification of lysine residues of P450c and P450b did not grossly disturb the protein conformation as revealed by several spectral studies. This differential effect of lysine modification on the P450 activity in the system reconstituted with reductase versus the system supported by cumene hydroperoxide suggested an important role for P450 lysine residues in the interaction with reductase. Using $\rm\sp{14}C$-acetic anhydride, P450 lysine residues were labelled and further identified on P450c and P450b. Those lysine residues are at position 97, 271, 279, and 407 for P450c, and 251, 384, 422, 433, and 473 for P450b. Alignment of those identified lysine residues on P450c and P450b with amino acid residues identified in other studies indicated those residues reside in three major sequence areas. Modification of arginine residues of P450b by phenylglyoxal and 2, 3-butanedione have no significant effect on P450 activity either supported by NADPH and reductase or supported by cumene hydroperoxide. Further studies using $\rm\sp{14}C$-phenylglyoxal reveals that no incorporation of phenylglyoxal into P450b was found. These results demonstrated a predominant role of lysine residues of P450 in the electrostatic interaction with reductase. To understand the protein binding sites on each of P450 and reductase, I generated three anti-peptide antibodies against regions on reductase and five anti-peptide antibodies against five putative reductase binding sites on P450c. These anti-peptide antibodies were affinity purified and characterized on ELISA and by Western blot analysis. Inhibition experiments using these antibodies demonstrated that regions 109-120 and 204-220 of reductase are probably the two major binding sites for P450. The association of reductase with cytochromes P450 and cytochrome c may rely on different mechanisms. The data from experiments using anti-peptide (P450c) antibodies supports the important role of P450c lysine residues 271/279 and 458/460 in the interaction with reductase. ^

Cytochrome P-450 reductase FAD binding domain: Cloning, enzymatic activity and flavin binding characterization

The Mixed Function Oxidase System metabolizes a wide range of biochemicals including drugs, pesticides and steroids. Cytochrome P450 reductase is a key enzymatic component of this system, supplying reducing equivalents from NADPH to cytochrome P450. The electrons are shuttled through reductase via two flavin moieties: FAD and FMN. Although the exact mechanism of flavins action is not known, the enzymatic features of reductase greatly depleted of either FMN of FAD have been characterized. Additionally, flavin location within reductase has been proposed by homology and chemical modification studies. This study seeks to extend the flavin depletion analysis in a more controlled system by eliminating the proposed FMN binding domain with recombinant DNA techniques and biochemical analysis. Two P450 reductase cDNA clones containing only the FMN and NADPH binding domain were isolated, expressed and the protein products purified and analysed. This study confirms the proposed FAD binding site, role of FAD in electron shuttling pathway and provides new methods to study the FAD binding domain. ^

Characterization of NARJ: A molecular chaperone involved in assembly of nitrate reductase

The major goal of this work was to define the role of accessory protein, NARJ, in assembly of nitrate reductase which is a membrane-bound multisubunit enzyme that can catalyze the reduction of nitrate to nitrite under anaerobic growth in E. coli. Nitrate reductase is encoded by the nar GHJI operon under control of the narG promoter. The purified nitrate reductase is composed of three subunits: $\alpha,\ \beta,$ and $\gamma.$ The NARJ protein which is encoded by the third gene (narJ) is not found to be associated with any of the purified preparations of the enzyme, but is required for active nitrate reductase. In this study the product of the narJ gene was identified. NARJ appeared to be produced at a reduced level, compared to the other proteins encoded by the nar operon. Since NARJ could not be overexpressed to a level for an efficient purification, NARJ was expressed and purified as a recombinant protein with polyhistidine tag. The recombinant protein NARJ-6His could functionally replace native NARJ. Purified NARJ-6His is a dimeric protein which contains no identifiable cofactors or unique secondary structure. NARJ was localized in the cytoplasm, and was not associated with nitrate reductase in the membrane. In vivo NARJ activated the $\alpha\beta$ complex and stabilized the $\alpha$ subunit against protease degradation. In the absence of the membrane-bound $\gamma$ subunit, NARJ formed an intermediate complex with $\alpha\beta$ in the cytosol. Based on these studies, NARJ fits the formal definition of a molecular chaperone. It appears to be required only for the biogenesis of nitrate reductase and, therefore, is defined as a private chaperone specifically involved in the assembly of nitrate reductase system. ^

