Cytochrome P-450 of adrenal mitochondria. Spin states as detected by difference spectroscopy.

Adrenal mitochondrial cytochrome P-450 which functions in cholesterol side chain cleavage (P-450scc) exhibited type I (lambdamax 385, lambdamin 420 nm) and inverse type I (lambdamin 385, lambdamax 420 nm) difference spectra with several steroids. The magnitude and type of response were dependent on the particular steroid and on the extent to which cholesterol was bound to the cytochrome in the intact mitochondrion. the inverse type I difference spectrum induced by 3beta-hydroxy-pregn-5-ene-20-one (pregnenolone) was dependent on the proportion of high spin cholesterol-cytochrome P-450scc complexes. With rat adrenal mitochondria cholest-5-ene-3beta, 20alpha-diol (20alpha-hydroxycholesterol) invariably induced a smaller inverse type I response and, under conditions where cytochrome P-450scc was nearly free of cholesterol, even produced a small type I response. Two distinct steroid binding sites on cytochrome P-450scc were detected by, respectively, the slow type I response to cholest-5-ene-3beta, 25-diol (25-hydroxycholesterol) and the rapid type I response to a subsequent addition of cholest-5-ene-3beta, 20alpha, 22 R-triol (20alpha, 22R-dihydroxycholesterol). The relative proportions of the spectral responses to these steroids were dependent on the previous extent of adrenal activation by adrenocorticotropic hormone (ACTH), because this stimulatory process altered the combination of mitochondrial cholesterol with cytochrome P-450scc. It is proposed that the two steroid binding sites on cytochrome P-450scc interact with steroids in the following way: site I binds cholesterol, 25-hydroxycholesterol, and 20alpha, 22R-dihydroxycholesterol with formation of a partially high spin cytochrome; site II binds both pregnenolone and 20alpha-OH cholesterol resulting in a low spin cytochrome. Interactions between sites I and II are not competitive, and occupancy of site II ensures a low spin state irrespective of the occupancy of site I. A second mode of interaction by 20alpha, 22R-dihydroxycholesterol stabilizes a high spin cytochrome and is competitive with site II binding by 20alpha-hydroxycholesterol or pregnenolone. Formation of a maximally high spin cytochrome follows occupancy by 20alpha, 22R-dihydroxycholesterol at both sites.     

Hepatic microsomes from freshwater fish--I. In vitro cytochrome P-450 chemical interactions

1. Of 87 chemicals tested for their ability to interact with oxidized hepatic cytochrome P-450 from mature male brook trout (Salvelinus fontinalis), 21 formed detectable type I or type II binding spectra. 2. When 8 of these 21 chemicals were tested with cytochrome P-450 of nine other species of freshwater fish, wide species variation in hepatic microsomal cytochrome P-450 was evident, since the spectral size of chemical interactions as related to the carbon monoxide spectrum and the ratio of type II to type I binding were not alike. 3. These spectral data suggest that hepatic microsomal cytochrome P-450 of freshwater fish exists in different forms.     

A comparative study on the influence of cysteamine and metyrapone on mixed-function oxygenase activities in variously pretreated liver microsomes from rats and mice.

It has been found that metyrapone can inhibit both type I and type II mixed-function oxygenase reactions, while cysteamine inhibits only type I activity in this mammalian system. Following pretreatment with phenobarbital and 3-methylcholanthrene the half-maximal inhibiting concentrations for the O-demethylation of paranitranisol are increased for cysteamine and decreased for metyrapone. Both cysteamine and metyrapone give type II binding spectra with oxidized cytochrome P-450. The negative and positive peaks are at 393 and 426 nm respectively for metyrapone, and 410 and 434 nm for cysteamine. Cysteamine showed no binding comparable to that of metyrapone for reduced cytochrome P-450. Metyrapone showed little or no inhibition of the NADH cytochrome-c reductase (EC 1.6.1.1) or NADPH (EC 1.6.2.3) cytochrome-c reductase while cysteamine had a more or less strong inhibiting effect depending on the pretreatment of animals. Neither the binding to P-450 heme nor the inhibition of NADH and NADPH cytochrome-c reductase correlates well with cysteamine inhibition of total activity. It is therefore suggested that cysteamine reacts with an intermediate electron carrier of non-heme iron or glycoprotein character thus inhibiting mixed-function oxygenase activity.     

