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M J Coon

Publications and source records attributed to M J Coon.

At least 127 records · Page 7Linked to original sources
PubMed1982

Monoclonal antibodies to rabbit liver cytochrome P-450LM2 and cytochrome P-450LM4.

Monoclonal antibodies were prepared from hybridoma clones isolated by the fusion of myeloma cells and spleen cells derived from mice immunized with either purified rabbit liver microsomal cytochrome P-450LM2 or cytochrome P-450LM4. Seven hybridoma clones produced three kinds of monoclonal antibodies to P-450LM2. The first class bound, precipitated, and inhibited the enzyme activity of P-450LM2 for both benzo[a]pyrene hydroxylation and 7-ethoxycoumarin deethylation. The other two classes either bound and precipitated or only bound the enzyme. These monoclonal antibodies to P-450LM2 showed a precipitin reaction and inhibition of enzyme activity that was specific for cytochrome P-450LM2. Thus, they did not react with or inhibit the enzyme activity of the other isozyme cytochrome P-450LM4, Fraction 1 or Fraction 7. All of the monoclonal antibodies formed against P-450LM2 were mouse immunoglobulin (Ig) subclass IgG1. The most effective monoclonal antibody strongly inhibited the formation of oxygenated metabolites of benzo[a]pyrene at various positions as well as the deethylation of 7-ethoxycoumarin. Four hybridomas were isolated which produced monoclonal antibodies to P-450LM4. One of the four was of the IgM class and three were of the IgG1 type. The four monoclonal antibodies bound to P-450LM4 but did not precipitate the enzyme, and did not bind to P-450LM2. The monoclonal antibody P-450LM4 complexes interacted with protein A, and the enzyme activity for benzo[a]pyrene hydroxylation could be removed by centrifugation. The high specificity and monoclonality of these antibodies suggest their potential usefulness for studying the genetics, regulation, and roles of the different isozymes of P-450LM in drug and carcinogen metabolism.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1982

Molecular mechanisms of interactions between phospholipids and liver microsomal cytochrome P-450 LM2.

The interactions between cytochrome P-450 LM2, NADPH-dependent P-450 reductase and different phospholipids have been analysed in reconstituted systems utilizing spin equilibrium and the reduction reaction as sensitive probes. The results have been correlated with structural data derived from second derivative spectroscopic measurements. The data obtained indicate that phospholipids with acidic groups are of special importance in shifting the spin equilibrium to the high spin state. The detergent substitution of phospholipids, on the other hand, in the reduction reaction provides evidence that the capability to shift the spin state is not strictly correlated with the functional integrity of the system. The results imply that the phospholipids exert a linker function orienting the proteins within the bilayer into a proper interaction state. This function can be substituted by detergents. The phospholipid effect is specified by their head groups and increases with negative charges.

Animals↗
PubMed1981

Specificity in the activation and inhibition by flavonoids of benzo[a]pyrene hydroxylation by cytochrome P-450 isozymes from rabbit liver microsomes.

The effect of flavone and 7,8-benzoflavone on the metabolism of benzo[a]pyrene to fluorescent phenols by five cytochrome P-450 isozymes obtained from rabbit liver microsomes was determined. Benzo[a]pyrene metabolism was stimulated more than 5-fold by the addition of 600 microM flavone to a reconstituted monooxygenase system consisting of NADPH, cytochrome P-450 reductase, dilauroylphosphatidylcholine, and cytochrome P-450LM3c or cytochrome P-450LM4. In contrast, an inhibitory effect of flavone on benzo[a]pyrene metabolism was observed when cytochrome P-450LM2, cytochrome P-450LM3b, or cytochrome P-450LM6 was used in the reconstituted system. 7,8-Benzoflavone (50-100 microM) stimulated benzo[a]pyrene metabolism by the reconstituted monooxygenase system about 10-fold when cytochrome P-450LM3c was used, but benzo[a]pyrene hydroxylation was strongly inhibited when 7,8-benzoflavone was added to the cytochrome P-450LM6-dependent system. Smaller effects of 7,8-benzoflavone were observed on the metabolism of benzo[a]pyrene by the cytochrome P-450LM2-, cytochrome P-450LM3b-, and cytochrome P-450LM4-dependent monooxygenase systems. These results demonstrate that the activating and inhibiting effects of flavone and 7,8-benzoflavone on benzo[a]pyrene metabolism depend on the type of cytochrome P-450 used in the reconstituted monooxygenase system.

Animals↗
PubMed1981

Separate roles for FMN and FAD in catalysis by liver microsomal NADPH-cytochrome P-450 reductase.

Rat liver microsomal NADPH-cytochrome P-450 reductase was prepared free of detectable amounts of FMN by a new procedure based on the exchange of this flavin into apoflavodoxin. The resulting FMN-free reductase binds NADP in the oxidized state with the same affinity (Kd = 5 microM) and stoichiometry (1:1 molar ratio) as does the native enzyme. Both the native and FMN-free reductase catalyze rapid reduction of ferricyanide, but the ability to reduce th 5,6-benzoflavone-inducible form of the liver microsomal cytochrome P-450 (P-450LM4) is lost upon removal of FMN. The FMN-free enzyme was reconstituted with artificial flavins which, in the free state, have oxidation-reduction potentials ranging from -152 to -290 mV, including 5-carba-5-deaza-FMN and several FMN analogs with a halogen or sulfur substituent on the dimethylbenzene portion of the ring system. Enzyme reconstituted with 5-carba-5-deaza-FMN has catalytic properties which are not significantly different from those of the FMN-free reductase, and is unable to reduce P-450LM4. On the other hand, the ability to reduce P-450LM4 and the other FMN-dependent activities of the native reductase are restored by substitution of several other analogs for FMN, but the kinetics of P-450LM4 reduction, studied under anaerobic conditions by stopped flow spectrophotometry, are significantly altered. The oxidation-reduction behavior of enzyme reconstituted with 7-nor-7-Br-FMN is substantially different from that of the native enzyme, and less thermodynamic stabilization of the semiquinone is observed with this flavin analog. In contrast, the oxidation-reduction properties of enzyme containing 8-nor-8-mercapto-FMN are similar to those of the native enzyme, but the spectral properties are significantly different. As shown in a stopped flow experiment, reduction of this FMN analog precedes reduction of P-450LM4 when a complex of the flavoprotein and P-450LM4 is allowed to react with NADPH. Our experiments support a sequence of electron transfer in this enzyme system as follows: NADPH leads to FAD leads to FMN leads to P-450. We propose that the enzyme cycles between a le- and a 3e-reduced state during turnover and that electrons are donated to acceptors via the reaction, FMNH2 leads to FMNH ..

