Biomedical subjects

E F Johnson

Publications and source records attributed to E F Johnson.

At least 91 records · Page 5Linked to original sources

PubMed1987

Intraspecies individuality for the metabolism of steroids.

A variety of regulatory factors contribute to differences in the rates of 6 beta-hydroxylation, 16 alpha-hydroxylation and 21-hydroxylation of progesterone as catalysed by liver microsomes prepared from individual rabbits. It is likely that the 6 beta-hydroxylation of progesterone is catalysed primarily by cytochrome P-450 3c, an enzyme that exhibits allosteric activation by alpha-napthoflavone, and by a form of P-450 3b, 6 beta+, that is expressed in some rabbits in an autosomal dominant manner. The mechanism of activation for P-450 3c appears to reflect an effector mediated increase of the affinity of the enzyme for substrate as judged by substrate binding studies. A second form of P-450 3b, 6 beta-, catalyses a major portion of hepatic progesterone 16 alpha-hydroxylation and exhibits activation by a variety of C21 steroids of which 5 beta-pregnane-3 beta,20 alpha-diol is the most efficacious. P-450 1, which catalyses the 21-hydroxylation of progesterone, is expressed at 10-fold higher levels in the 21H phenotype than the 21L phenotype, and the former is inherited as an autosomal dominant characteristic. A cDNA encoding a P-450 1-related gene product exhibits a predicted amino acid sequence that is 95% homologous to that of P-450 1. The P-450 1-related gene product is expressed in liver to a similar degree in both 21H and 21L rabbits.

Allosteric Regulation↗

PubMed1987

A Minority Health Careers Exposure Program.

The University of Tennessee-Memphis (UT-M) Health Careers Exposure Program was initiated in response to the complaint of minority college students that basic medical science laboratories and clinical centers at private and state-supported health-science organizations in Tennessee were inaccessible to them as career-motivating, summer work experiences. The preceptor-apprentice relationship was the means utilized to expose and stimulate minority college students to enter health careers in this study. The formal activity was usually conducted for eight to ten weeks of summer vacation in the basic science and clinical facilities on the UT-M campus.A survey of the current career activities of former apprentices suggests that direct exposure of academically talented, minority college students to health careers is a factor that increases the number entering the health professions.

Black or African American↗

PubMed1986

Evidence that rabbit cytochrome P-450 K is encoded by the plasmid pP-450 PBc2.

The complete coding sequence for P-450 PBc2 was inserted into a T7-phage promoter system, and a capped cRNA was generated using T7 RNA polymerase. The P-450 PBc2 cRNA was translated in a rabbit reticulocyte lysate. The in vitro translation product was indirectly immunoprecipitated by the monoclonal antibodies 2F5 and 3C3 that recognize P-450 K. SDS-polyacrylamide gel electrophoresis revealed that the translated protein product exhibits the same relative electrophoretic mobility as P-450 K. The N-terminal amino acid sequence was determined to be MDLVVVLGL-LS-LLLLSL- for P-450 K immunopurified from rabbit kidney using the monoclonal antibody 2F5. This sequence agrees with the predicted amino acid sequence derived from the P-450 PBc2 cDNA. These results indicate that P-450 K or a protein closely related to P-450 K is encoded by the plasmid pP-450 PBc2.

Amino Acid Sequence↗

PubMed1986

Active site-directed inhibition of rabbit cytochrome P-450 1 by amino-substituted steroids.

