The cytochromes in microsomal fractions of germinating mung beans.

Detailed studies of microsomal cytochromes from mung-bean radicles showed the presence of cytochrome P-420, particularly in dark-grown seedlings, accompanied by smaller quantities of cytochrome P-450. Similar proportions of cytochrome P-420 to cytochrome P-450 were found spectrophotometrically in vivo with whole radicles and hypocotyls. Assayed in vitro, maximum concentrations of both cytochromes were attained after 4 days of growth, before undergoing rapid degradation. Illumination of seedlings stabilized cytochrome P-450 and decreased the amount of cytochrome P-420. Three b cytochromes were present in the microsomal fraction, namely cytochromes b-562.5 (Em + 105 +/- 23 mV), b-560.5 (Em + 49 +/- 13 mV) and b5 (Em - 45 +/- 14 mV), all at pH 7.0. Of the b cytochromes, cytochrome b5 alone undergoes a rapid degradation after day 4, Changes in cytochrome b concentrations were confined to the microsomal fraction: mitochondrial b cytochrome concentrations were unaltered with age. Protohaem degradation (of exogenous methaemalbumin) was detected in microsomal fractions of mung beans. The rates of degradation were highest in extracts of young tissue and declined after day 4. The degradation mechanism and products did not resemble those of mammalian haem oxygenase.     

Modulation in cytochrome P-420 and P-450 content in Saccharomyces cerevisiae according to physiological conditions and genetic background

The diploid strain D5 of Saccharomyces cerevisiae, relative to other strains of yeast, has a large amount of cytochrome P-450 present during the logarithmic phase of growth and a low amount of cytochrome P-420. As the stationary phase of growth is approached, an increasing intensity of absorbance is observed at 420 nm. If the cells are suspended in buffer during mid-logarithmic growth, the absorbance at 450 nm disappears and absorbance at 420 nm is increased after the cells have been held in buffer for 24 h. At late logarithmic growth, the absorbance at 450 nm is still retained after the cells have been held in buffer for 24 h. Within 44 h of the time of harvest, the absorbance at 450 nm disappears completely and the absorbance at 420 nm is intense. Cytoplasmic petite variants of strain D5 have less of both cytochromes P-450 and P-420 than does the grande D5 strain; the absorbance at 450 and 420 nm are retained up to 96 h when the cells are held in buffer. Haploid spores of strain D5 exhibit absorbances at 450 and 420 nm during the logarithmic phase of growth, and these absorbances are retained after the cells are held in buffer for 24 h.

An hypothesis is proposed which states that cytochrome P-450 is the membrane-bound form and cytochrome P-420 is free in the cytosol; the cytochromes interconvert and are active in either state until the associated enzymes disassociate.     

High pressure induced inactivation of ferrous cytochrome P-450 LM2 (IIB4) CO complex: evidence for the presence of two conformers in the oligomer.

The effect of high pressure on the spectral properties of cytochrome P-450 LM2(Fe2+)-CO complex was studied. The application of high pressure was shown to induce the conversion of cytochrome P-450 to P-420. In the solution when P-450 was oligomeric only about 65% of the total converted to P-420. The remaining portion of cytochrome P-450 was stable at pressures up to 6 kbar. When P-450 was incorporated into membranes or when it was succinylated, the proportion of the pressure sensitive fraction was slightly higher (about 75%). Dissociation of P-450 oligomers into monomers was made by addition of 0.2% Triton N-101. Monomers were the most sensitive to pressure; they could be completely converted to P-420. These results have been interpreted as evidence for the existence of two different conformers of P-450 LM2, which differ in pressure stability. Splitting between these two states appears to be a result of the oligomeric organization of cytochrome P-450 in solution and in the membrane.     

