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J B Schenkman

Publications and source records attributed to J B Schenkman.

At least 55 records · Page 3Linked to original sources
PubMed1987

Purification to homogeneity and enzymological characterization of a functional covalent complex composed of cytochromes P-450 isozyme 2 and b5 from rabbit liver.

A covalent complex between rabbit hepatic microsomal cytochromes P-450 isozyme 2 (LM2) and b5 was created and purified to greater than 95% homogeneity. The purified complex was largely comprised of the two cytochromes covalently attached at the interface of the functional electron transfer-effector complex as shown by the following evidence. The spin state of the LM2 within the complex was greater than the spin state of free LM2, and the addition of free cytochrome b5 (cyt b5) did not further increase the spin state of the LM2 within the complex. The spectral binding parameters (Kd and delta Amax) for the association of benzphetamine with LM2 in the complex were identical to those observed with free LM2 in the presence of saturating concentrations of free cyt b5 and much different from those observed for LM2 in the absence of cyt b5. Reconstituted monooxygenase activity of the covalent LM2-cyt b5 complex (LM2-cyt b5) in the presence of NADPH-cytochrome P-450 reductase was much higher than the activity of free LM2 and approached the activity of free LM2 in the presence of optimal concentrations of free cyt b5. Furthermore, the Km for the flavoprotein in supporting either free LM2 or LM2-cyt b5-dependent p-nitroanisole demethylation were similar. (iv) Less than 20-25% of the cyt b5 within the complex could be reduced by free NADH-cytochrome b5 reductase (NADH-cyt b5 reductase) albeit at a slow rate. The implications of this data to the current understanding of the mechanism and stoichiometry of protein interactions in the hepatic mixed function oxidase system are discussed.

Animals↗
PubMed1986

Differences in the spectral interactions between NADPH-cytochrome P-450 reductase and a series of cytochrome P-450 enzymes.

The interaction between NADPH-cytochrome P-450 reductase and a series of cytochrome P-450 isozymes was investigated using UV-visible spectrophotometry. In the absence of substrate the interactions between the reductase and RLM3, RLM5, and RLM5a were tight, exhibiting sub-micromolar dissociation constants and resulted in type I spectra of varying magnitude from which the following increases in the proportion of high spin hemoprotein were calculated; RLM3 (7%), RLM5 (36%), RLM5a (6%), LM2 (29%), RLM2 (0%). Preincubation of LM2 with its type I substrate benzphetamine increased the affinity of the cytochrome for the reductase. Using initial estimates of the P-450 spin states in the absence of reductase in conjunction with the spectral binding data and equations relating these parameters to the microequilibria for the association of reductase with high or low spin P-450, RLM3, RLM5, RLM5a and LM2 were shown to bind significantly more tightly to high spin P-450. The relevance of this data to the understanding of spin state influence on P-450 reduction is discussed.

Animals↗
PubMed1986

Evidence of binary complex formations between cytochrome P-450, cytochrome b5, and NADPH-cytochrome P-450 reductase of hepatic microsomes.

Water-soluble carbodiimide-catalyzed cross-linking of purified cytochrome P-450 LM2, cytochrome b5, and NADPH-cytochrome P-450 reductase was used to identify stable complexes formed between these proteins. High yields of P-450-b5 and P-450 reductase-b5 dimers, and lower yields of P-450 reductase-LM2 dimers were obtained. Substitution of native b5 and P-450 reductase with fully amidinated derivatives showed that LM2 and b5 were cross-linked exclusively through their respective amino and carboxyl groups. However, there appeared to be two complexation sites on the reductase which cross-link to b5 through amino groups and to LM2 through carboxyl groups respectively. A heterotrimer could not be identified following incubation of all three proteins together with EDC.

Animals↗
PubMed1986

Mechanism of interaction between cytochromes P-450 RLM5 and b5: evidence for an electrostatic mechanism involving cytochrome b5 heme propionate groups.