A variety of amino-substituted steroids were investigated as inhibitors of the rabbit hepatic, steroid 21-hydroxylase, cytochrome P-450 1. We reasoned that a steroid analog of pregnenolone capable of mimicking the binding of C21-steroids to the enzyme at the active site and bearing an amine moiety on the 17 beta-side chain would be a potent inhibitor if the amine were free to interact with the heme iron. Our studies reveal that 22-amino-23,24-bisnor-5-cholen-3 beta-ol (22-ABC) is a tightly-bound inhibitor of cytochrome P-450 1-catalyzed reactions (Ki less than 1 nM). Spectral differences elicited by 22-ABC indicate that when bound to the enzyme, the amine moiety of 22-ABC is coordinated to the heme iron. In contrast, several other hepatic cytochrome P-450s which mediate distinct regio-specific routes of metabolism for progesterone or pregnenolone remain largely unaffected at concentrations of 22-ABC that exceed by 2 orders of magnitude that required to inhibit cytochrome P-450 1. 22-ABC also inhibited the metabolism of benzo(a)pyrene attributable to cytochrome P-450 1 but did not inhibit that induced by treatment with rifampicin or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Analogs of 22-ABC bearing a hydroxyl group or a methylamine in place of the amine moiety exhibit lower affinities for cytochrome P-450 1. In addition, either increasing or decreasing the number of carbons of the side chain reduced the affinity of the inhibitor for cytochrome P-450 1.

Animals↗

PubMed1986

Secretory cells in the nucleus pulposus of the adult human intervertebral disc. A preliminary report.

A light microscopical study was conducted to ascertain the type of cells in the nucleus pulposus of the adult human intervertrebral disc. Three lumbar intervertebral discs were removed from each of 15 male and female adults at autopsy (ages ranged from 19 to 62 years). The tissue was fixed in formalin, decalcified in formic acid, dehydrated in a graded series of ethanol, embedded in paraffin, and serially sectioned at 7-10 micron. Tissue sections were affixed to albuminized glass slides and stained either by hematoxylin and eosin or hematoxylin and Van Gieson's stain. The cells of the bulk of the nucleus pulposus consisted of chondrocytes and a few fibroblasts; however, the subchondral matrix of the nucleus pulposos contained numerous stellate cells with (from 1 to 8) unusually long (up to 80 micron) primary cytoplasmic processes that often branch into secondary processes. The cell processes contained cytoplasmic varicosities at various intervals along their lengths; and their endings often expanded into bulbous, vesicle-filled process terminals. The surrounding extracellular matrix usually contained numerous, vesicle-filled, eosinophil matrix bodies. Morphological similarities of cytoplasmic varicosities, process terminals, and matrix bodies, as well as the apparent budding of process terminals, suggest that these previously unidentified cells are secreting an unknown matrix component into the subchondral matrix of the nucleus pulposus of the adult human.

Adult↗

PubMed1985

Two catalytically distinct subforms of cytochrome P-450 3b as obtained from inbred rabbits.

Cytochrome P-450 3b has been shown previously to exist in one of two catalytically and structurally distinct forms. Preparations of P-450 3b obtained from outbred New Zealand White rabbits exhibit both high-efficiency (Vmax/Km) progesterone 6 beta- and 16 alpha-hydroxylases and a low-efficiency 16 alpha-hydroxylase. In contrast, P-450 3b prepared from a genetically defined strain of rabbits, IIIVO/J, does not display the high-efficiency 6 beta- and 16 alpha-hydroxylase activities. This appears to reflect the inheritance of one of two catalytically distinct subforms of P-450 3b. In the present study, we provide information with respect to the characterization of these progesterone hydroxylases as they occur in two highly inbred strains of rabbits, B/J and III/J. The identification of these strains as sources of these subforms was achieved by utilizing a monoclonal antibody to P-450 3b that produced a distinct pattern of inhibition for each phenotype. The functional and structural attributes of these subforms of P-450 3b indicate that whereas only the low-efficiency 16 alpha-hydroxylase is expressed in B/J rabbits, both of the catalytically distinct subforms of P-450 3b are expressed in the III/J rabbits. Thus, it is unlikely that these enzymic variants reflect allomorphic forms of P-450 3b.

Animals↗

PubMed1985

Multiple gene-like sequences related to the rabbit hepatic progesterone 21-hydroxylase cytochrome P-450 1.