Identification of various hydrophobic zones in microsomal membranes containing cytochrome P-450 binding sites for type I and II substrates

Two different phospholipid hydrophobic zones in the membrane associated with cytochrome P-450 were identified. One of them exhibits selective sensitivity towards the protonated influence of carbonylcyanide-m-chlorophenyl hydrazone and possesses binding sites for type I substrates. The second one includes the heme group of cytochrome P-450 and herein the binding sites for type II substrates. Sodium deoxycholate (0.75-2.0 mM) disrupts both zones. Subsequent incorporation of fluorine atoms into a type II substrate (aniline) results in the increase in the hydrophobicity of aniline halogenated derivatives and in changes in the substrate-binding sites for tetra- and pentafluoroanilines from the heme-iron sphere to the apoenzyme of cytochrome P-450.     

A prostaglandin omega-hydroxylase cytochrome P-450 (P-450PG-omega) purified from lungs of pregnant rabbits

Cytochrome P-450-dependent prostaglandin omega-hydroxylation is induced over 100-fold during late gestation in rabbit pulmonary microsomes (Powell, W.S. (1978) J. Biol. Chem. 253, 6711-6716). Purification of cytochromes P-450 from lung microsomes of pregnant rabbits yielded three fractions. Two of these fractions correspond to rabbit lung P-450I (LM2) and P-450II (LM5), which together constitute 70-97% of total cytochrome P-450 in lung microsomes from nonpregnant rabbits. The third form, which we designate rabbit cytochrome P-450PG-omega, regioselectively hydroxylates prostaglandins at the omega-position in reconstituted systems with a turnover of 1-5 min-1. Titration with purified pig liver cytochrome b5, demonstrated a 4-fold maximum stimulation at a cytochrome b5 to a P-450 molar ratio of 1-2. Rabbit lung P-450PG-omega formed a typical type I binding spectrum upon the addition of prostaglandin E1 with a calculated K8 of 1 microM, which agreed reasonably well with the kinetically calculated Km of 3 microM. Cytochrome P-450PG-omega was isolated as a low-spin isozyme with a lambda max (450 nm) in the CO-difference spectrum distinguishable from P-450I (451 nm) and P-450II (449 nm). Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis demonstrated that although purified P-450PG-omega had a relatively low specific content (12.1 nmol mg-1), it appeared homogeneous with a calculated minimum Mr of 56,000, intermediate between rabbit LM4 and LM6. When lung microsomes from pregnant and nonpregnant rabbit were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a protein band, with a Mr identical to P-450PG-omega, was observed in the pregnant rabbit, whereas this band appeared to be very faint or absent in microsomes from the nonpregnant rabbit. Purification of cytochromes P-450 from nonpregnant rabbit lung yielded only P-450I and P-450II. P-450PG-omega appears to be a novel rabbit P-450, possessing high activity towards omega-hydroxylation of prostaglandins, and is greatly induced during pregnancy in rabbit lung.     

Cytochrome P-450 of adrenal mitochondria. In vitro and in vivo changes in spin states.