The role of menaquinone in the nitrate reductase complex

Membrane bound, respiratory nitrate reductase in Escherichia coli is composed of three subunits, αβγ. The active complex is anchored to the membrane by membrane-integrated γ subunit and can reduce nitrate to nitrite with membrane quinones, (ubiquinone or menaquinone) as physiological electron donors. The transfer of electrons through the complex is thought to involve the sequence: membrane quinols → b-type hemes (γ subunit) → Fe-S centers (β subunit) → molybdopterin (α subunit) → nitrate. The enzyme can be assayed with the artificial electron donor reduced methyl viologen (MVH) which transfers electrons directly to the molybdopterin cofactor. These studies have focused on the possible role of protein-bound menaquinone in the structure and function of this multisubunit complex. ^ Nitrate reductase was purified as two distinct forms; after solubilization of membrane proteins with detergents, purification rendered an αβγ complex (holoenzyme) which catalyzes nitrate reduction with MVH or the quinols analogs, menadiol and duroquinol, as electron donors. Alternatively, heat-treatment of the membranes in the absence of detergents and subsequent purification of the active enzyme produced an αβ complex, which reduces nitrate only with MVH as electron donor. The active αβ dimer was also separated from γ subunit by heat treatment of the holoenzyme. ^ Menaquinone-9 was isolated directly from the purified αβ complex, and identified by mass spectrometry. Based on the composition of the membrane quinone pool, it was concluded that menaquinone-9 is sequestered from the membrane pool in a specifically protein-bound form. ^ The role of the bound menaquinone in the structure-function of nitrate reductase was also investigated, along with its participation in UV-light inactivation of the enzyme. Menaquinone-depleted nitrate reductase from a menaquinone deficient mutant retained activity with all electron donors and it remained sensitive to UV inactivation. However, the MVH-nitrate reductase activity and the rate of UV inactivation of the enzyme were significantly reduced and the optical properties of the enzyme were modified by the absence of the bound menaquinone-9. ^ Menaquinone-9 is not absolutely required for electron transfer in nitrate reductase but it appears to be specifically-bound during assembly of the complex and to enhance the transfer of electrons through the complex. The possible plasticity of the functional electron transfer pathway in nitrate reductase is discussed. ^

Structural studies of cytochrome P4501A1: Identification of substrate and cumene hydroperoxide binding regions in the active site of P4501A1 and characterization of the P4501A1 interaction with cytochrome P450 reductase

Cytochromes P450 are a superfamily of heme-thiolate proteins that function in a concert with another protein, cytochrome P450 reductase, as terminal oxidases of an enzymatic system catalyzing the metabolism of a variety of foreign compounds and endogenous substrates. In order to better understand P450s catalytic mechanism and substrate specificity, information about the structure of the active site is necessary. Given the lack of a crystal structure of mammalian P450, other methods have been used to elucidate the substrate recognition and binding site structure in the active center. In this project I utilized the photoaffinity labeling technique and site-directed mutagenesis approach to gain further structural insight into the active site of mammalian cytochrome P4501AI and examine the role of surface residues in the interaction of P4501A1 with the reductase. ^ Four crosslinked peptides were identified by photoaffinity labeling using diazido benzphetamine as a substrate analog. Alignment of the primary structure of cytochrome P4501A1 with that of bacterial cytochrome P450102 (the crystal structure of which is known) revealed that two of the isolated crosslinked peptides can be placed in the vicinity of heme (in the L helix region and β10-β11 sheet region of cytochrome P450102) and could be involved in substrate binding. The other two peptides were located on the surface of the protein with the label bound specifically to Lys residues that were proposed to be involved in reductase-P450 interaction. ^ Alternatively, it has been shown that some of the organic hydroperoxides can support P450 catalyzed reactions in the absence of NADPH, O2 and reductase. By means of photoaffinity labeling the cumene hydroperoxide binding region was identified. Using azidocumene as the photoaffinity label, the tripeptide T501-L502-K503 was shown to be the site where azidocumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A1 with cytochrome P450102 predicts that this region might correspond to β-sheet structure localized on the distal side of the heme ring near the I helix and the oxygen binding pocket. The role of Thr501 in the cumene hydroperoxide binding was confirmed by mutations of this residue and kinetic analysis of the effects of the mutations. ^ In addition, the role of two lysine residues, Lys271 and Lys279, in the interaction with reductase was examined by means of site-directed mutagenesis. The lysine residues were substituted with isoleucine and enzymatic activity of the wild type and the mutants were compared in reductase- and cumene hydroperoxide-supported systems. The lysine 279 residue has been shown to play a critical role in the P4501A1-reductase interaction. ^

CYTOPLASMIC MALATE DEHYDROGENASE IS THE AROMATIC ALPHA-KETO ACID REDUCTASE IN MAN (PHENYLKETONURIA, PKU, TYROSINE)