Rabbits exhibit phenotypic differences, 21H and 21L, in the rate of hepatic progesterone 21-hydroxylation that reflect 10-fold higher microsomal concentrations of cytochrome P-450 1 in 21H rabbits. A cDNA library in pBR322 was prepared from liver mRNA isolated from a 21H rabbit. A clone, p1-8, producing a hybrid protein resulting from the insertion of the cDNA into the beta-lactamase gene of the plasmid expressed 5 distinct epitopes that were recognized by a panel of monoclonal antibodies developed toward P-450 1. RNAs selected from total hepatic mRNA by filter hybridization with p1-8 yield at least two electrophoretically distinct proteins when translated in vitro and immunoprecipitated with the 3C3 monoclonal antibody. Only one of the two proteins is recognized by the 1F11 monoclonal antibody, which is highly specific for P-450 1, and the immunoprecipitated protein exhibits the electrophoretic mobility of P-450 1. The other protein remains unidentified. Northern blot analysis indicates that the 3' noncoding portion of p1-8 hybridizes to higher steady state concentrations of polyadenylated RNA in the 21H as compared to 21L rabbits. This correspondence in expression with that of P-450 1 in the 21H and 21L phenotypes further suggests that p1-8 encodes P-450 1 or a closely related protein. The cDNA is 1871 base pairs in length and encodes a protein of 487 amino acids. Southern blot analysis indicates that several independent, gene-like sequences hybridize with the 3' noncoding region of p1-8 under conditions of high stringency. These results indicate that P-450 1 is a member of an extensive multigene family.

Amino Acid Sequence↗

PubMed1985

Characterization of variations in rabbit hepatic progesterone 21-hydroxylase activity by serial biopsy.

Earlier work has shown that the 21-hydroxylation of progesterone in the hepatic microsomal fraction of outbred New Zealand White rabbits varies over a 10-fold range. To determine whether the differences in 21-hydroxylase activity were due to a transient inductive effect, livers from a group of 28 rabbits were serially biopsied at least three times over a minimum period of two months. Both progesterone 21- and 16 alpha-hydroxylase activities were determined in the post-8700g supernatant of homogenates prepared from these biopsy samples. A substantial variability in both the 21- and 16 alpha-hydroxylase activity was observed for serial biopsy samples from individual rabbits. Each animal was found, however, to maintain relatively constant ratios of 21/16 alpha-hydroxylase activity throughout the course of the study. Previous studies have indicated that the 21-hydroxylase activity does not correlate with the 16 alpha-hydroxylase activity and that the 21-hydroxylase phenotype could be determined from the ratio of these activities. On the basis of this ratio, two groups of animals could be distinguished in the present study. Approximately 25% of the animals exhibited an elevated 21/16 alpha-hydroxylase ratio (greater than 1.5), the remainder were below this level. Furthermore, the expression of elevated levels of the 21-hydroxylase activity were found to be consistent within this subpopulation suggesting that a transient inductive effect is not responsible for the differences in 21-hydroxylase activity among populations of outbred New Zealand White rabbits. This study demonstrates the determination of the hepatic enzymatic phenotype while maintaining the animal for long periods of time and for subsequent investigations.

Animals↗

PubMed1985

The complete amino acid sequence of a constitutive form of liver microsomal cytochrome P-450.

The complete covalent structure of the constitutive cytochrome P-450, form 3b, from rabbit liver microsomes was determined. The apocytochrome contains 490 amino acid residues in a single polypeptide chain, Mr = 55,860. Peptides from selective chemical and proteolytic cleavages were isolated by a combination of gel filtration and high performance liquid chromatography and sequenced by automated Edman degradation. Overlapping peptide sequences were used to deduce the complete sequence. The constitutive form is only 46% homologous to the phenobarbital-induced cytochrome P-450 (Heinemann, F. S., and Ozols, J. (1983) J. Biol. Chem. 258, 4195-4201) and contains a deletion at position 22. Strongly conserved regions include Cys435 and a previously identified tryptic peptide, residues 345-357. The distribution of hydrophobic segments is used to predict the membrane topology of the protein, and four possible orientations of this protein in the membrane are presented.