Steroid-induced difference spectra have been used to examine the combination of cholesterol with adrenal mitochondrial cytochrome P-450 which participates in cholesterol side chain cleavage (P-450scc) and the depletion of cholesterol from the cytochrome which results from turnover of the enzyme system. Type I difference spectra-induced by cholest-5-ene-3beta, 25-diol (25-hydroxycholesterol) and cholest-5-ene-3beta, 20 alpha, 22R-triol (20alpha, 22R dihydroxycholesterol) have been used to quantitate binding of cholesterol to two sites (I and II) on cytochrome P-450scc. The action of adrenocorticotropic hormone (ACTH) in vivo and the action of calcium or phosphate ions on isolated mitochondria stimulate the combination of cholesterol with site I but not site II. Cholesterol derived from lecithin-cholesterol micelles, however, binds to both sites. Malate-induced cholesterol depletion occurred at a comparable rate to the transfer of cholesterol from lecithin-cholesterol micelles. However, a residual proportion of cholesterol-cytochrome P-450scc complexes remained, even after 10 min of exposure to malate, and was of similar magnitude in mitochondria from both cycloheximide-treated and stressed rats. It is suggested that this reflects a less reactive form of cholesterol-cytochrome complex. Steroid-induced difference spectra indicate that sites I and II on cytochrome P-450scc are similarly depleted after metabolism of mitochondrial cholesterol in vitro and after inhibition of the action of ACTH in vivo. Anaerobiosis of adrenal cells after excision of the accumulation of cholesterol at cytochrome P-450cc. When anaerobiosis was prevented, cytochrome P-450scc in the freshly isolated mitochondria was apparently essentially free of complexed cholesterol, irrespective of the extent of ACTH action. For 30 min after suspension of the mitochondria in 0.25 M sucrose at 4 degrees, cholesterol combines with cytochrome P-450scc. The extent of this process was not affected by the presence of cycloheximide during ether stress treatment of the rats. It is concluded that there are at least two pools of mitochondrial cholesterol with access to cytochrome P-450scc but that ACTH stimulates only the pool which most readily interacts with the cytochrome.     

Role of phospholipids in reconstituted cytochrome P450 3A form and mechanism of their activation of catalytic activity.

Cytochrome P-450 coded for by the 3A gene family requires specific conditions in a reconstituted system, if its catalytic activity is to be efficient. We investigated the mechanism of activation of the catalytic activity of cytochrome P450 3A by phospholipids. Rat P450 PB-1 (3A2), human P450NF (3A4), and rabbit P450 3c (3A6) were used. They had low activity in a reconstituted system (system I) with dilauroylphosphatidylcholine (DLPC) but had high activity with a mixture of phospholipids (DLPC, dioleoylphosphatidylcholine, and phosphatidylserine) and sodium cholate (system II). P450 3A forms are cationic (having a high content of lysine residues) and needed the anionic phospholipid phosphatidylserine to have sufficient activity. Double-reciprocal plots of the metabolic rate of cytochrome P-450 versus the concentration of NADPH-cytochrome P-450 reductase showed that cytochrome P-450 and the reductase interacted more in system II than in system I. P450 PB-1 did not absorb at 450 nm in the presence of reductase, CO, DLPC, and NADPH, although other cytochrome P-450s absorbed at around 450 nm in such a mixture. However, P450 PB-1 was reduced in the presence of the phospholipid mixture and sodium cholate instead of DLPC. These results suggested that the stimulation of catalytic activity by phospholipids involved increased interaction between cytochrome P-450 and the reductase. Studies of proteolytic digestion and chemical cross-linking in systems I and II showed that a P450 3A form needed disaggregation of cytochrome P-450 and/or the reductase, not the formation of an aggregated complex necessary for the catalytic activity of other cytochrome P-450s.     

Hepatic cytochrome P-450-dependent monooxygenase systems of the trout, frog and snake--I. Components

1. Components of the hepatic monooxygenase systems (cytochrome P-450, cytochrome b5, NADPH cytochrome P-450- or c-reductase) of the brown trout (Salmo trutta), leopard frog (Rana pipiens) and garter snake (Thamnophis) were considerably lower than those found in the rat. 2. Reactivity of snake NADPH-cytochrome P-450-reductase with cytochrome P-450 was about twice that of the rat reductase; reactivities of trout and frog reductases were similar, but lower than that of the rat. The optimal temperature for the rat, frog and snake reductase activity was 37 degrees C, but 26 C for the trout reductase, regardless of whether cytochrome P-450 or cytochrome c was the electron acceptor for the reaction. 3. A type I substrate (benzphetamine) and a type II substrate (aniline) were less reactive with P-450 cytochrome from the trout, frog and snake than with P-450 cytochrome from the rat. 4. Qualitative differences were seen in the ethylisocyanide spectrum of microsomes from the rat, trout, frog and snake; these differences reflect qualitative differences in the populations of P-450 cytochromes among each of the four species.     