The role of cytochrome b5 heme propionate groups in the functional interactions between cytochromes P-450 RLM5 and b5 has been investigated by comparing the capacity of RLM5 to interact with both native b5 and a b5 derivative in which the native heme was replaced with ferric protoporphyrin IX dimethyl ester (DME-b5). Both forms of b5 interacted with RLM5 causing an increase in the RLM5 spin state from 28 to 68% high-spin RLM5 at saturation, as judged using uv-visible spectrophotometry. However, DME-b5 exhibited a 7-fold weaker affinity for RLM5. The apparent dissociation constant (Kd) for the interaction between RLM5 and b5 was also shown to be a strong function of ionic strength, in a manner consistent with the involvement of electrostatic attraction in complex formation. Reconstitution of b5 into an RLM5-dependent monooxygenase system stimulated the p-nitroanisole demethylase rate about 25-fold and 7-ethoxycoumarin deethylase about 6-fold. DME-b5, however, produced only 30% of the stimulation of RLM5-dependent turnover of p-nitroanisole observed at equivalent concentrations of native b5 without a change in Km. With 7-ethoxycoumarin, turnover was 50% diminished. The diminished capacity of DME-b5 to stimulate RLM5-dependent substrate turnover was shown not to be due to impairment of electron flow between NADPH-cytochrome P-450 reductase and DME-b5, since the Km of reductase for DME-b5 is 2.5-fold lower, and the Vmax is actually increased, but rather to an impairment of some aspect of functional interaction between the DME-b5 and RLM5. The data show that complex formation between cytochrome P-450 and b5 involves electrostatic attraction mediated in part by cytochrome b5 heme propionate groups.

Animals↗
PubMed1986

Differences in the mechanism of functional interaction between NADPH-cytochrome P-450 reductase and its redox partners.

Selective methylamidation of NADPH-cytochrome P-450 reductase (EC 1.6.2.4) carboxyl groups was used to assess the relative importance of these groups in the enzyme-catalyzed reduction of cytochromes c, b5, and P-450. Methylamidation of as few as 7 mol of carboxyl groups per mol of reductase caused 80% inhibition of cytochrome c reduction, 50% inhibition of rat liver microsomal RLM3 reduction, and up to 90% inhibition in the capacity of the reductase to support reconstituted monooxygenase activities of RLM3, RLM5, and LM2. In marked contrast, cytochrome b5 reduction measured under comparable conditions was stimulated by 50%. The impaired interactions between the reductase and cytochromes P-450 LM2 and RLM5 were shown not to arise from an impaired capacity for the proteins to bind each other but more likely to be due to an inhibition of a step(s) subsequent to complex formation between the oxidized proteins. These results show that the reductase interacts functionally with cytochrome c and cytochromes P-450 on the one hand and cytochrome b5 on the other through different mechanisms.

Animals↗
PubMed1985

Kinetics of cytochrome P-450 reduction: evidence for faster reduction of the high-spin ferric state.

Results are presented that support our hypothesis [Backes, W. L., Sligar, S. G., & Schenkman, J. B. (1980) Biochem. Biophys. Res. Commun. 97, 860-867] that the multiphasic reduction kinetics of cytochrome P-450 are, in part, due to the spin equilibrium of the ferric hemoprotein. The disappearance of the high-spin charge-transfer band at 650 nm during reduction of the hemoprotein by NADPH was fast, exhibiting a rate constant greater than that of the fast phase of reduction measured by formation of the carbon monoxide adduct. In contrast, the disappearance of the ferric low-spin form of the cytochrome was at a considerably slower rate. A mathematical expression of the fractional content of high-spin cytochrome P-450 was obtained by comparing the ratio of the initial rate of change in the fraction of total oxidized cytochrome remaining to the initial rate of change in the fraction of high-spin ferric P-450 remaining. Results supporting the model were obtained by using both microsomes and purified cytochrome P-450 RLM5. The calculation from experimental data yielded results that were similar to those obtained by different extrapolation methods used for estimation of the amount of high-spin cytochrome P-450, supporting further the proposed relationship between the spin equilibrium and the reduction kinetics of this hemoprotein.

Animals↗
PubMed1985

Purification and characterization of a new form (RLM2) of liver microsomal cytochrome P-450 from untreated rat.

A new cytochrome P-450 isozyme (RLM2) has been purified to electrophoretic homogeneity from liver microsomes of the untreated rat. It has an apparent minimum molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 49,000. Absolute spectrum of the oxidized form indicates that this isozyme is essentially all in the low spin state. The maximum of the reduced CO complex is at 449 nm. Amino-terminal partial amino acid sequence and amino acid composition are different from those of RLM3 and RLM5, two other native forms of cytochrome P-450 previously reported from this laboratory as well as other forms reported in the literature. RLM2 is capable of oxidizing a variety of drug substrates, like benzphetamine and aminopyrine, and to a lesser extent ethoxycoumarin. With the steroid substrate multiple isomeric products are formed differentially. Progesterone is preferentially hydroxylated at the 15-position (15 beta-hydroxylation (34%) and 15 alpha-hydroxylation (13%) of the total) and at the 6 beta-position (21%). The major metabolite when testosterone was the substrate, 15 alpha-hydroxytestosterone, comprised 43% of the total, while a modest amount of 6 beta-hydroxytestosterone (12%) is formed. