Separation of acetanilide and its hydroxylated metabolites and quantitative determination of "acetanilide 4-hydroxylase activity" by high-pressure liquid chromatography.

A simple and very sensitive method for the separation of 4-hydroxyacetanilide, 3-hydroxyacetanilide, 2-hydroxyacetanilide, and acetanilide was developed with the use of high-pressure liquid chromagraphy. Each of these phenolic derivatives can be separated completely from acetanilide and from one another. A simple assay for “acetanilide 4-hydroxylase activity” is thus described. The limit of sensitivity for cytochrome P-450-mediated acetanilide 4-hydroxylase activity is estimated to be 1.0 pmol/min/mg microsomal protein, thereby allowing this assay to be useful in detecting monooxygenase activity in “low level” nonhepatic tissues. Hepatic acetanilide 4-hydroxylase activity is induced about fourfold in $\text{C57BL}\text{6N}$ mice by 3-methylcholanthrene. Although acetanilide 2-hydroxylase activity is about seven times lower than the 4-hydroxylase activity, the 2-hydroxylase is also induced about three- or fourfold in $\text{C57BL}\text{6N}$ mice by 3-methylcholanthrene. The “2-hydroxylase activity” cannot, however, be strictly quantitated under the conditions described herein. The K_m values of both the 3-methylcholanthrene-induced and control 4-hydroxylase activity are about 0.55 mm; V_max values for 3-methylcholanthrene-treated and control mice, respectively, are 4.9 ± 1.1 and 1.1 ± 0.31 nmol/min/mg microsomal protein. The 4-hydroxylase in the liver of both 3-methylcholanthrene-treated and control mice appears to represent two or more catalytic activities, i.e., two or more forms of P-450 having widely differing affinities for the substrate acetanilide.     

Ring-andN-hydroxylation of 2-acetamidofluorene by rat liver reconstituted cytochrome P-450 enzyme system.

The N- and ring-hydroxylation of 2-acetamidofluorene were studied with a reconstituted cytochrome P-450 enzyme from microsomal fractions of liver from both control and 3-methylcholanthrene-pretreated rats. Proteinase treatment and Triton X-100 solubilization were two important steps for partial purification of the cytochrome P-450 fraction. Both cytochrome P-450 and NADPH-cytochrome c reductase fractions were required for optimum N- and ring-hydroxylation activity. Hydroxylation activity was determined by the source of cytochrome P-450 fraction; cytochrome P-450 fraction from pretreated animals was severalfold more active than the fraction from controls. Formation of N-hydroxylated metabolites with reconstituted systems from both control and pretreated animals was greater than that with their respective whole microsomal fractions.     

Metabolism of benzo[c]phenanthrene by rat liver microsomes and by a purified monooxygenase system reconstituted with different isozymes of cytochrome P-450

Metabolism of the environmental pollutant and weak carcinogen benzo[c]-phenanthrene (B[c]Ph) by rat liver microsomes and by a purified and reconstituted cytochrome P-450 system is examined. B[c]Ph proved to be one of the best polycyclic aromatic hydrocarbon substrates for rat liver microsomes. It is metabolized by microsomes from control rats and by rats treated with phenobarbital or 3-methylcholanthrene at 3.9, 4.2 and 7.8 nmol/nmol cytochrome P-450/min, respectively. Principal metabolites are dihydrodiols along with small amounts (less than 10%) of phenols. The K-region 5,6-dihydrodiol is the major metabolite and accounts for 77-89% of the total metabolites. The 3,4-dihydrodiol with a bay-region 1,2-double bond is formed in much smaller amounts and accounts for only 6-17% of the total metabolites, the highest percentage being formed by microsomes from control rats. Highly purified monooxygenase systems reconstituted with cytochrome P-450a, P-450b and P-450c and epoxide hydrolase form predominantly the 5,6-dihydrodiol (95-97% of total metabolites) and only a small percentage of the 3,4-dihydrodiol (3-5% of total metabolites). The 3,4-dihydrodiol is formed with higher enantiomeric purity by microsomes from 3-methylcholanthrene-treated rats (88%) than by microsomes from control rats (78%) or phenobarbital-treated rats (60%). In each case the (3R,4R)-enantiomer predominates. B[c]Ph 5,6-dihydrodiol formed by all three microsomal preparations is nearly racemic.     