Selective loss of pulmonary cytochrome P-450I in rabbits pretreated with polychlorinated biphenyls

The polychlorinated biphenyls mixture, Aroclor 1254, generally considered a powerful inducer of rat hepatic and pulmonary microsomal monooxygenases, caused a 70% decrease in ethylmorphine N-demethylase activity, a 31% decrease in benzo(a)pyrene hydroxylase activity, and a 42% decrease in cytochrome P-450 content in rabbit lung microsomes. When pulmonary cytochrome P-450 was solubilized and subjected to column chromatography, the elution profiles of the two forms of the hemeprotein showed a marked decrease in cytochrome P-450I in treated rabbits, with no significant alteration in cytochrome P-450II content. These data were confirmed by subjecting the two cytochromes to gel electrophoresis and staining the electrophoretic bands for protein and heme-associated peroxidase activity. Cytochromes P-450I and P-450II isolated from Aroclor 1254-treated rabbits showed differences in spectral properties as well as in their stabilities. The CO difference spectral determinations showed absorbance maxima at 452 and 450 nm for cytochromes P-450I and P-450II, respectively. At room temperature, cytochrome P-450II was much more stable than P-450I. The present studies provide evidence not only for species differences in the biological actions of the polychlorinated biphenyls but also demonstrate differential effects of the environmental pollutant on the two major forms of cytochrome P-450 and associated enzymic activities in rabbit lungs.     

The rabbit pulmonary monooxygenase system. Immunochemical and biochemical characterization of enzyme components.

The enzymatic components of the rabbit pulmonary monooxygenase system, cytochromes P-450I and P-450II and NADPH-cytochrome P-450 reductase, are immunochemically distinct proteins. In pulmonary microsomes, the N-demethylation of benzphetamine, amino-pyrine, and ethylmorphine, and the O-deethylation of 7-ethoxycoumarin are dependent only on cytochrome P-450I, and the hydroxylation of coumarin is apparently catalyzed by both cytochromes. Cytochrome P-450II is immunochemically distinct from the major forms of hepatic cytochrome P-450 induced by phenobarbital or 3-methylcholanthrene, whereas cytochrome P-450I is indistinguishable from the former on the basis of physical and catalytic as well as immunochemical characteristics. Pulmonary and hepatic NADPH-cytochrome P-450 reductases also have identical physical, catalytic, and immunochemical properties. The lack of response of the lung monooxygenase system to phenobarbital, therefore, is apparently not due to an inability of the lung to synthesize the enzymes induced by phenobarbital in the liver. The relatively high proportion of cytochrome P-450I in the lung appears to be responsible for the higher rates (per nmol of P-450) of N-demethylation that have been observed in rabbit pulmonary as compared to hepatic microsomal fractions.     

Limitations on the metyrapone assay for the major phenobarbital inducible form of cytochrome P-450

Limitations on the determination of the concentration of the major phenobarbital inducible form of cytochrome P-450 (P-450b) in hepatic microsomes by the metyrapone assay of Luu-The $\text{et al.}$ (1) are reported. Compounds which bind to the Type I, II and IR binding sites, or convert cytochrome P-450 to P-420, decrease the apparent concentration of cytochrome P-450b by 20 to 100% in hepatic microsomes from untreated and pregnenolone-16α-carbonitrile or phenobarbital treated rats. It is calculated that errors of greater ca. 40% in the concentration of cytochrome P-450b can arise in the presence of appreciable quantities of the major pregnenolone-16α-carbonitrile or polycyclic hydrocarbon inducible forms of cytochrome P-450.     