Amino Acid Sequence↗

PubMed1985

Variation in hepatic microsomal cytochrome P-450 1 concentration among untreated rabbits alters the efficiency of estradiol hydroxylation.

The metabolism of 17 beta-estradiol was examined using both rabbit liver microsomes and highly purified forms of rabbit liver microsomal cytochrome P-450. The predominant microsomal metabolite of 17 beta-estradiol is the 2-hydroxylated product. 2-Hydroxyestradiol is also the principal metabolite in reconstitution experiments in which P-450 1 exhibits the greatest Vmax, ca. 6 mol min-1 mol P-450 1(-1), vs less than 0.6 mol min-1 mol P-450(-1) for forms 2, 3b-, 3b+, 3c, 4, and 6. In addition P-450 1 has the lowest Km, ca. 2 microM. This suggested that microsomes which differ in their content of P-450 1 would also differ in the kinetic parameters characterizing the 2-hydroxylation of 17 beta-estradiol. Microsomes containing low amounts of P-450 1, less than 0.1 nmol/mg protein, exhibit a low-efficiency (Vmax/Km) 2-hydroxylase activity. Microsomes containing elevated concentrations of P-450 1, greater than 0.3 nmol/mg protein, exhibit a substrate dependence suggestive of an additional high-efficiency enzyme. The latter is specifically inhibited by a monoclonal antibody that recognizes P-450 1. These results indicate that the elevated expression of P-450 1 in microsomes leads to a marked increase in the apparent first-order rate constant for the 2-hydroxylation of 17 beta-estradiol, as it does for the 21-hydroxylation of progesterone. This should have a marked effect on the metabolism of these two steroid hormones at concentrations that are likely to occur in vivo.

Animals↗

PubMed1985

Variations in hepatic progesterone 21-hydroxylase activity reflect differences in the microsomal concentration of rabbit cytochrome P-450 1.

A monoclonal antibody specific for cytochrome P-450 1 that extensively (greater than 95%) inhibits the hepatic 21-hydroxylation of progesterone was used in a two-site immunoradiometric assay to estimate the concentration of cytochrome P-450 1 in microsomes prepared from 24 individual, untreated New Zealand White rabbits. The progesterone 21-hydroxylase activities of these microsomes ranged from 0.2 to 5.8 nmol min-1 mg microsomal protein-1. Scatchard analysis revealed similar slopes and thus apparent affinities between the antibody and microsome samples that varied greater than 10-fold in 21-hydroxylase activity. The maximal extent of binding of the antibody to different microsomal preparations was greater for microsomes exhibiting high as compared to low 21-hydroxylase activity, suggesting that the level of binding reflects the microsomal content of P-450 1. Quantitation was based on the extent of binding of the 125I-labeled monoclonal antibody to P-450 1 sequestered from a sample by a heterologous monoclonal antibody adsorbed to the wells of a microtiter plate. These results indicate that the microsomal content of P-450 1 varies from less than 0.05 to 0.5 nmol/mg microsomal protein. The microsomal content of this antigen as determined in the two-site immunoradiometric assay was highly correlated (r = 0.97) with progesterone 21-hydroxylase activity. Linear regression analysis was used to estimate the turnover number for progesterone in situ, yielding a value of 11 nmol deoxycorticosterone formed min-1 nmol microsomal P-450 1(-1). This is similar to the value of 14 nmol deoxycorticosterone formed min-1 nmol-1 obtained for the reconstituted, purified P-450 1 used as a standard in the immunoquantitation assay.

Animals↗

PubMed1985

Cloning and characterization of cDNAs encoding 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible rabbit mRNAs for cytochrome P-450 isozymes 4 and 6.