Partial inhibition of hepatic microsomal aminopyrine N-demethylase by caffeine in partially purified cytochrome P450

Cytochrome P-450 substrate interactions were studied with cytochrome P-450 partially purified from livers of untreated, phenobarbital-treated, benzo[a]pyrene-treated and caffeine-treated rats. Partial inhibition of aminopyrine N-demethylase in presence of in vitro caffeine observed with intact microsomes was further investigated in a reconstituted system composed of partially purified cytochrome c reductase. Caffeine addition (in vitro) to partially purified cytochrome P-450 altered the hexobarbital, aniline and ethylisocyanide induced spectral change, and decreased NADPH oxidation in presence of substrates aminopyrine and acetanilide. NADPH oxidation was found to be increased in presence of aminopyrine and unaltered in presence of acetanilide in reconstituted system having partially purified cytochrome P-450 from caffeine-treated rats. Our studies suggest that caffeine acts as a true modifier of cytochrome P-450 and is possibly responsible for the formation of abortive complexes with aminopyrine.     

Microbial conversion of acetanilide to 2'-hydroxyacetanilide and 4'-hydroxyacetanilide

Approximately 700 cultures of various types were examined for their ability to hydroxylate acetanilide. The major product formed by unidentified Streptomyces species RJTS-539 was identified as 4′-hydroxyacetanilide (N-acetyl-p-aminophenol). This culture gave a peak yield of 405 mg per liter from 1,000 mg of acetanilide per liter. Considerably lower yields of 4′-hydroxyacetanilide were isolated from S. cinnamoneus NRRLB-1285. The major conversion product of acetanilide formed by Amanita muscaria F-6 was identified as 2′-hydroxyacetanilide, with a peak yield of 433 mg per liter from 1,000 mg per liter of substrate. A small amount of 4′-hydroxyacetanilide was also formed. Six other Streptomyces cultures formed small amounts of one or two products identical or similar to 2′-hydroxyacetanilide or 4′-hydroxyacetanilide as determined by thin-layer chromatography and ultraviolet spectra.     

Microbial Conversion of Acetanilide to 2′-Hydroxyacetanilide and 4′-Hydroxyacetanilide

Approximately 700 cultures of various types were examined for their ability to hydroxylate acetanilide. The major product formed by unidentified Streptomyces species RJTS-539 was identified as 4′-hydroxyacetanilide (N-acetyl-p-aminophenol). This culture gave a peak yield of 405 mg per liter from 1,000 mg of acetanilide per liter. Considerably lower yields of 4′-hydroxyacetanilide were isolated from S. cinnamoneus NRRLB-1285. The major conversion product of acetanilide formed by Amanita muscaria F-6 was identified as 2′-hydroxyacetanilide, with a peak yield of 433 mg per liter from 1,000 mg per liter of substrate. A small amount of 4′-hydroxyacetanilide was also formed. Six other Streptomyces cultures formed small amounts of one or two products identical or similar to 2′-hydroxyacetanilide or 4′-hydroxyacetanilide as determined by thin-layer chromatography and ultraviolet spectra.     

Dimethylnitrosamine demethylation by reconstituted liver microsomal cytochrome P-450 enzyme system.

Oxidative demethylation of dimethylnitosamine was studied with both reconstituted and unresolved liver microsomal cytochrome P-450 enzyme systems from rats and hamsters. Proteinase treatment of liver microsomal preparations yielded cytochrome P-450 particulate fractions. Both cytochrome P-450 and NADPH- cytochrome c reductase fractions were required for optimum demethylation activity. Particulate cytochrome P-450 fractions were more effecient than either Triton X-100- or cholatesolubilized preparations of these particles in demethylation activity with rat and hamster liver preparations appear to be due to differences in specificity in their cytochrome P-450 fractions.     

