Ouchterlony Double Diffusion Research Articles

Isolation of immunochemically distinct form of cytochrome P-450 from microsomes of tulip bulbs

A highly purified cytochrome P-450 was obtained from the microsomes of tulip bulbs (Tulipa gesneriana L.). The molecular weight (Mr=52,500) and amino acid composition of this plant cytochrome P-450 are similar to those reported for rat livers. On the contrary, Ouchterlony double diffusion analyses indicated that cytochrome P-450 isolated from tulip bulbs shares no common antigenic determinants with those of 9 other plants, in spite of the presence of comparable contents of cytochrome P-450 and/or trans-cinnamate 4-monooxy-genase with tulip bulbs.

Assessment of health effects from ozone exposure by using an integrated dosimetric biologic model

Pancreatic islets contained insulin-degrading activity that was completely removed by antisera to purified microsomal thiol-protein disulfide oxidoreductase from rat liver. In Ouchterlony double-diffusion experiments with these antisera, extracts of islet homogenates showed a single precipitation band of identity with the purified liver enzyme. Twodimensional immunoelectrophoresis also gave a single precipitate peak like that of the liver enzyme. The concentration of the enzyme in rat islets as determined by quantitation of the precipitates obtained in the electroimmunodiffusion analysis was in the order of 1.0% of total islet protein. The results suggest that, in vitro, cleavage of insulin into its polypeptide chains is catalyzed by the thiol-protein disulfide oxidoreductase. This enzyme promoting thiol-protein disulflde interchange may be important for regulating the content of pancreatic insulin.

Rotation of cytochrome P-450. Complex formation of cytochrome P-450 with NADPH-cytochrome P-450 reductase in liposomes demonstrated by combining protein rotation with antibody-induced cross-linking.

Purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase were co-reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles by a cholate dialysis technique. Rotational diffusion of cytochrome P-450 was measured by detecting the decay of absorption anisotropy r(t), after photolysis of the heme X CO complex by a vertically polarized laser flash. All cytochrome P-450 was found to be rotationally mobile when co-reconstituted with equimolar amounts of NADPH-cytochrome P-450 reductase in lipid to cytochrome P-450 ((L/P450)) = 1 (w/w] vesicles. Antibodies against NADPH-cytochrome P-450 reductase were raised. Their specificity was demonstrated by Ouchterlony double diffusion analysis. Antireductase Fab fragments were prepared from antireductase IgG by papain digestion. The N-demethylation of benzphetamine, catalyzed by the proteoliposomes, was significantly inhibited by antireductase IgG and by antireductase Fab fragments. Cross-linking of NADPH-cytochrome P-450 reductase by antireductase IgG resulted in complete immobilization of cytochrome P-450 in L/P450 = 1 vesicles. Antireductase IgG also immobilized cytochrome P-450 in L/P450 = 5 vesicles, although the degree of immobilization was slightly smaller. No immobilization of cytochrome P-450 in L/P450 = 1 vesicles was detected in the presence of antireductase Fab fragments or preimmune IgG. These results further support the proposal of the formation of monomolecular complexes between cytochrome P-450 and NADPH-cytochrome P-450 reductase in liposomal membranes (Gut, J., Richter, C., Cherry, R.J., Winterhalter, K.H., and Kawato, S. (1982) J. Biol. Chem. 257, 7030-7036).

Bacillopeptidase F: two forms of a glycoprotein serine protease from Bacillus subtilis 168.

Bacillopeptidase F is a serine endopeptidase excreted by Bacillus subtilis 168 after the end of exponential growth. As a step toward discovering a physiological function for this protease, an enzymological and immunological study was undertaken. When bacillopeptidase F was purified at pH 10, a number of enzymically active, rapidly moving electrophoretic forms were observed, as had been previously reported. Rabbit antiserum was prepared against one form. When the enzyme was purified at pH 6.0 in the presence of the covalent inhibitor phenylmethylsulfonyl fluoride, using the rabbit antiserum to detect the bacillopeptidase F protein, no fast-moving electrophoretic forms were observed. Instead, only two forms of the enzyme were isolated. One form had a molecular weight of 33,000, and the other had a molecular weight of 50,000, as determined by equilibrium sedimentation methods. Both forms appeared to be glycoproteins, both contained compounds, released on acid hydrolysis, which cochromatographed with phosphoserine and galactosamine, and the two gave identical immunoprecipitin lines in Ouchterlony double-diffusion tests. The smaller form had a pI of 4.4, whereas the larger had a pI of 5.4. The data suggest that bacillopeptidase F is distinct from all other proteases of B. subtilis.