Monoclonal antibodies toward rabbit liver cytochrome P-450 isozyme 6 (P-450 6) were used to identify several recombinant clones from a pBR322 cDNA library that express beta-lactamase-P-450 6 hybrid proteins. The nucleic acid sequence and predicted amino acid sequence of a rabbit P-450 6 cDNA shows a high degree of homology with rat P-450c and mouse P1-450. When used as a probe to rescreen the library, the P-450 6 cDNA hybridized to several heterologous classes of cDNAs. One such class was shown to encode P-450 4 by comparison of its predicted amino acid sequence to amino acid sequences of cysteine-containing tryptic peptides derived from P-450 4. DNA sequence analysis of a cDNA clone belonging to a third class demonstrated that it contained a 131-base-pair intervening sequencing when compared to the cDNA coding for P-450 6. This sequence corresponds in location to intron E of the rat P-450c gene. Blot-hybridization analysis demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dramatically induced P-450 4 and 6 mRNAs, which differ in size. The sizes of these mRNAs differ from their analogs in the mouse as a result of divergence in the 3' untranslated portions of the mRNAs.

Amino Acid Sequence↗

PubMed1985

Evidence that variation among untreated rabbits in hepatic progesterone 21-hydroxylase activity is indicative of enzyme heterogeneity rather than a transient inductive effect.

Previous work has shown that the 21-hydroxylation of progesterone in the hepatic microsomal fraction of outbred NZW rabbits varies over a tenfold range. In contrast, the 16-hydroxylase activity is relatively constant and is not correlated to the activity of the 21-hydroxylase. The distribution of the 21-hydroxylase activity is roughly bimodal with about one-third of the animals (21-H) exhibiting 21-hydroxylase activity exceeding 1 nmol/min/mg microsomal protein, whereas the remainder (21-L) generally exhibit an activity that is less than 1 nmol/min/mg protein. To determine if this was due to a transient phenomenon, liver punch biopsies were collected from 28 rabbits at intervals of approximately three weeks for at least three serial samples. The 21- and 16-hydroxylase activities were determined in the postmitochondrial fractions of these biopsy samples. A substantial variability in both 21- and 16-hydroxylase activities was observed for serial biopsy samples from individual rabbits. The variation of the 16-hydroxylase activity paralleled, however, that of the 21-hydroxylase, thus suggesting that the variation between biopsy samples for individual rabbits was due to factors such as contamination with blood and connective tissue, which would affect both activities equally. Rabbits, therefore, were phenotyped as 21-H or 21-L on the basis of the 21/16-hydroxylase ratio. The mean ratio was 3.2 +/- 1.2 and 0.8 +/- 0.3 for rabbits phenotyped as 21-H and 21-L, respectively. Similar values for this ratio were obtained for the other group of rabbits phenotyped as 21-H and 21-L, 3.8 +/- 1.6 and 0.5 +/- 0.2, respectively, following the isolation of microsomes from whole liver homogenates. The ratio of 21/16-hydroxylase activity was found to be relatively constant for biopsy samples obtained from the same animal over the course of this study, thus indicating that the elevated 21-hydroxylase activity is not a transient phenomenon.

Animals↗

PubMed1985

Elastic fibres in the anulus fibrosus of the adult human lumbar intervertebral disc. A preliminary report.

Two intervertebral discs were removed at autopsy from the lumbar region of the spine from male or female adult human cases at the following ages: 26, 34, 43, 50 and 62 years. The discs were prepared by paraffin wax embedding, sectioned serially and stained with haematoxylin and eosin or by Verhoff's elastic fibre method. Four light microscopy photomicrographs were made of randomly selected 400 micron 2 areas of anulus fibrosus matrix (two each from different levels of a single disc). Each photomicrograph was examined for elastic fibres, and the areas occupied by them were quantitated by manual planimetry. Elastic fibres were observed in all lamellae of the anulus fibrosus at lumbar levels and they were circularly, obliquely and vertically oriented. Approximately 10% of the matrix of the anulus fibrosus consisted of elastic fibres and their numbers tended to decrease slightly with increasing age of the individual.

Adult↗

PubMed1985

Variations among untreated rabbits in benzo(a)pyrene metabolism and its modulation by 7,8-benzoflavone.

The metabolism of benzo(a)pyrene by rabbit liver microsomes can be stimulated or inhibited by 7,8-benzo(a)flavone (ANF) depending on the distribution of specific P-450 enzymes present within the microsomes. Treatment of rabbits with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or rifampicin leads to an increase of hepatic microsomal metabolism of benzo(a)pyrene. ANF stimulates the rate of benzo(a)pyrene metabolism catalyzed by microsomes isolated from rabbits treated with rifampicin by 3-fold. In contrast, ANF moderately inhibits the activity of microsomes from TCDD-treated rabbits. Variations in the benzo(a)pyrene hydroxylase activity of microsomes from untreated rabbits apparently reflect differences in the expression of P-450 1, a constitutive form of P-450. Thus, the benzo(a)pyrene hydroxylase activity of microsomes from untreated rabbits, which varies from 0.40 to 1.5 nmol/min/mg of protein, is directly correlated with the microsomal concentration of P-450 1. The metabolism of benzo(a)pyrene by microsomes containing high concentrations of P-450 1 is inhibited by a monoclonal antibody specific for this cytochrome to approximately the rate exhibited by microsomes with a low concentration of P-450 1. The benzo(a)pyrene activity stimulated by ANF in microsomes with a low concentration of P-450 1 is not inhibited by the monoclonal antibody. The activity of P-450 1 is inhibited by ANF at concentrations that stimulate other constitutive forms of P-450. Thus, ANF produces offsetting effects on benzo(a)pyrene metabolism in microsomes from untreated animals by stimulating the activity of at least one cytochrome and inhibiting P-450 1-mediated activity.

Animals↗

PubMed1984

Use of a monoclonal antibody specific for rabbit microsomal cytochrome P-450 3b to characterize the participation of this cytochrome in the microsomal 6 beta- and 16 alpha-hydroxylation of progesterone.

A monoclonal antibody was developed that is specific for the 3b electrophoretic class of rabbit liver microsomal cytochrome P-450 as judged by immunoprecipitation and subsequent electrophoretic analysis. The antibody is inhibitory of catalytically distinct, variant forms of P-450 3b prepared from New Zealand White or IIIVO/J rabbits, respectively. Peptide mapping of the immunopurified P-450 3b from NZW and IIIVO/J microsomes indicates that a characteristic difference between the variant forms is exhibited by the antigen. In addition, a competitive assay indicates that the binding properties of the antibody do not differ substantially toward the variant forms of P-450 3b. The inhibitory antibody was used to examine the contribution of P-450 3b to the microsomal 16 alpha- and 6 beta-hydroxylation of progesterone. The antibody inhibits 40-70% of the 16 alpha-hydroxylase activity of microsomes from either New Zealand White or IIIVO/J rabbits. In contrast, it does not inhibit 6 beta-hydroxylation catalyzed by microsomes prepared from strain IIIVO/J but does inhibit this reaction as catalyzed by microsomes from most New Zealand White rabbits. The antibody also inhibits the increased 16 alpha-hydroxylase activity of IIIVO/J microsomes observed in the presence of 5 beta-pregnane-3 beta,20 alpha-diol, an allosteric effector of this variant form of P-450 3b. Use of this monoclonal antibody provides a link between the observed properties of the purified, variant forms of P-450 3b and microsomal metabolism. These results indicate that the antibody can be used to phenotype variant forms of P-450 3b in microsomal fractions.

Animals↗

PubMed1984

Three monoclonal antibodies to rabbit microsomal cytochrome P-450 1 recognize distinct epitopes that are shared to different degrees among other electrophoretic types of cytochrome P-450.