Stereoselective metabolism of the (+)-(S,S)- and (-)-(R,R)-enantiomers of trans-3,4-dihydroxy-3,4-dihydrobenzo[c]-phenanthrene by rat and mouse liver microsomes and by a purified and reconstituted cytochrome P-450 system

Metabolism of (+)-, (-)-, and (+/-)-trans-3,4-dihydroxy-3, 4-dihydrobenzo[c]phenanthrenes by liver microsomes from rats and mice and by a purified monooxygenase system reconstituted with cytochrome P-450c has been examined. Bay-region 3,4-diol 1,2-epoxides are minor metabolites of both enantiomers of the 3,4-dihydrodiol with liver microsomes from 3-methylcholanthrene-treated rats or with the reconstituted system (less than 10% of total metabolites). Microsomes from control and phenobarbital-treated rats and from control mice form higher percentages of these diol epoxides (13-36% of total metabolites). Microsomes from 3-methylcholanthrene-treated rats and cytochrome P-450c in the reconstituted system form exclusively the diol expoxide-1 diastereomer, in which the benzylic hydroxyl group and oxirane oxygen are cis to each other, from the (+)-(3S,4S)-dihydrodiol. The same enzymes selectively form the diol expoxide-2 diastereomer, with its oxirane oxygen and benzylic hydroxyl groups trans to each other, from the (-)-(3R,4R)-dihydrodiol (77% of the total diol epoxides). Liver microsomes from control rats show similar stereoselectivity whereas liver microsomes from phenobarbital-treated rats and from control mice are less stereoselective. Three bis-dihydrodiols and three phenolic dihydrodiols are also formed from the enantiomeric 3,4-dihydrodiols of benzo[c]phenanthrene. A single diastereomer of one of these bis-dihydrodiols with the newly introduced dihydrodiol group at the 7,8-position accounts for 79-88% of the total metabolites of the (-)-(3R,4R)-dihydrodiol formed by liver microsomes from 3-methylcholanthrene-treated rats or by the reconstituted system containing epoxide hydrolase. In contrast, the (+)-(3S,4S)-dihydrodiol is metabolized to two diastereomers of this bis-dihydrodiol, a third bis-dihydrodiol, and two phenolic dihydrodiols.     

Immunochemical characterization of some monooxygenase activities in liver microsomes from untreated and phenobarbital-treated rats

Two major forms of liver microsomal cytochrome P 450, one from untreated rats (P 450 A₂NI) and the other from phenobarbital-treated rats (P 450 B₂PB), were partially purified. Reconstitution of monooxygenase activities of purified enzymes and inhibition patterns of these activities by antibodies in microsomes gave the following results: 1) aniline hydroxylase activity is mainly supported by cytochrome P 450 A₂NI. This form is the major one in microsomes from control rats, but is also found at minute amounts in microsomes from phenobarbital-treated rats. It behaves as a constitutive form. 2) 4-nitroanisole-and benzphetamine-demethylase activities are mainly supported by cytochrome P 450 B₂PB which is predominant in phenobarbital-treated rats but is also present in control microsomes at low levels. 3) 4-nitroanisole-O-demethylase activity is less specific than benzphetamine-N-demethylase activity towards cytochrome P 450 B₂PB.     