Rat liver cytochrome P-450b, P-420b, and P-420c are degraded to biliverdin by heme oxygenase

In this report we provide data, for the first time, demonstrating the conversion of the heme moiety of certain cytochrome P-450 and P-420 preparations, to biliverdin, catalyzed by heme oxygenase. We have used purified preparations of cytochromes P-450c, P-450b, P-450/P-420c, or P-450/P-420b as substrates in a heme oxygenase assay system reconstituted with heme oxygenase isoforms, HO-2 or HO-1, NADPH-cytochrome c (P-450) reductase, biliverdin reductase, NADPH, and Emulgen 911. With cytochrome P-450b or P-450/P-420b preparations, a near quantitative conversion of degraded heme to bile pigments was observed. In the case of cytochrome P-450/P-420c approximately 70% of the degraded heme was accounted for as bilirubin but only cytochrome P-420c was appreciably degraded. The role of heme oxygenase in this reaction was supported by the following observations: (i) bilirubin formation was not observed when heme oxygenase was omitted from the assay system; (ii) the rate of degradation of the heme moiety was at least threefold greater with heme oxygenase and NADPH-cytochrome c (P-450) reductase than that observed with reductase alone; and (iii) the presence of Zn- or Sn-protoporphyrins (2 microM), known competitive inhibitors of heme oxygenase, resulted in 70-90% inhibition of bilirubin formation.     

Light-dependent cytochrome P-450 changes in mung beans (Phaseolus aureus).

Maximum concentrations of microsomal cytochrome P-450 are present in 3-4 day-old mung beans (Phaseolus aureus). On illumination of dark-grown seedlings, cytochrome P-450 and later cytochrome P-450 undergo a rapid decrease in concentration in vivo, with an apparent half-time of about 6 h. Conversely light-grown seedlings, transferred to darkness, show a slow accumulation of cytochrome P-450, doubling time of about 30 h, with a later accumulation of cytochrome P-420. Microsomal cytochromes b559, b560.5 and b562.5 do not significantly alter on light-dark transitions. Possible functions for dark-induced cytochrome P-450 are discussed.     

A study of the interaction of substrates with cytochrome P-450 by a method of UV- and 1H-NMR spectroscopy

Acceleration of substrate longitudinal relaxation (T1) was used to study cytochrome P-450-aminopyrine (1st type substrate) and P-450-4-methoxypyridine (2nd type substrate) complexes. Dissociation constant, T1 and/or residence time of substrate in the complex can be obtained from the dependence of T1 of substrate protons on substrate concentration. Basing on the relaxation times, distances between Fe3+ ion in the active site and protons of the substrate moiety were determined. For aminopyrine all the distances proved to be about 8 A. In the P-450-4-methoxypyridine complex the pyridine nitrogen is directed towards Fe3+ ion. Cytochrome P-450 is compared with its denatured form, cytochrome P-420, and metmyoglobin.     

In vitro effects of trans-4-hydroxy-2-alkenals on mouse liver cytochrome P-450

Under in vitro conditions, trans-4-hydroxy-2-hexenal (t-4HH), trans-4-hydroxy-2-nonenal (t-4-HN) and trans-2-hexenal (t-2H) significantly reduced the levels of mouse liver microsomal cytochrome P-450. Incubation of trans-4-hydroxy-alkenals, under anaerobic conditions in the absence of an NADPH-generating system indicated that these compounds were converting cytochrome P-450 to cytochrome P-420. Prior activation by the mixed function oxidase system was not required for trans-4-hydroxy-alkenals to alter cytochrome P-450 concentrations. trans-4-Hydroxy-alkenals and non-hydroxylated alpha,beta-unsaturated aldehydes may be exerting their effects on cytochrome P-450 by binding to sulfhydryl groups in a similar manner as reported for sulfhydryl reagents such as p-chloromercuriphenylsulfonic acid and p-chloromercuribenzoate.     

Induction of multiple cytochrome P-450 species in housefly microsomes--SDS-gel electrophoresis studies.