Animals↗
PubMed1980

Analysis of interactions among purified components of the liver microsomal cytochrome P-450-containing monooxygenase system by second derivative spectroscopy.

Second derivative spectroscopy together with the respective difference spectroscopy offers an effective methodical tool to resolve overlapping bands and shoulders into distinct bands at eliminated background absorption. The improved resolution allows attribution of the distinct bands to individual amino acid residues. Both methods have been utilized to analyze interactions between the three essential components of the liver microsomal cytochrome P-450-containing monooxygenase system. The improved resolution of the aromatic amino acid residues in the derivative spectra of cytochrome P-450LM2 and reductase allows one to determine that in the interactions of the essential components tyrosine residue(s) are involved. The participation of phenylalanine is likely and the participation of tryptophan residues is excluded. The pH-dependent decrease of the tyrosine absorption bands in the medium ultraviolet region with increasing pH is accompanied by a concurrent decrease of the heme absorption in the Soret region. Based on this concurrence, the existence of a heme-linked tyrosine as one of the axial heme iron ligands in cytochrome P-450 is postulated.

Amino Acids↗
PubMed1980

On the mechanism of action of cytochrome P-450. Spectral intermediates in the reaction of P-450LM2 with peroxy compounds.

In previous studies in this laboratory, highly purified liver microsomal cytochrome P-450 was shown to catalyze the hydroperoxide-dependent hydroxylation of a variety of substrates in the absence of NADPH, NADPH-cytochrome P-450 reductase, and molecular oxygen, and evidence was obtained that the oxygen atom in the product was derived from the peroxide. To determine whether the cytochrome functions in such reactions by a peroxidase-type mechanism, the kinetics of its interactions with a variety of substituted hydroperoxides and peroxy acids have been determined by stopped flow spectrophotometry. The reaction of P-450LM2 with various peroxy compounds yields an intermediate with an absorption maximum at about 436 nm in the difference spectrum, with pseudo-first order or biphasic kinetics depending upon the individual rate constants and the concentration of the oxidant used. The results are in accord with a reversible two-step mechanism, as follows: P-450 + oxidant in equilibrium C in equilibrium D, where C represents a transient intermediate which is detected spectrally only under certain conditions and is probably an enzyme . oxidant complex, and D is the complex with an absorption maximum at about 436 nm in the difference spectrum. The absolute and difference spectra of C and D vary in magnitude and in the positions of maxima and minima with the organic moiety of the peroxy compound used. Whereas the kinetics of the reaction with cumene hydroperoxide and benzyl hydroperoxide is unchanged in the pH range 5.0 to 9.0, that of perbenzoic acids decreases markedly at higher pH, thus indicating that only the unionized compound reacts with the enzyme. Experiments with a variety of substituted cumene hydroperoxides, benzyl hydroperoxides, and perbenzoic acids indicated that the first equilibrium is driven to the right by hydrophobic bonding of the oxidant to P-450LM2 and that the rate of conversion of C to D is increased by electron-withdrawing substituents in the oxidant and decreased by electron donating substituents. Following the formation of Complex D, irreversible heme destruction occurs slowly. These results indicate that the reaction of liver microsomal P-450 with peroxides differs in two important respects from that of typical peroxidases; the intermediates arising from the reaction of P-450LM2 with peroxy compounds are formed reversibly, and the spectra of these intermediates vary with structural differences in the peroxy compounds. Furthermore, the absence of a common isosbestic point in the spectra observed with P-450LM2 rules out the possibility that they represent mixtures of Compounds I and II as reported for peroxidases.

Animals↗
PubMed1980

On the possible relationship of cytochrome P-450 to alcohol metabolism: fundamental aspects of the microsomal hydroxylation system, including properties and interactions of the components.

Recent studies on the cytochrome P-450-containing enzyme system of liver microsomes have shown that the cytochrome is present as a number of distinct isozymes, one of which is induced in vivo by the administration of aromatic hydrocarbons, as well as several forms which are less well characterized and not known to be inducible. The various cytochromes exhibit partially selective but overlapping activities with a variety of substrates. Additional components are NADPH-cytochrome P-450 reductase, which binds to the cytochrome to form a tight 1:1 complex, phosphatidylcholine, and cytochrome b5, the role of which is still not clear. Recent evidence indicates that some of the components are mutually beneficial in favoring formation of a functional complex. For example, phosphatidylcholine enhances the binding of substrate and reductase to P-450LM2, reductase enhances the binding of phosphatidylcholine, and substrate (benzphetamine) facilitates the binding of reductase. The possible effect of ethanol on these interactions should be considered in evaluating the reported inhibition by high concentrations of ethanol of reactions catalyzed by liver microsomal cytochrome P-450.

Animals↗