This dissertation presents evidence to support the hypothesis that cytoplasmic malate dehydrogenase (MDH-1) is the enzyme in humans which catalyzes the reduction of aromatic alpha-keto acids in the presence of NADH, and the enzyme which has been described in the literature as aromatic alpha-keto acid reductase (KAR; E.C. 1.1.1.96) is actually a secondary activity of cytoplasmic malate dehydrogenase.^ Purified MDH and purified KAR have the same molecular weight, subunit structure, heat-inactivation profile and tissue distribution. After starch gel electrophoresis, and using p-hydroxyphenylpyruvic acid (HPPA) as substrate, KAR activity co-migrates with MDH-1 in all species studied except some marine animals. Inhibition with malate, the end-product of malate dehydrogenase, substantially reduces or totally eliminates KAR activity. Purified cytoplasmic MDH from human erythrocytes has an alpha-keto acid reductase activity with identical mobility. All electrophoretic variants of MDH-1 seen in the fresh-water bony fish Xiphophorus, the amphibians Rana and humans exhibited identical variation for KAR, and the two traits co-segregated in the small group of offspring from one Rana heterozygote studied. Both enzymes show almost no electrophoretic variation among humans from many ethnic groups, and among several inbred strains of mice both MDH-s and KAR co-migrate with no variation. MDH-1 and KAR in mouse and Chinese hamster fibroblasts show identical mobility differences between species. Antisera raised against purified chicken cytoplasmic MDH totally inhibited both MDH-1 and KAR in chickens and humans. Mitochondrial MDH from tissue homogenates has no detectable KAR activity but purified MDH-2 does.^ The previous claim that the gene for KAR is on human chromosome 12 is disputed because both MDH-1 and LDH bands appear with slightly different mobility approximately midway between the human and hamster controls in somatic cell hybrid studies, and the meaning of this artifact is discussed. ^

REGULATION OF THE EXPRESSION OF NAR OPERON ENCODING NITRATE REDUCTASE IN ESCHERICHIA COLI

The nar operon, which encodes the nitrate reductase in Escherichia coli, can be induced under anaerobic conditions without nitrate to a low level and with nitrate to a maximum level. The anaerobic formation of nitrate reductase is dependent upon the fnr gene product while the narL gene product is required for further induction by nitrate. The sequence was determined across the entire promoter and regulatory region of the nar operon. The translational start site of the first structural gene of the nar operon, narG gene, was established by identifying the nucleotide sequence for the first 20 N-terminal amino acid residues of the alpha subunit of nitrate reductase. The transcriptional start site and the level of the transcript was determined by S1 mapping procedure. One major transcript was identified which was initiated 50 base pair (bp) upstream from the translational start site of the first structural gene. The synthesis of the transcript was repressed aerobically, fully induced by nitrate anaerobically, and greatly reduced in a ${\rm Fnr\sp-}$ mutant. Deletions were created in the 5$\sp\prime$ nar regulatory sequence with either an intact nar operon or a nar::lacZ fusion. The expression of the plasmids with deletions were determined in a strain with wild type fnr and narL loci, a Fnr- mutant strain and a NarL- mutant strain. These experiments demonstrated that the $5\sp\prime$ limit of the nar operon lies at about $-210$ bp from the transcription start site. The region required for anaerobic induction by the fnr gene product is located around $-60$ bp. Two putative narL recognition sites were identified, one of which is around $-200$ and another immediately adjacent to the fnr recognition region. The deletion of the sequences around $-200$ rendered the remaining narL complex repressive and thus decreased the expression of nar operon, suggesting that the two potential narL sites interact with each other over a significant length of DNA. ^

NADPH-CYTOCHROME P-450 REDUCTASE: EVIDENCE FOR TWO SEPARATE STRUCTURAL DOMAINS AND ISOLATION AND CHARACTERIZATION OF THE MEMBRANE ASSOCIATED SEGMENT