1. Microsomal fractions isolated from various housefly strains have been characterized with respect to multiple forms of cytochrome P-450 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 2. Susceptible NAIDM houseflies were pretreated with known inducers of cytochrome P-450, and their microsomal electrophoretic profiles were compared to control NAIDM microsomes, using as standards partially purified cytochrome P-450s from noninduced NAIDM houseflies. 3. Tentatively, at least five different species of cytochrome P-450 may exist in the NAIDM housefly strain. 4. A comparison of the microsomal electrophoretic profile of different housefly strains also indicates the presence of at least two additional cytochrome P-450 species. 5. Induction with alpha-pinene and phenobarbital was expressed by a shift of the maximum absorbance at 452 nm in the CO-difference spectrum to lower wavelengths in the NAIDM strain; whereas, beta-naphthoflavone, although increasing the amount of cytochrome P-450, did not change the wavelength of maximum absorbance. 6. Cytochromes of the P-452 type appear to predominate in the susceptible NAIDM strain, while cytochromes of the P-450 and P-448 types predominate in resistant strains.     

Studies on the maintenance of cytochromes P-450 and b5, monooxygenases and cytochrome reductases in primary cultures of rat hepatocytes

The cytochrome P-450 content of rat hepatocytes declined rapidly over 72 h in culture, due primarily to denaturation to cytochrome P-420. Six different media were investigated for their ability to conserve cytochrome P-450 during culture, and the most successful was a modified Earle's medium. After 72 h culture in this medium, cytochromes P-450 and b5, NADH-cytochrome b5- and NADPH-cytochrome c-reductases were maintained at 40, 100, 35 and 52% of fresh cell values, respectively. Cytochrome P-450 showed differential functional stability during culture with ethoxyresorufin O-deethylation being more stable than either pentoxyphenoxazone O-depentylation or biphenyl 4-hydroxylation. Monooxygenase than did cytochrome P-450 content. This discrepancy was not explained by loss of flavin nucleotides, FMN or FAD.     

The formation of cytochrome P-450 from cytochrome P-420 is promoted by spermine

This paper is concerned with camphor-bound bacterial cytochrome P-450 and processes that alter its spin-state equilibrium and influence its transition to the nonactive form, cytochrome P-420, as well as its renaturation to the native camphor-bound cytochrome P-450. Spermine, a polycation carrying a charge of 4 +, and potassium, a monovalent cation, were shown to differently cause an increase of high-spin content of camphor-bound cytochrome P-450. The spermine-induced spin transition saturates around 75% of the high spin; a further addition of KCl to the spermine-containing sample shifted the spin state to 95% of the high spin. The volume change of these spin transitions as measured by the use of high pressure indicated an excess of -40 mL/mol for the sample containing potassium as compared to that containing spermine. These results suggest that the proposed privileged site for potassium has not been occupied by spermine and that pressure forces both the camphor and the potassium ion from its sites, allowing solvent movement into the protein as well as ordering of solvent by the excluded camphor and potassium. Cytochrome P-420 was produced from cytochrome P-450 by hydrostatic pressure in the presence of potassium, spermine, and cysteine. Potassium cation shows a bigger effect on the stability of cytochrome P-450 than spermine or cysteine, as revealed by a higher value of the pressure of half-inactivation, P1/2, and a bigger inactivation volume change. However, potassium cation did not promote renaturation of cytochrome P-420 to cytochrome P-450 while the presence of spermine did.(ABSTRACT TRUNCATED AT 250 WORDS)     

B-type cytochromes in plasma membranes isolated from rat liver, in comparison with those of endomembranes

Fractions of plasma membranes, Golgi apparatus, endoplasmic reticulum (ER), and nuclear envelope were isolated from rat liver and were characterized by electron microsocpe and biochemical methods. The purity of the fractions was controlled by morphometry and by marker enzyme activities. Amounts of cytochromes b5, P-450, and P-420 were measured, as well as the NADPH- and NADPH-cytochrome c reductase activities. The pigments of the microsomal electron transport system were found in all membrane fractions in relatively high amounts, thus excluding an origin by microsomal contamination. Purified preparations of plasma membrane and Golgi apparatus contained approximately 30% of the cytochrome b5 and cytochrome P-450 + P-420 found in ER membranes. Plasma membranes were also characterized by a high ratio of P-420/450. Degradation of cytochromes P-450 and P-420 was relatively rapid in all fractions, except in the ER. Cytochrome b5 extracted from plasma membranes was spectrophotometrically and enzymatically indistinguishable from ER cytochrome b5. However, immunnlogical characterization with rabbit antibodies against the trypsin-resistant core of microsomal cytochrome b5 showed the presence of at least two types of cytochrome b5 in ER membranes, in contrast to the plasma membranes in which only one of these components was detected. This immunological differentiation also demonstrates that the plasma membrane-bound cytochrome b5 is endogenous to this membrane and does not reflect contamination by ER elements. We conclude that cytochromes b5, P-450, and P-420 are not confined only to ER and nuclear membranes but also occur in signficant amounts in Golgi apparatus and plasma membranes. The findings are discussed in relation to observations of similar redox components in Golgi apparatus, secretory vesicles, and plasma membranes of other cells.     