Biosynthesis of Cholesterol Side-Chain Cleavage Cytochrome P-450 in Human Fetal Adrenal Cells in Culture*

An immunoglobulin G fraction from antiserum raised against cholesterol side-chain cleavage cytochrome P-450 (cytochrome P-450sec) purified from bovine adrenocortical mitochondria cross-reacted with human fetal adrenal cytochrome P-450sec in an Ouchterlony double diffusion system. This property of the immunoglobulin G fraction was used for the immunoisolation of cytochrome P-450sec of human fetal adrenal cells in culture. The molecular weight of the immunoisolate from [35S] methionine-labeled human fetal adrenal cells was similar to that of purified bovine adrenocortical cytochrome P-450sec (49,000). However the molecular weight of cytochrome P-450sec immuno-isolated from an in vitro translation system programmed by human fetal adrenal RNA was approximately 54,000, about 5,000 larger than that of the immunoisolate from radiolabeled cells. The rate of synthesis of cytochrome P-450sec by human fetal adrenal cells in culture, when incubated with ACTH (10(-6) M) for 36 h, was 2.5-fold greater than that by fetal adrenal cells incubated in the absence of ACTH. This result suggests that, as in the case of bovine adrenal cells, cytochrome P-450sec of human fetal adrenal tissue is synthesized as a larger molecular weight precursor which presumably undergoes processing to the mature form during translocation into the mitochondria. This synthesis is stimulated by ACTH, which is the putative trophic factor regulating human fetal adrenal steroidogenesis.

Electroimmunochemical quantification of UDP-glucuronosyltransferase in rat liver microsomes.

Microsomal UDPglucuronosyltransferase(1-naphthol), an enzyme form previously shown to be selectively inducible in rat liver by 3-methylcholanthrene-type inducers, was purified to apparent homogeneity. Rabbit antibodies against this enzyme form precipitated UDPglucuronosyltransferase activities towards 1-naphthol and 4-methylumbelliferone faster and to greater extents than enzyme activities towards bilirubin, oestrone and 4-hydroxybiphenyl. Ouchterlony double-diffusion analysis showed immunochemical similarity of the rat liver enzyme with the enzymes from other organs of the rat (kidney, testes) and the mouse liver but not with the enzyme from cat and human liver. Electroimmunochemical quantification of the enzyme indicated that its level was enhanced 1.3-fold and 2.5-fold in liver microsomes from phenobarbital-treated and 3-methylcholanthrene-treated rats, respectively. The results indicate that 3-methylcholanthrene treatment increases the enzyme level of rat liver microsomal UDPglucuronosyltransferase(1-naphthol). Despite phospholipid-dependence of its catalytic activity microsomal enzyme activity appears to be a good index of the enzyme level.

Characterization of periplasmic hydrogenase from Desulfovibrio vulgaris Miyazaki K.

Periplasmic hydrogenase [hydrogen:ferricytochrome c3 oxidoreductase, EC 1.12.2.1] from Desulfovibrio vulgaris Miyazaki K (MK) was purified to homogeneity. Its chemical and immunological properties were examined and compared with those of other Desulfovibrio hydrogenases. The pure enzyme showed a specific activity of 1,000 mumol H2 evolution min-1 (mg protein)-1. The enzyme had a molecular weight of 50,000 as estimated by gel filtration and consisted of a single polypeptide chain. The absorption spectrum of the enzyme was characteristic of an iron-sulfur protein and the extinction coefficients at 400 and 280 nm were 34 and 104 mM-1. cm-1, respectively. It contained 9.4 mol iron and 6.9 mol of acid-labile sulfide per mol. The amino acid composition of the preparation was very similar to the value reported for D. desulfuricans NRC 49001 hydrogenase. Rabbit antisera were prepared against the enzyme of D. vulgaris MK. Ouchterlony double diffusion and immunotitration tests of crude extracts from several strains of Desulfovibrio revealed that the enzyme from MK cells was immunologically identical with those from D. vulgaris Hildenborough and D. desulfuricans NRC 49001, but different from those from D. vulgaris Miyazaki F (MF) and Miyazaki Y, and D. desulfuricans Essex 6 strains. It is concluded that among Desulfovibrio hydrogenases, those from D. vulgaris MK, D. vulgaris Hildenborough and D. desulfuricans NRC 49001 form one group in terms of both subunit structure and antigenicity.