Homogenous detergent-solubilized NADPH-Cytochrome P-450 reductase was incorporated into microsomes and liposomes. This binding occurred spontaneously at temperatures between 4(DEGREES) and 37(DEGREES) and appeared to involve hydrophobic forces as the binding was not disrupted by 0.5 M sodium chloride. This exogenously-added reductase was active catalytically towards native cytochrome P-450, suggesting an association with the microsomal membrane similar to endogenous reductase. Homogeneous detergent-solubilized reductase was disaggregated by Renex-690 micelles, confirming the presence of a hydrophobic combining region on the enzyme. In contrast to these results, steapsin protease-solubilized reductase was incapable of microsomal attachment and did not interact with Renex-690 micelles. Detergent-solubilized reductase (76,500 daltons) was converted into a form with the electrophoretic mobility of steapsin protease-solubilized reductase (68,000 daltons) and a 12,500 dalton peptide (as determined by polyacrylamide-SDS gel electrophoresis) when the liposomal-incorporated enzyme was incubated with steapsin protease. The 68,000 dalton fragment thus obtained had properties identical with steapsin protease-solubilized reductase, i.e. it was catalytically active towards cytochrome c but inactive towards cytochrome P-450 and did not bind liposomes. The 12,500 dalton fragment remained associated with the liposomes when the digest was fractionated by gel filtration, suggesting that this is the segment of the enzyme which is embedded in the phospholipid bilayer. Thus, detergent-solubilized reductase appears to contain a soluble catalytic domain and a separate and separable membrane-binding domain. This latter domain is required for attaching the enzyme to the membrane and also to facilitate the catalytic interaction between the reductase and its native electron acceptor, cytochrome P-450. The membrane-binding segment of the reductase was isolated by preparative gel electrophoresis in SDS following its generation by proteolytic treatment of liposome-incorporated reductase. The peptide has a molecular weight of 6,400 as determined by gel filtration in 8 M guanidine hydrochloride and has an amino acid composition which is not especially hydrophobic. Following removal of SDS and dialysis out of 6 M urea, the membrane-binding peptide was unable to inhibit the activity of a reconstituted system containing purified reductase and cytochrome P-450. Moreover, when reductase and cytochrome P-450 were added to liposomes which contained the membrane-binding peptide, it was determined that mixed function oxidase activity was reconstituted as effectively as when vesicles without the membrane-binding peptide were used. Thus, the membrane-binding peptide was ineffective as an inhibitor of mixed function oxidase activity, suggesting perhaps that it facilitates catalysis by anchoring the catalytic domain of the reductase proximal to cytochrome P-450 (i.e. in the same mixed micelle) rather than through a specific interaction with cytochrome P-450. ^

DRUG METABOLISM IN LIVER TUMORS: PURIFICATION AND CHARACTERIZATION OF NADPH-CYTOCHROME-P450 REDUCTASE AND THE RESOLUTION OF MULTIPLE FORMS OF CYTOCHROME P-450

The hydroxylation of N- and O-methyl drugs and a polycyclic hydrocarbon has been demonstrated in microsomes prepared from two transplantable Morris hepatomas (i.e., 7288C. t.c. and 5123 t.c.(H). The hydroxylation rates of the drug benzphetamine and the polycyclic hydrocarbon benzo {(alpha)} pyrene by tumor microsomes were inducible 2 to 3-fold and 2-fold, respectively by pretreatment of rats with phenobarbital/hydrocortisone. Hepatoma 5123t.c.(h) microsomal hydroxylation activities were more inducible after these pretreatments than hepatoma 7288C.t.c. Two chemotherapeutic drugs (cyclophosphamide and isophosphamide) were shown to be mutagenic after activation by the tumor hemogenate with the TA100 strain of Salmonella typhimurium bacteria. NADPH-cytochrome P-450 was purified from phenobarbital/hydrocortisone treated rat hepatoma 5123t.c.(H) microsomes 353-fold with a specific activity 63.6 nmol of cytochrome c reduced per min per mg of protein. The purified enzyme, has an apparent molecular weight of 79,500 daltons, and contained an equal molar ratio of FMN and FAD, with a total flavin content of 16.4 nmol per mg of protein. The purified enzyme also catalyzed electron transfer to artificial electron acceptors with the K(,m) values of the hepatoma reductase similar to those of purified liver reductase. The K(,m) value of the hepatoma reductase (13 uM) for NADPH was similar to that of purified liver reductase (5.0 uM). In addition the purified hepatoma reductase was immunochemically similar to the liver reductase.^ Hepatoma cytochrome P-450, the hemeprotein component of the hepatoma microsomes of rats pretreated with phenobarbital/hydrocortisone. The resolution of the six forms was achieved by the DE-53 ion-exchange chromatography, and further purified by hydroxyapatite. The six different fractions that contained P-450 activity, had specific contents from 0.47 to 1.75 nmol of cytochrome P-450 per mg of protein, and indicated a 2 to 9-fold purification as compared to the original microsomes. In addition, difference spectra, molecular weights and immunological results suggest there are at least six different forms of cytochrome P-450 in hepatoma 5123 t.c.(H). ^