An anomaly in the resonance Raman spectra of cytochrome P-450cam in the ferrous high-spin state.

Resonance Raman spectra of cytochrome P-450cam (P-450cam) and its enzymatically inactive form (P-420) in various oxidation and spin states were measured for the first time. The Raman spectrum of reduced P-450cam was unusual in the sense that the "oxidation-state marker" appeared at an unexpectedly lower frequency (1346 cm-1) in comparison with those of other reduced hemoproteins (approximately 1355-approximately 1365 cm-1), whereas that of oxidized P-450cam was located at a normal frequency. This anomaly in the Raman spectrum of reduced P-450cam can be explained by assuming electron delocalization from the fifth ligand, presumably a thiolate anion, to the antibonding pi orbital of the porphyrin ring. The corresponding Raman line of reduced P-420 appeared at a normal frequency (1360 cm-1), suggesting a status change or replacement of the fifth ligand upon conversion from P-450cam to P-420. The Raman spectrum of reduced P-450cam-metyrapone complex was very similar to that of ferrous cytochrome b5.     

Partial purification and characterization of phenobarbital-induced house fly cytochrome P-450

Two forms of phenobarbital-induced cytochrome P-450 were partially purified from the Rutgers diazinon-resistant strain of house fly using cholate solubilization, polyethylene glycol 6000 precipitation, and chromatography on DEAE cellulose. The preparation of highest purity had an absorbance maximum of 452 nm, a specific content of 10.0 nmol/mg protein, and an apparent molecular weight of 60,000 when examined by sodium dodecyl sulfate polyacrylamide electrophoresis. The yield of the highly purified cytochrome P-450 was 2–3%. This form contained proportionately less cytochrome P-420 than the original cholate solubilized microsomes, and is thus apparently more stable. A second form of cytochrome P-450 having a specific content of 0.50–0.89 nmol/mg protein was eluted from DEAE cellulose with a 0-0.25 M salt gradient. This is consistent with a previously reported elution pattern for Emulgen 913-solubilized house fly microsomes. Several methods of solubilizing house fly microsomes were examined. High salt, 2M KCI, in the absence of detergents effectively solubilized cytochrome P-450 (50–70% recovery) with little or no conversion to cytochrome P-(420).     

The involvement of cytochrome P-450 in hepatic microsomal steroid hydroxylation reactions supported by sodium periodate, sodium chlorite, and organic hydroperoxides.