Purification and some properties of a non-o1 Vibrio cholerae enterotoxin that is identical to cholera enterotoxin.

Cholera-like enterotoxin was isolated and purified from the culture supernatant of a non-O1 strain of Vibrio cholerae, E8498, isolated from the environment. Enterotoxin was purified by aluminum hydroxide absorption and elution and successive gel filtrations on Sephadex G-100, Bio-Gel A-5m, and Sephadex G-75. Purified enterotoxin gave a single stained band on polyacrylamide gel disc electrophoresis, and the mobility was the same as that of cholera enterotoxin. The specific biological activity of the purified enterotoxin was almost the same as that of cholera enterotoxin in the Chinese hamster ovary cell assay, fluid accumulation in mouse ligated intestine, increase in vascular permeability in rabbit skin, and passive immune hemolysis. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis showed that the purified enterotoxin consisted of subunits A and B, identical to those of cholera enterotoxin, and Ouchterlony double gel diffusion tests indicated that the two toxins were immunologically identical. Enterotoxins prepared from several non-O1 strains isolated from human patients were also immunologically identical to cholera enterotoxin.

Physiological and pathological characteristics of Erwinia chrysanthemi isolates from potato tubers

Nine isolates of Erwinia chrysanthemi from rotting potato tubers were compared with six type or reference strains of this species. Phenotypic properties of the potato isolates closely agreed with those of Erw. chrysanthemi pv. zeae and with the characteristics proposed for Dickey's infrasubspecific subdivision IV (1979) and Samson & Nassan‐Agha's biovar 3 (1978), where Zea mays was among the most common host species. Pathogenicity tests on 20 ornamental and agricultural species showed only Cyclamen sp. and Z. mays to be susceptible. In Ouchterlony double diffusion tests, antisera to whole live cells of one potato strain reacted with four of the six pathovars of Erw. chrysanthemi. Tuber isolates did not produce blackleg symptoms in inoculated stems. The rationale of intensive pathogenicity testing is discussed.

A form of rabbit liver cytochrome P-450 that catalyzes the 7α-hydroxylation of cholesterol

A form of cytochrome P-450 which comigrates with cytochrome P-450 LM4 (molecular weight, 55000) on SDS-polyacrylamide gel was purified from liver microsomes of cholestyramine-treated rabbits. This form of cytochrome P-450 catalyzed the 7α-hydroxylation of cholesterol with an activity of 37.5 pmol/min per nmol cytochrome P-450 in the reconstituted enzyme system containing cytochrome P-450 and NADPH-cytochrome P-450 reductase. The substrate specificity of this form of cytochrome P-450 was compared with cytochrome P-450 LM4 isolated from phenobarbital- and β-naphthoflavone-treated rabbit liver microsomes. The latter two isoenzymes do not catalyze 7α-hydroxylation of cholesterol, but are more active in O-deethylation of 7-ethoxycoumarin and p-nitrophenetole. Ouchterlony double diffusion revealed cross-reactivity between anti- P-450 LM4 (phenobarbital) IgG and cytochrome P-450 isolated from cholestyramine- or β-naphthoflavone-treated rabbit liver microsomes. A two-dimensional iodinated tryptic peptide fingerprint indicated only minor structural differences among these three cytochrome P-450 LM4 preparations.