Use of spin-labelled substrate analogs for a comparative study of microsomal cytochrome P-450 and P-448

The previously described, iodine-labeled alkylating stable nitroxyl radicals located at different distances between the N-O. group and the iodine atom were used for a comparative study of the structure of microsomal cytochromes P-450 and P-448 active centers. The radicals were shown to change the optical spectra of Fe3+ located in the active site of the enzyme that are similar to those induced by cytochrome P-450 substrates. Some differences in the type of the radicals binding to control, phenobarbital- and 3-methylcholanthrene-induced microsomes were revealed. The alkylating radical substrate analogs covalently bound to microsomal cytochrome P-450 in the vicinity of the active center, resulting in the inhibition of oxidation of type I and II substrates (e. g., aniline and naphthalene). The value of the spectral binding constant (Ks) for naphthalene in the presence of the radical covalently bound to the cytochrome P-450 active center showed a tendency to increase. Using the ESR technique, the interaction between Fe3+ and the radical localized in the active site of cytochrome P-450 was demonstrated. The contribution of Fe3+ to the relaxation of the radicals covalently bound to cytochrome P-450 was evaluated from the values of the spin label ESR spectra saturation curves at 77K. The distances between the N-O. group of these radicals and Fe3+ in the enzyme active center for the three types of microsomes were determined. The data obtained point to structural peculiarities of the active center of cytochrome P-450, depending on the microsomal type.     

Positive effectors of the binding of an active site-directed amino steroid to rabbit cytochrome P-450 3c

The binding of the amino steroid, 22-amino-23,24-bisnor-5-cholen-3 beta-ol (22-ABC), to rabbit liver cytochrome P-450 3c was studied using purified P-450 3c and liver microsomes prepared from rifampicin-treated B/J rabbits. 22-ABC binds to purified cytochrome P-450 3c producing a type II spectral change reflecting the coordination of the amine with the heme iron of the protein. In the absence of allosteric effectors, the binding is characterized by a Ks of 5 microM. In the presence of alpha-naphthoflavone or progesterone, the Ks decreases to 0.8 microM, indicating that these two compounds serve as positive effectors of the binding of 22-ABC to cytochrome P-450 3c. The antibiotic rifampicin induces cytochrome P-450 3c in rabbit liver microsomes, and the benzo(a)pyrene hydroxylase, estradiol 2-hydroxylase, and progesterone 6 beta-hydroxylase activities of these microsomes are stimulated by alpha-naphthoflavone. Moreover, the progesterone 6 beta-hydroxylase activity catalyzed by these microsomes exhibits a dependence on substrate concentration that is consistent with activation of the enzyme by the substrate, progesterone. The magnitude of the type II spectral change elicited by 22-ABC for microsomes prepared from rifampicin-treated B/J rabbits is greater than that observed for microsomes from untreated rabbits. For microsomes from rifampicin-treated rabbits, the apparent binding constant for 22-ABC was decreased 5-fold in the presence of alpha-naphthoflavone. We propose that the effects of alpha-naphthoflavone and progesterone on the binding of 22-ABC to cytochrome P-450 3c mimic the effects of the two positive effectors on the metabolism of substrates by increasing the affinity of the enzyme for substrate.     

Studies on the microsomal electron-transport system of anaerobically grown yeast. III. Spectral characterization of cytochrome P-450.

A carbon monoxide-binding pigment which shows an absorption peak at about 450 nm in the reduced carbon monoxide difference spectrum was purified from the microsomal fraction of yeast grown anaerobically. The spectral characteristics of the pigment were practically identical with those of cytochrome P-450 of hepatic microsomes, especially from polycyclic hydrocarbon-induced animals. The pigment was denatured to P-420, and bound with ethyl isocyanide in the reduced state. Although Type I spectral change was not evident, the pigment showed Type II and modified Type II spectral changes upon binding with some organic compounds, as in the case of hepatic cytochrome P-450. These observations clearly indicate that the carbon monoxide-binding pigment of yeast microsomes may be designated as cytochrome P-450 of yeast.     