ROLE OF RIBONUCLEASE ACTION IN PHENYLALANINE-INDUCED DISAGGREGATION OF RAT CEREBRAL POLYRIBOSOMES

—l -phenylalanine (1 mg/g body wt) or physiological saline (0.9% NaCl) was given intraperitoneally to infant (7-day old), immature (14-day old), and adult (42-day old) rats. The state of ribosomal aggregation was determined in the cerebral postmitochondrial supernatant and purified polyribosome fractions prepared in the presence of rat liver ribonuclease inhibitor. Polyribosomes isolated from cerebral cortices of infant and immature rats 30 or 60 min after administration of phenylalanine were partially disaggregated, whereas the state of aggregation of polyribosomes from mature cerebrum was unchanged. In contrast, little or no evidence of phenylalanine-induced polyribosome disruption was noted in the postmitochondrial supernatant fractions, from which the cerebral polyribosomes were prepared, in any of the animals. Omission of the ribonuclease inhibitor resulted in polyribosome disaggregation in the postmitochondrial supernatant fractions prepared from saline-treated as well as phenylalanine-treated infant rats, but the disruption was more profound in the latter group. Ribonuclease activities in cerebral postmitochondrial supernatant preparations from infant and immature rats were higher than the corresponding values in preparations from adult animals. In addition, the administration of phenylalanine resulted in increases in ribonuclease activities in cerebral postmitochondrial supernatant preparations from the younger animals, but had no effect on these activities in adult animals. These results suggest that alterations in structure and function of polyribosomes from the infant rat cerebrum following a loading dose of phenylalanine were related to exposure of the polyribosomes during isolation to elevated activities of cerebral ribonucleases resulting from this treatment. This hypothesis was supported by the finding that phenylalanine treatment had no effect on the incorporation in vivo of intracisternally-administered radioactive lysine into total, soluble or ribosomal protein of infant cerebrum. However, when cerebral ribosomal RNA was differentially labelled in phenylalanine-treated and saline-treated infant rats by the intracisternal administration of [³H] or [¹⁴C]uridine, and polyribosome fractions were then prepared from the pooled cerebral cortices of both groups, radioactive ribosomes derived from saline-treated rats were more highly aggregated than those derived from phenylalanine-treated animals. It is concluded that gross alterations in cerebral polyribosome structure and function do not occur in vivo in young rats given a large amount of phenylalanine intraperitoneally. However, this treatment, in addition to increasing ribonuclease activity in cerebral cell-free preparations, also sensitizes cerebral polyribosomes to subsequent breakdown upon exposure to ribonucleases during isolation.     

Formation of phenols from aromatic subtrates by plant and animal mono-oxygenases: The effect of adjacent deuteriums on the magnitude of the NIH shift of tritium

Hepatic metabolism of 4-[³H]acetanilide in vivo and in vitro yields 4-hydroxyacetanilide which retains, respectively, 40 and 62% of the tritium. When the 4-tritio-substrate contains adjacent deuteriums the retention of tritium is reduced to 26 and 40%. Hepatic metabolism of 4-[³H]anisole in vivo and in vitro yields 4-hydroxyanisole with 78% of the tritium. This retention is reduced to 62% in the corresponding 3,5-[²H₂]-4[³H]anisole. Similarly, the retention of tritium in trans-4-hydroxycinnamic acid derived by metabolism of trans-[4-³H]cinnamic acid with chick pea microsomes is reduced from 91% to 68% by the presence of adjacent deuteriums in the substrate. Hydroxylation at the 4-position does not result in selective loss of tritium from the 3-position of acetanilide, anisole, or cinnamic acid. The above isotope effects indicate that isomerization of the probable arene oxide intermediates proceeds mainly via the keto-tautomer of the phenolic product.     

Inactivation of cytochrome P450 and inhibition of ferrochelatase by analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine with 4-nonyl and 4-dodecyl substituents.

Cytochrome P450- and heme-destructive effects of the 4-nonyl and 4-dodecyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) were determined using hepatic microsomal preparations obtained from untreated, beta-naphthoflavone-treated, and phenobarbital-treated chick embryos. The 4-nonyl analogue of DDC was less efficacious than 4-ethyl DDC and 4-hexyl DDC, but more efficacious than 4-dodecyl DDC with respect to cytochrome P450-destructive activity. In all hepatic microsomal preparations, cytochrome P450 destruction by 4-nonyl DDC was accompanied by loss of microsomal heme. In contrast, 4-dodecyl DDC caused loss of heme only in hepatic microsomal preparations obtained from phenobarbital-treated chick embryos. The ability of 4-nonyl DDC and 4-dodecyl DDC to lower ferrochelatase activity was compared with that of 4-ethyl DDC and 4-hexyl DDC in cultured chick embryo hepatocytes. As the length of the 4-alkyl group was increased, the ferrochelatase-lowering efficacy and potency of the DDC analogue decreased. The 4-dodecyl DDC analogue was unable to lower ferrochelatase activity, which accorded with the finding that the administration of 4-dodecyl DDC to phenobarbital-treated rats did not lead to the accumulation of an N-alkylprotoporphyrin. The ability of 4-nonyl DDC to lower ferrochelatase activity was attributed to the formation of N-nonylprotoporphyrin IX following the administration of 4-nonyl DDC to phenobarbital-treated rats.     