Immunochemical studies on cytochrome P-450 in adrenal microsomes

An antibody was prepared against electrophoretically homogeneous cytochrome P-450 C21 purified from bovine adrenal microsomes. This antibody was used to compare various cytochromes P-450 in bovine and guinea pig adrenal microsomes. In an Ouchterlony double diffusion test, a spur formation was observed between the precipitin lines of the purified bovine cytochrome P-450 C21 and guinea pig adrenal microsomes against anti-cytochrome P-450 C21 IgG. Anti-cytochrome P-450 C21 IgG inhibited 21-hydroxylation both of bovine and guinea pig adrenal microsomes but the inhibition was much more effective in the bovine microsomes than in the guinea pig microsomes. These results suggest that the 21-hydroxylase in the guinea pig microsomes has some molecular similarities to the bovine cytochrome P-450 C21 and a part of the antibodies cross-reacts with the 21-hydroxylase in the guinea pig microsomes. Anti-cytochrome P-450 C21 IgG did not inhibit the activities of 17α-hydroxylase and C 17,20-lyase in the bovine and guinea pig microsomes but stimulated these activities. This result shows that different species of cytochrome P-450 other than cytochrome P-450 C21 catalyzes the 17α-hydroxylation and C 17,20 bond cleavage. The stimulation of 17α-hydroxylation and C 17,20 bond cleavage by blocking 21-hydroxylation indicates that the electron transfer systems for various cytochromes P-450 are intimately linked in adrenal microsomes.

Molecular structure and subcellular localization of spinach leaf glycolate oxidase

Glycolate oxidase (E.C. 1.1.3.1) was purified from spinach leaves (Spinacia oleracea). The molecular weight of the native protein was determined by sucrose density gradient centrifugation to be 290,000 daltons (13S), whereas that of the monomeric form was 37,000 daltons. The quaternary structure of the holoenzyme is likely to be octameric, analogous to pumpkin cotyledon glycolate oxidase [Nishimura et al, 1982]. The subcellular localization of the enzyme was studied using linear sucrose density gradient centrifugation, and it was found that glycolate oxidase activity is detectable in both leaf peroxisomal and supernatant fractions, but not in chloroplasts and mitochondria; the activity distribution pattern is essentially similar to that for catalase, a known leaf peroxisomal enzyme. Ouchterlony double diffusion and immunotitration analyses, demontrated that the rabbit antiserum against purified spinach leaf glycolate oxidase cross-reacted, identically, with the enzyme molecules present in two different subcellular fractions, i.e, the leaf peroxisome and supernatant fractions. It is thus concluded that the enzyme present in the supernatant is due to the disruption of leaf peroxisomes during the isolation, and hence glycolate oxidase is exclusively localized in leaf peroxisomes in spinach leaves.

Molecular structure and subcellular localization of spinach leaf glycolate oxidase

Glycolate oxidase (E.C. 1.1.3.1) was purified from spinach leaves (Spinacia oleracea). The molecular weight of the native protein was determined by sucrose density gradient centrifugation to be 290,000 daltons (13S), whereas that of the monomeric form was 37,000 daltons. The quaternary structure of the holoenzyme is likely to be octameric, analogous to pumpkin cotyledon glycolate oxidase [Nishimura et al, 1982]. The subcellular localization of the enzyme was studied using linear sucrose density gradient centrifugation, and it was found that glycolate oxidase activity is detectable in both leaf peroxisomal and supernatant fractions, but not in chloroplasts and mitochondria; the activity distribution pattern is essentially similar to that for catalase, a known leaf peroxisomal enzyme. Ouchterlony double diffusion and immunotitration analyses, demonstrated that the rabbit antiserum against purified spinach leaf glycolate oxidase cross-reacted, identically, with the enzyme molecules present in two different subcellular fractions, i.e, the leaf peroxisome and supernatant fractions. It is thus concluded that the enzyme present in the supernatant is due to the disruption of leaf peroxisomes during the isolation, and hence glycolate oxidase is exclusively localized in leaf peroxisomes in spinach leaves.

Purification and characterization of pulmonary cytochrome P-450 from 3-methylcholanthrene-treated rats.

A major form of pulmonary cytochrome P-450 (pulmonary P-450MC) was purified approximately 313 fold from lung microsomes of 3-methylcholanthrene (MC)-treated rats. The purified preparation contained 12.5 nmol of cytochrome P-450 per mg protein and was essentially free from NADPH-cytochrome c-reductase, NADH-cytochrome b5-reductase, and cytochrome b5. By SDS-polyacrylamide gel electrophoresis, the molecular weight of pulmonary P-450MC was estimated to be 54,000, which is smaller than that of a major form of hepatic cytochrome P-450 (hepatic P-450MC) purified from MC-treated rats. The peptide patterns of the purified pulmonary P-450MC were apparently different from those of hepatic P-450MC on partial proteolysis with either S. aureus V8 protease or papain, indicating that the primary structure of pulmonary P-450MC is different from that of hepatic P-450MC. In reconstituted systems, pulmonary P-450MC efficiently catalyzed benzo(a)pyrene hydroxylation and ethoxycoumarin O-deethylation, but showed a low activity for benzphetamine N-demethylation. Pulmonary P-450MC formed a single precipitation line with the antibody prepared against hepatic P-450MC in Ouchterlony double diffusion analysis. The pulmonary and hepatic P-450MC activities of benzo(a)pyrene hydroxylation and ethoxycoumarin O-deethylation were inhibited in the same manner by the antibody against hepatic P-450MC. These results suggest that pulmonary P-450MC is immunologically very similar to hepatic P-450MC.