The mechanism of steroid hydroxylation in rat liver microsomes has been investigated by employing NaIO4, NaClO2, and various organic hydroperoxides as hydroxylating agents and comparing the reaction rates and steroid products formed with those of the NADPH-dependent reaction. Androstenedione, testosterone, progesterone, and 17beta-estradiol were found to act as good substrates. NaIO4 was by far the most effective hydroxylating agent followed by cumene hydroperoxide, NADPH, NaClO2, pregnenolone 17alpha-hydroperoxide, tert-butyl hydroperoxide, and linoleic acid hydroperoxide. Androstenedione was chosen as the model substrate for inducer and inhibitor studies. The steroid was converted to its respective 6beta-, 7alpha, 15-, and 16alpha-hydroxy derivatives when incubated with microsomal fractions fortified with hydroxylating agent. Evidence for cytochrome P-450 involvement in androstenedione hydroxylation included a marked inhibition by substrates and modifiers of cytochrome P-450 and by reagents which convert cytochrome P-450 to cytochrome P-420. The ratios of the steroid products varied according to the type of hydroxylating agent used and were also modified by in vivo phenobarbital pretreatment. It was suggested that multiple forms of cytochrome P-450 exhibiting different affinities for hydroxylating agent are responsible for these different ratios. Horse-radish peroxidase, catalase, and metmyoglobin could not catalyze androstenedione hydroxylation. Addition of NaIO4, NaClO2, cumene hydroperoxide and other organic hydroperoxides to microsomal suspensions resulted in the appearance of a transient spectral change in the difference spectrum characterized by a peak at about 440 nm and a trough at 420 nm. The efficiency of these oxidizing agents in promoting steroid hydroxylation in microsomes appeared to be related to their effectiveness in eliciting the spectral complex. Electron donors, substrates, and modifiers of cytochrome P-450 greatly diminished the magnitude of the spectral change. It is proposed that NaIO4, NaClO2, and organic hydroperoxides promote steroid hydroxylation by forming a transient ferryl ion (compound I) of cytochrome P-450 which may be the common intermediate hydroxylating species involved in hydroxylations catalyzed by cytochrome P-450.     

Inactivation of sodium dithionite reduced cytochromes P-450 of different origins

The inactivation of five dithionite reduced soluble cytochrome P-450 isoforms has been studied. The inactivation of microsomal rabbit liver isoform LM2 and bacterial linalool cytochrome P-450 is followed by its conversion into cytochrome P-420. Microsomal rabbit liver isoform LM4, bacterial camphor and p-cymene cytochromes P-450 were not inactivated under these conditions. The inactivation of linalool cytochrome P-450 and LM2 isoform is a first order reaction; the rate constants for linalool cytochrome P-450 and LM2 are 0.3 and 0.1 min-1, respectively. In the case of linalool cytochrome P-450 its carboxycomplex (Fe2+-CO) is inactivated, while in the case of LM2 the inactivation affects its oxycomplex (Fe2+-O2). The amino acid residues of linalool cytochrome P-450 are probably modified due to a direct electron transfer in its carboxycomplex. The amino acid residues of LM2 isoform are modified, presumably due to oxidation by oxygen active species which are released during the oxycomplex decay.     

The optical interconversion of the P-450 and P-420 forms of neuronal nitric oxide synthase: effects of sodium cholate, mercury chloride and urea

We investigated whether or not neuronal nitric oxide synthase (nNOS) (EC 1.14.13.39) was converted to the P-420 form on exposure to sodium cholate, mercury chloride or urea, and the reconversion of the P-420 to the P-450 form. Sodium cholate and mercury chloride induced the conversion of nNOS from the P-450 to the P-420 form in concentration- and incubation time-dependent manners, and the nNOS activity decreased. In the presence of glycerol, L-arginine and/or tetrahydrobiopterin, the sodium cholate-treated P-420 form could be reconverted to the P-450 form under constant experimental conditions, and the nNOS activity could also be restored. The mercury chloride-treated P-420 form of nNOS could be reconverted to the P-450 form on incubation with reduced glutathione (GSH) or L-cysteine, and the nNOS activity was recovered. However, no reconversion of the mercury chloride-treated P-420 form to the P-450 form was observed in the presence of glycerol, L-arginine, or tetrahydrobiopterin. Urea (4.0 M) dissociated nNOS into its subunits, but nNOS remained in the P-450 form. The nNOS monomer was more susceptible to sodium cholate. After removing the urea by dialysis, and supplementation of the nNOS solution with glycerol, L-arginine or BH(4), the P-420 was reconverted to the P-450 form, and the reassociation of nNOS monomers was also observed. These results suggested that nNOS was more stable as to exposure to sodium cholate, mercury chloride or urea in comparison to microsomal cytochrome P-450, which may be due to the different heme environment and protein structure.     

The microsomal metabolism of the organometallic derivatives of the group-IV elements, germanium, tin and lead.