Partial purification and properties of cytochromes P-450 and P-448 from rat liver microsomes

Cytochromes P-450 and P-448 in rat liver microsomes were solubilized with sodium cholate and were partially purified. The preparations contained 5.0–5.5 nmoles of cytochrome P-450 or P-448 per mg of protein; contamination with cytochrome P-420 and cytochrome b₅, was less than 10% of the total heme content. The absolute spectra of Cytochromes P-450 and P-448 differed only slightly; both hemoproteins had a Soret peak at 418–419 nm in the oxidized absolute spectra and at 448 and 450 nm in the reduced plus CO absolute spectra. Both hemoproteins showed typical type I (benzphetamine) and type II (aniline) binding spectra but differed in their binding of hexobarbital (another type I substrate). The total phospholipid content of the preparation (per mg protein) has been reduced by approximately 90% relative to microsomes and the hemoprotein has been purified 20–25 fold with respect to phospholipid. The partially purified hemoprotein fractions, after combination with a reductase and lipid fraction, were capable of oxidizing a variety of substrates inluding drugs, steroids, and chemical carcinogens.     

Purification and characterization of seven distinct forms of liver microsomal cytochrome P-450 from untreated and inducer-treated male Wistar rats

A total of nine forms of cytochrome P-450 were purified to homogeneity from liver microsomes of male Wistar rats. They were P-451 I and P-451 II from untreated rats, P-450 II and P-450 III from phenobarbital-treated rats, MC-P-448 L and MC-P-448 H from 3-methylcholanthrene-treated rats, and P-452, P-448 L, and P-448 H from 3,4,5,3',4'-pentachlorobiphenyl-treated rats. Among them, MC-P-448 L and MC-P-448 H were indistinguishable from P-448 L and P-448 H, respectively, with regard to electrophoretic, spectral, catalytic and immunochemical properties, and thus seven forms were distinct hemoproteins. The minimal molecular weight of each form was as follows: P-451 I (49,000), P-451 II (52,000), P-450 II (52,000), P-450 III (53,500), P-452 (48,000), P-448 L (56,000), P-448 H (54,000). Judging from the oxidized absolute spectra, P-448 H was a high-spin form and the others were of low-spin type. In a reconstituted system, N-demethylations of benzphetamine and aminopyrine were catalyzed by most of the forms at comparable rates. On the other hand, the activities for the oxidations of benzo[a]pyrene, 7-ethoxycoumarin, biphenyl, and estradiol-17 beta varied greatly among the forms of cytochrome P-450. The most efficient catalysts were as follows: P-448 L and P-451 II for benzo[a]pyrene 3-hydroxylation; P-448 L for 7-ethoxycoumarin O-deethylation; P-448 L, P-451 II, and P-448 H for biphenyl 4-hydroxylation; P-448 L and P-448 H for biphenyl 2-hydroxylation; and P-451 II and P-448 H for estradiol 2-hydroxylation. P-451 I, P-450 II, and P-450 III were somewhat poorer catalysts in metabolizing all the substrates except for benzphetamine and aminopyrine, but their substrate specificities were still distinguishable from one another. Of all the purified cytochrome P-450's, P-452 showed the least ability to metabolize all the substrates. Judging from the properties, it appears that six forms in male Wistar rats correspond to the distinct forms of cytochrome P-450 in Long-Evans and/or Sprague-Dawley rats reported by other workers, but P-451 I is a new constitutive isozyme in Wistar rats.     

Studies on the substrate-induced spectral change of cytochrome P-450 in liver microsomes.

The spectral changes of cytochrome P-450 caused by the addition of small molecules to liver microsomes were investigated precisely and the following conclusions were reached. 1. The Type I spectral change was entirely due to the interaction of the cytochrome with a hydrocarbon residue in a ligand. To induce the modified Type II spectral change, the presence of a hydroxyl group in a ligand was required. Compounds which contain a basic amino group induced the Type II spectral change. 2. The Type I spectral change was caused by the interaction of a ligand with the 419-nm form of cytochrome P-450, with its concomitant conversion to the 394-nm form. Whereas, compounds inducing modified Type II spectral change interacted with the 394nm form of the cytochrome. In this case, however, the 394-nm form was not converted back to the 419-nm form but was converted to a new state showing an absorption peak at 416 nm. The Type II spectral change-inducing interaction of a ligand with the cytochrome could occur with all forms of the cytochrome. 3. Both Type II and modified Type II compounds bound to the cytochrome at heme iron, and converted the cytochrome into modified ferrihemochromes. On the other hand, the Type I interaction occurred ina protein moiety of the cytochrome, and probably caused a conformational change of the cytochrome accompanied either by weakening of the internal ligand interaction or by displacement of the ligand with another one having a weaker field at the heme iron. 4. Type I and each of other two types of binding of compounds with cytochrome P-450 could occur simultaneously.     