Three monoclonal antibodies, 1F11, 1G11, and 2F5, were developed toward rabbit microsomal cytochrome P-450 1. Each was linked to Sepharose in order to indirectly immunoprecipitate microsomal proteins. All three antibodies bind polypeptides from solubilized microsomes that exhibit the same electrophoretic mobility as P-450 1. However, 2F5 recognizes an additional microsomal protein that exhibits a relative electrophoretic mobility between that of P-450 1 and 3b that does not appear to be P-450 2. 1G11 also reacts with several microsomal proteins that include both P-450 1 and P-450 3b. These results indicate that at least three electrophoretically distinct forms of microsomal P-450 share one or more antigenic determinants with P-450 1. Binding studies demonstrate that each antibody can react independently with P-450 1, indicating that the three antigenic determinants recognized by the respective monoclonal antibodies are spatially distinct and nonoverlapping. Reconstituted P-450 1 isolated from rabbit liver microsomes that exhibit 10-fold higher progesterone 21-hydroxylase than most preparations of liver microsomes obtained from outbred New Zealand White rabbits catalyzes this reaction with relatively high rates compared to that exhibited by microsomes or to that catalyzed by five electrophoretically distinct forms of P-450. Both the 1F11 and 2F5 antibodies extensively inhibit the liver microsomal 21-hydroxylation of progesterone. When the 1F11 antibody is used to indirectly immunoprecipitate the antigens it recognizes in microsomes displaying a high rate of progesterone 21-hydroxylase activity, a single electrophoretic band corresponding in mobility to P-450 1 was observed.

Animals↗

PubMed1984

Metabolic processing of 2-acetylaminofluorene by microsomes and six highly purified cytochrome P-450 forms from rabbit liver.

Kinetic analysis of oxidative metabolism of 2-acetylaminofluorene (AAF) was studied in control and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced microsomes and with six highly purified cytochrome P-450 isoenzymes from rabbit liver. Kinetic parameters were defined for 7-, 5-, 3-, 1- and N-hydroxylations of AAF. 7-Hydroxylation was best defined by a two enzyme system, displaying a high affinity and relatively low capacity and a low affinity high capacity components in both control and TCDD induced microsomes. All the purified cytochrome P-450 isoenzymes were capable of catalyzing the 7-hydroxylation of AAF and, with the exception of form 4, this was the only oxidation on the AAF molecule catalyzed by these forms. It is probable that forms 1, 4 and 6 accounted for a substantial part (greater than or equal to 25%) of total metabolic capacity corresponding to the high affinity component of 7-hydroxylation, whereas forms 3b and 3c accounted for less than 5% of the metabolic capacity displayed by the low affinity component in control microsomes. However, forms 4 and 6 could account for greater than 90% of the metabolic capacity of the high affinity component of 7-hydroxylation in TCDD microsomes, whereas the form(s) responsible for the metabolic capacity of the low affinity component were not identified. Each of the 1-, 3-, 5- and N-hydroxylations were best defined by a single enzyme system in both control and TCDD microsomes (3- and 5-hydroxylations could not be defined in TCDD microsomes). Close agreements were found between the apparent Km for N-hydroxylation in control, TCDD induced microsomes and with form 4. alpha-Naphthoflavone inhibited AAF N-hydroxylation to a similar extent in control and TCDD microsomes and in form 4. These date indicate that: a subpopulation of cytochrome P-450 isoenzymes, which includes all the purified P-450 forms tested in the present study, is solely involved in detoxification (i.e., 7-hydroxylation) of AAF, and as such probably behave as a functional unit in vivo; modulation of cytochrome P-450 content by inducers such as TCDD results in emergence of relatively few cytochrome P-450 isoenzymes that can account for most of the oxidative metabolism of AAF; and a single cytochrome P-450 isoenzyme (i.e., form 4) is responsible for catalyzing N-hydroxylation of AAF, the first and the obligatory step in the metabolic activation of this carcinogen.

2-Acetylaminofluorene↗