Cleavage of nascent UDP glucuronosyltransferase from rat liver by dog pancreatic microsomes

Antibody to mouse UDP glucuronosyltransferase, previously shown to cross-react with rat transferase [1], immunoadsorbed 3 electrophoretically distinct transferase forms from the microsomes of untreated and phenobarbital-treated rats and 4 forms from 3-methylcholanthrene treated animals. The forms from phenobarbital-treated or control animals ranged in molecular weights from 49,000 to 52,000 daltons, and those from 3-methylcholanthrene-treated rats ranged from 51,000 to 57,000 daltons. The intensity of the electrophoretic bands indicated that the levels of at least two forms were increased by the administration of either compound.In contrast, only a 52,000-dalton electrophoretic band was observed after immunoadsorption of in vitro translated products using poly(A) RNA isolated from either control, phenobarbital-, or 3-methylcholanthrene-treated rats. When dog pancreatic microsomes were included in the in vitro translation assay for either of the poly(A) RNA preparations, part of the 52,000-dalton band remained and a new band of about 50,000 daltons was generated. This processed transferase form(s) appeared to be inserted into or sequestered by the microsomes. These results indicate that some of the electrophoretic variants of rat liver transferase arise by posttranslational modifications and that at least one rat transferase form undergoes proteolytic cleavage of an approximate 2,000-dalton peptide fragment during insertion into the membrane.     

7 Alpha-hydroxylation of cholesterol by reconstituted systems from rat liver microsomes

A reconstituted system from rat liver microsomes, consisting of partially purified fractions of cytochrome P-450 and NADPH-cytochrome P-450 reductase was shown to catalyze 7α-hydroxylation of cholesterol in the presence of NADPH and a synthetic phosphatidylcholine. The rate of 7α-hydroxylation of added [4-¹⁴C] cholesterol was linear with the concentration of cytochrome P-450 and increased with the concentration of NADPH-cytochrome P-450 reductase up to a certain level and then remained constant. Omission of phosphatidylcholine resulted only in a 20% decrease in cholesterol 7α-hydroxylase activity of the system. The rate of 7α-hydroxylation was 2–3 times higher in reconstituted systems with cytochrome P-450 from cholestyramine-treated rats than in those with cytochrome P-450 from untreated rats.     

Aldrin epoxidation catalyzed by purified rat-liver cytochromes P-450 and P-448. High selectivity for cytochrome P-450

Aldrin epoxidation was studied in monooxygenase systems reconstituted from purified rat liver microsomal cytochrome P-450 or P-448, NADPH-cytochrome c reductase, dilauroylphosphatidylcholine and sodium cholate. Cytochrome P-450, purified from hepatic microsomes of phenobarbital-treated rats, exhibited a high rate of dieldrin formation. The low enzyme activity observed in the absence of the lipid and sodium cholate was increased threefold by addition of dilauroylphosphatidylcholine and was further stimulated twofold by addition of sodium cholate. The apparent Km for aldrin in the complete system was 7 +/- 2 microM. SKF 525-A, at a concentration of 250 microM, inhibited aldrin epoxidation by 65%, whereas 7,8-benzoflavone had no inhibitory effect at concentrations up to 250 microM. Addition of ethanol markedly increased epoxidase activity. The increase was threefold in the presence of 5% ethanol. When cytochrome P-448 purified from hepatic microsomes of 3-methylcholanthrene-treated rats was used, a very low rate of epoxidation was observed which was less than 3% of the activity mediated by cytochrome P-450 under similar assay conditions. Enzyme activity was independent of the lipid factor dilauroylphosphatidylcholine. The apparent Km for aldrin was 27 +/- 7 microM. The modifiers of monooxygenase reactions, 7,8-benzoflavone, SKF 525-A and ethanol, inhibited the activity mediated by cytochrome P-448. The I50 was 0.05, 0.2 and 800 mM, respectively. These results indicate that aldrin is a highly selective substrate for cytochrome P-450 species present in microsomes of phenobarbital-treated animals and is a poor substrate for cytochrome P-448. The two forms of aldrin epoxidase can be characterised by their turnover number, their apparent Km and their sensitivity to modifiers, like 7,8-benzoflavone and ethanol.     