Hepatic microsomes from freshwater fish--II. Reduction of benzo(a)pyrene metabolism by the fish anesthetics quinaldine sulfate and tricaine

1. A single in vivo exposure of brook trout (Salvelinus fontinalis) to a 30.0 mg/l solution of quinaldine sulfate or a 112.5 mg/l solution of tricaine for 5 min significantly reduced the in vitro hydroxylation of benzo(a)pyrene. 2. Since quinaldine sulfate and tricaine formed type I and II binding spectra, respectively, with brook trout hepatic cytochrome P-450, these chemicals probably reduced benzo(a)pyrene hydroxylase enzyme activity by altering the form(s) of cytochrome P-450 responsible for this activity. 3. Hepatic microsomal cytochrome P-450 from brook trout treated with tricaine for 5 min and then placed into fresh water for 24 hr had returned to control levels. 4. Caution should be exercised in the use of quinaldine sulfate or tricaine to anesthetize fish prior to analysis of hepatic microsomal mixed function oxidases.     

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces changes in the heme spin state of microsomal cytochrome P-450

In vitro studies on the nature of interaction of the neurotoxin MPTP with hepatic microsomal cytochrome P-450 were carried out. Spectral perturbation studies showed nitrogenous ligand type binding between MPTP and cytochrome P-450 with a peak at 423 nm and a broad trough at 400 nm. Scatchard analysis of MPTP-cytochrome P-450 binding suggested that MPTP binds to at least 2 species of cytochrome P-450--a high affinity binding species with an apparent spectral dissociation constant (Ks) of 372 microM and a low affinity species with Ks of 37.6 mM. EPR studies confirmed that MPTP is a type II substrate for the forms of cytochrome P-450 with which it interacts and causes a shift from the high spin state of cytochrome P-450 to the low spin state. MPTP is, thus, likely to be an effective inhibitor of cytochrome P-450.     

Cytochrome P-450cam and putidaredoxin interaction during electron transfer.

Cytochrome P-450cam, the bacterial hemeprotein which catalyzes the 5-exo-hydroxylation of d-camphor, requires two electrons to activate molecular oxygen for this monooxygenase reaction. These two electrons are transferred to cytochrome P-450cam in two one-electron steps by the physiological reductant, putidaredoxin. The present study of the kinetics of reduction of cytochrome P-450cam by reduced putidaredoxin has shown that the reaction obeys first order kinetics with a rate constant of 33 s-1 at 25 degrees C with respect to: 1) the appearance of the carbon monoxide complex of Fe(II) cytochrome P-450cam; 2) the disappearance of the 645 nm absorbance band of high-spin Fe(III) cytochrome P-450cam; and 3) the disappearance of the g = 1.94 EPR signal of reduced putidaredoxin. This data was interpreted as indicative of the rapid formation of a bimolecular complex between reduced putidaredoxin Fe(III) cytochrome P-450cam. The existence of the complex was first shown indirectly by kinetic analysis and secondly directly by electron paramagnetic resonance spectroscopic analysis of samples which were freeze-quenched approximately 16 ms after mixing. The direct evidence for complex formation was the loss of the EPR signal of Fe(III) cytochrome P-450cam upon formation of the complex while the EPR signal of reduced putidaredoxin decays with the same kinetics as the appearance of Fe(II) cytochrome P-450. The mechanism of the loss of the EPR signal of cytochrome P-450 upon formation of the complex is not apparent at this time but may involve a conformational change of cytochrome P-450cam following complex formation.