Preparation of homogeneous NADPH-cytochrome P-450 reductase from rat hepatoma

NADPH-cytochrome P-450 reductase was purified to homogeneity from hepatoma 5123t.c.(H) microsomes from phenobarbital and hydrocortisone-treated rats by detergent solubilization and affinity chromatography with an overall 8% recovery. The purified enzyme has a minimum subunit molecular weight of 79000 and contains one molecule each of FMN and FAD per 79000 molecular weight. The purified hepatoma cytochrome P-450 reductase catalyzes electron transfer to artificial electron acceptors with K m. values similar to those of purified liver reductase. The K m value of the hepatoma reductase for NADPH, 13 μM, is also similar to that of purified liver reductase. The tumor reductase appears immunochemically identical to liver reductase by Ouchterlony double-diffusion analysis and inhibition of activity. Peptide maps of the hepatoma and hepatic enzymes after proteolysis demonstrate the identity of the two proteins.

Development of hybridomas secreting monoclonal antibodies to the chicken intestinal 1 alpha,25-dihydroxyvitamin D3 receptor.

Four hybridomas that secrete monoclonal antibodies to the chicken intestinal cytoplasmic 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor have been obtained. Splenic lymphocytes, derived from two male Lewis rats expressing serum antireceptor activity after repeated immunization with a partially purified preparation of this protein, were fused with two nonsecreting murine myeloma cell lines, SP2/0-Ag14 and P3-X63-Ag8.653. Viable hybrids were screened for anti-chicken intestinal 1,25-(OH)2D3 receptor activity by incubation of hybrid media with receptor-hormone complex; this was followed by immunoprecipitation with rabbit anti-rat IgG. Of 1,724 hybridomas assayed by this technique, 4 were positive (2 derived from each animal) for the secretion of an antireceptor immunoglobulin molecule. After cloning by limiting dilution, the cell lines (designated SP2/0-4A5, P3-8C8, SP2/0-8D3, and SP2/0-9A7) were expanded into suspension culture. Antibody-induced alterations in the sedimentation pattern of the native 1,25-(OH)2D3 receptor, coupled with Ouchterlony double-diffusion techniques, indicate that SP2/0-4A5 secretes an IgG2a, SP2/0-9A7 produces an IgG2b, and P3-8C8 secretes an IgG. In contrast, SP2/0-8D3 was found to synthesize an IgM. The monoclonal antibodies react with both occupied and unoccupied chicken intestinal receptor and nuclear receptors, and they crossreact with 1,25-(OH)2D3 receptors from a wide variety of tissue and cultured cell types, including human 1,25-(OH)2D3 receptors. These immunological reagents should prove valuable in the elucidation of the molecular action of 1,25-(OH)2D3.

Three step purification of C1q by DNA precipitation, ion exchange and lectin affinity chromatography.

The difficulties associated with the isolation of pure C1q in sufficient amounts are reflected by the substantial number of isolation procedures, which are being published. The two major problems are a low yield and contaminating immunoglobulins. In addition, some isolation protocols appear to produce C1q contaminated with an inhibitor (C1q-INH). The present isolation protocol involves precipitation of C1q by DNA, chromatography using Sephadex QAE A 50 followed by Con A affinity chromatography. By this combination of purification steps maximal advantage was taken of the cationic properties and high carbohydrate content of the C1q molecule. The yield was 1-2 mg C1q per 100 ml serum. The isolated C1q was free of any demonstrable contaminants as demonstrated by Ouchterlony double diffusion and polyacrylamide gel electrophoresis.