The NADPH- and oxygen-dependent microsomal metabolism of the di-, tri- and tetra-ethyl-substituted derivatives of germanium, tin and lead was shown to give rise to ethylene as a major product and ethane as a minor product. These reactions were shown to be catalysed by the liver microsomal cytochrome P-450-dependent mono-oxygenase. Since formation of ethane and ethylene was differentially inhibited by anaerobiosis, the results suggest that at least a large portion of the ethane produced may be derived by a reductive mechanism. Triethyltin bromide in both the absence and presence of NADPH was shown to convert cytochrome P-450 into cytochrome P-420 and to affect the function of the mono-oxygenase in vitro. Tetraethyltin caused the NADPH- and time-dependent formation of cytochrome P-420, suggesting that tetraethyltin is converted into triethyltin salts in significant concentrations. The order of potency in formation of cytochrome P-420 was closely paralleled by the ability of the tin derivatives to induce microsomal lipid peroxidation in vitro.     

Interactions of microsomal cytochrome P-450 with spin-labeled substrate analogs

The iodine-containing stable iminoxyl radicals with various distances between the N-O-group and the iodine atom are proposed to be used to study the structure of the active center of the microsomal cytochrome P-450. The radicals used induce changes in the optical spectra of the Fe3+ ion located in the active center of the enzyme, as in the case of type 1 substrates and inhibit essentially the microsomal oxidation of cytochrome P-450 substrates of type 1 and 2. This inhibition is neither due to suppression of the NADPH-cytochrome c reductase activity nor to cytochrome P-450 conversion to cytochrome P-420. Cytochrome P-450 substrates (aminopyrine) protect the enzyme against the radical-induced inactivation. The iodine-containing radicals are covalently bound to cytochrome P-450 in the vicinity of active center. The values of dissociation constants for the reversible enzyme-radical constants and the rate constants for the monomolecular transformation in the complex, k, were determined. The EPR method was used to detect the coupling between Fe3+ and the radical located in the active center of cytochrome P-450. The saturation curves of radical SPR spectra at 77 degrees K were employed to determine the contribution of Fe3+ to the relaxation time, T1, of the radicals covalently bound to cytochrome P-450 and to estimate the distances between the Fe3+ ion and the N-O-group of these radicals in the enzyme active center.     

Spectral and catalytic properties of cytochrome P-450 from a diazinon-resistant housefly strain

Microsomes from the diazinon-resistant Rutgers strain of housefly contain amounts of cytochrome P-450 that are larger than those reported for rat liver, but the specific activity expressed as nmole of cytochrome P-450 per mg protein is much lower. The hemoprotein shows that spectral changes type I, II and IV are essentially in the low-spin form as judged by the n-octylamine and ethyl isocyanide difference spectra, and is unstable at pH below 6.5 and above 8.0. Cytochrome P-420 is also produced with time when CO-difference spectra are recorded. This is accelerated at pH above 8.0. The presence of contaminating amounts of cytochrome P-420, due to denaturation during spectral analysis or to the method used to isolate the microsomes, makes questionable the practice of characterizing the hemoprotein on the basis of the 455 nm peak in the ethyl isocyanide spectra, since a 434 nm peak is produced with concomitant decrease of the 455 nm peak. Microsomes hydroxylate naphthalene, aminopyrine and aniline, but the activity when expressed as nmole of product per nmole of cytochrome P-450 is the same or lower than that reported for other resistant housefly strains.     

Reconversion of detergent- and sulfhydryl reagent-produced P-420 to P-450 by polyols and glutathione

After treatment with detergents or sulfhydryl reagents, the cytochrome pigment P-420 can be converted back to P-450 by treatment with polyols or reduced glutathione. This reconversion of P-420 to P-450 is highly dependent on temperature, pH, concentration of sodium cholate and incubation time, but ionic strength was of little influence. However, after treatment with alcohols or ketones, P-420 could no longer be reconverted to P-450 by polyols and reduced glutathione.

The presence of 20% (v/v) glycerol stabilizes P-450 for more than 1 week.