Aging-related decrease in hepatic cytochrome oxidase of the Fischer 344 rat

The effect of aging on the hepatic mitochondrial population has been determined using a rigorously defined group of Fischer 344 rats with known survivorship data. The age groups studied included mature adult controls (8.5 months; 100% survivorship), an intermediate aged group (17.5 months; 90% survivorship), and an aged group (29 months; 20% survivorship). Cytochrome oxidase activity and content were determined in homogenates and mitochondrial fractions. The mitochondrial fractions were characterized by determination of respiratory activity, and monoamine oxidase activity as well as evaluation of the polypeptide composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. The yield of protein in the isolated mitochondrial fraction as well as the mitochondrial specific content decreased significantly as a function of aging. Mitochondrial specific content was determined from the specific activities of cytochrome oxidase in the homogenate (per gram liver) and in the isolated mitochondrial fraction (per mg protein). Specific activity of hepatic cytochrome oxidase decreased approximately 15% (P = 0.035) in homogenates from the 17.5-month animals with a further, highly significant (P = 0.0002) decrease (29%) in the 29-month animals. In contrast, there was no statistically significant difference among the age groups in the cytochrome oxidase specific activity in the isolated hepatic mitochondrial fractions. However, the percentage of the total homogenate cytochrome oxidase activity recovered in the isolated mitochondrial fraction decreased significantly in the 29-month animals (P = 0.0063 vs the 8.5-month controls; P = 0.022 vs the 17.5-month group). Cytochrome aa₃ content of total liver homogenates from aged animals decreased (P = 0.00064) which is in agreement with the decline in cytochrome oxidase specific activity in this age group. In the mitochondrial fraction from the aged animals, cytochrome aa₃ content was essentially unchanged which is consistent with the lack of aging-related change in mitochondrial cytochrome oxidase specific activity. In freshly isolated mitochondrial fractions, no aging-related alterations were observed in respiratory control and $\text{ADP}\text{O}$ ratios. The addition of exogenous NADH and cytochrome c did not change significantly the respiratory rate of hepatic mitochondria from control or aged animals. These results demonstrate the integrity of freshly isolated mitochondrial preparations from both control and aged Fischer 344 rats. In addition, there was no aging-related alteration in either monoamine oxidase specific activity or polypeptide composition. The similarities observed in the specific activities of cytochrome oxidase and monoamine oxidase, as well as in the cytochrome aa₃ content and polypeptide composition of the isolated mitochondrial fraction, suggest a generalized decrease in hepatic mitochondrial content as a function of aging rather than a selective loss of mitochondrial components.     

A highly sensitive radiometric assay for zoxazolamine hydroxylation by liver microsomal cytochrome P-450 and P-448: properties of the membrane-bound and purified reconstituted system.

A radiometric assay for the in vitro metabolism of zoxazolamine has been developed which combines high sensitivity and rapid determination of product. [4,6-³H]zoxazolamine was metabolized to 6-hydroxyzoxazolamine, and the tritium released as ³H₂O was determined after treating the incubation mixture with activated charcoal. This treatment efficiently removes labeled substrate (99.98%), permitting enzymatically released tritium to be measured directly in the aqueous medium. Since the preponderant in vitro product of zoxazolamine metabolism by rat liver microsomes and the purified reconstituted mixed function oxidase system is 6-hydroxyzoxazolamine, and since this aryl hydroxylation occurs without significant NIH shift, the subsequent release of tritium from the 6-position accurately represents metabolism of the molecule. The use of [4,6-³H]zoxazolamine for a tritium release assay of mixed function oxidase activity is ideal since this compound shows no significant isotope effect or NIH shift during metabolic conversion to 6-hydroxyzoxazolamine. 3-Methylcholanthrene treatment of rats resulted in a fourfold induction of zoxazolamine hydroxylation while phenobarbital or pregnenolone 16α-carbonitrile pretreatment caused only a 20–50% increase in zoxazolamine metabolism. The use of a purified reconstituted system revealed that cytochrome P-448 from 3-methylcholanthrene-treated rats was approximately 10- to 15-fold more efficient than cytochrome P-450 from phenobarbital-treated rats in catalyzing the hydroxylation of zoxazolamine.     