Insulin-like activity of photochemically obtained monovalent monomeric concanavalin A in the presence of anti-concanavalin A antibodies: dependence on multivalency for stimulation of glucose oxidation of rat fat cells.

Insulin-like action of monovalent monomeric concanavalin A (m-Con A) was examined in rat adipocytes in the presence of anti-m-Con A antiserum. The antisera from rabbits injected with m-Con A reacted with not only monovalent monomeric but also tetravalent tetrameric concanavalin A (alpha-Con A) in Ouchterlony double diffusion analysis. m-Con A alone did not show any appreciable effect on glucose oxidation of adipocytes while it slightly inhibited glycerol release stimulated by epinephrine. In contrast, exposure of adipocytes to m-Con A in the presence of antibodies to m-Con A resulted in stimulation of glucose oxidation and inhibition of epinephrine-stimulated lipolysis. The stimulation and the inhibition with m-Con A in the presence of the antibodies were of the same degree as those with alpha-Con A. Both alpha- and m-Con A were slightly active in inhibiting 125I-labeled insulin binding. These results demonstrate that the ability of anti-m-Con A antiserum to aggregate m-Con A bound to receptors on the isolated-adipocyte plasma membrane allowed m-Con A to mimic the biological activity of insulin and that the aggregation of receptors for ligands other than insulin can induce insulin-like action in rat fat cells.

A search for trace expression of placental-like alkaline phosphatase in non-malignant human tissues: demonstration of its occurrence in lung, cervix, testis and thymus

A search for placental or placental-like human alkaline phosphatase (ALP) was made in human tissues. The tissue extracts were assayed for ALP before and after heating at 65°C for l h. Trace amounts of heat-stable ALP activity (> 0.01 IU/g) were found in lung, testis, cervix and thymus. The heat-stable ALP in these four tissues gave in Ouchterlony double diffusion plates lines of apparent identity with placental ALP when a rabbit anti-human placental antiserum was used. Inhibition studies with L-phenyl-alanine (Phe), L-homoarginine (Har), L-phenylalanylglycylglycine (Pgg), L-leucine (Leu) and levamisole (Leva), were carried out on the heat-stable ALP and on the total ALP. The heat-stable ALPs from cervix and lung gave [ I] 50 values with each inhibitor comparable to those of placental ALP. The heat-stable ALPs from testis and thymus gave [ I] 50 values for Leu and Pgg which were significantly different from the placental isoenzyme. Electrophoresis of heat-stable lung ALP from different individuals showed polymorphic differences similar to those seen with placental ALPs. Such differences were not seen with heat-stable testis ALP. We conclude that human non-malignant testis, cervix, lung and thymus tissues contain small amounts of placental or placental-like ALPs. The heat-stable ALPs in cervix and lung appear to have the same characteristics as placental ALP and are probably encoded by the same gene locus. The heat stable ALPs in testis and thymus, though immunologically very similar to placental ALP differ from it in inhibition profile and electrophoretically. The significance of the results in relation to the ‘ectopic’ expression of placental and placental-like ALPs in malignancy is discussed.

Comparison of cytochromes P-450 with high activity toward benzo[ a]pyrene purified from liver microsomes of β-naphthoflavone and 3-methylcholanthrene-pretreated rats

Cytochromes P-450 with high activity toward benzo[ a]pyrene were isolated from liver microsomes of rats treated with either β-naphthoflavone or 3-methylcholanthrene and examined for similarity using several physical and catalytic criteria. The β-naphthofla-vone-inducible cytochrome P-446 and the 3-methylcholanthrene-inducible cytochrome P-448 have the same subunit molecular weight (56,000 ± 1000) and electrophoretic mobility. Antibodies prepared to either form cross-react with each form without spurring in Ouchterlony double-diffusion experiments suggesting immunochemical identity. After proteolytic digestion with Staphylococcus aureus SV-8 protease and electrophoresis, both Cytochromes P-450 show the presence of the same bands. Both cytochromes have the same absorption maximum (446.5 ± 0.5 nm) in the CO-reduced absolute spectrum. The catalytic activity toward benzo[ a]pyrene of cytochrome P-446 is somewhat greater than that of cytochrome P-448. Thus, all the physical evidence suggests identity of the two cytochromes. The significance of the difference in catalytic activity remains to be defined.