HETEROGENEITY OF ROUGH-SURFACED LIVER MICROSOMAL MEMBRANES OF ADULT,PHENOBARBITAL-TREATED,AND NEWBORN RATS

Rough microsomes from the livers of adult, phenobarbital-treated, and newborn rats were subfractionated on a continuous sucrose gradient. Among the subfractions a marked heterogeneity in the distribution patterns of some enzyme activities appears. The isopycnic density of the various fractions in aqueous sucrose ranges from 1.17 to 1.25. The sedimentation coefficients (s₀) in 0.25 M sucrose lie between 0.4 x 10³ S and 1.2 x 10³ S. In adult animals, the NADH- and NADPH-cytochrome c reductase as well as the G6Pase activities are much higher in the slower sedimenting fractions than in the pellet. The increase in the level of G6Pase induced by fasting as well as the phenobarbital-induced changes are most prominent in the slowly sedimenting fractions. Three injections of phenobarbital have no effect on the specific NADPH-cytochrome c reductase activity in the pellet, but cause a significant increase of this enzyme activity in the light fractions. In the newborn animal, the NADH-ferricyanide reductase and NADPH-cytochrome c reductase activities are highest in the light fractions. On the other hand, the amount of cytochrome b ₅ is evenly distributed in all cases. Short-term incorporation of leucine-¹⁴C and glycerol-³H in vivo after phenobarbital treatment shows contrasting results, as the former is increased and the latter is decreased in the slowly sedimenting fractions. Leucine-¹⁴C incorporation into isolated, total membrane proteins is greater in both phenobarbital-treated and newborn animals than in untreated adults. The data support a multistep model for membrane biogenesis and indicate dynamic and individual behavior of the different parts of the rough-surfaced endoplasmic reticulum.     

Zwitterionic detergent mediated interaction of purified cytochrome P-450LM4 from 5,6-benzoflavone-treated rabbits with MADPH-cytochrome P-450 reductase

Hydroxylation of acetanilide catalyzed by purified cytochrome P-450LM4 and NADPH-cytochrome P-450 reductase was reconstituted with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The optimum rate of production of 4-hydroxyacetanilide was observed between 3 and 7 mM CHAPS and was about half that with 0.05 mM dilauroylglyceryl-3-phosphocholine (di-12-GPC). At higher detergent concentrations, hydroxylase activity decreased until at 15-20 mM CHAPS the system was inactive. The effect of CHAPS on the state of aggregation of P-450LM4 and on interaction between the cytochrome and P-450 reductase alone and under turnover conditions was investigated by ultracentrifugation. At 4 mM CHAPS, P-450LM4 was hexameric to heptameric (Mr 369,000). Neither reductase nor reductase plus acetanilide and NADPH altered the state of P-450LM4 aggregation, suggesting that a stable 1:1 P-450/reductase complex did not form under turnover conditions. Replacing CHAPS with 0.05 mM di-12-GPC resulted in formation of heterogeneous P-450 oligomers (Mr greater than 480,000). At CHAPS concentrations where substrate hydroxylation did not occur (15 and 22 mM), P-450LM4 was shown by sedimentation equilibrium measurements to be dimeric and monomeric, respectively. P-450 reductase was shown to reduce monomeric P-450LM4 in the presence of NADPH. Thus, the dependence of hydroxylase activity on [CHAPS] may be related to the state of aggregation of the cytochrome. An apparent correlation between P-450 aggregation state and NADPH-supported hydroxylation was also observed with phenobarbital-inducible P-450LM2 in the presence of detergents [Dean, W.L., & Gray, R.D. (1982) J. Biol. Chem. 257, 14679-14685; Wagner, S.L., Dean, W.L., & Gray, R.D. (1984) J. Biol. Chem. 259, 2390-2395].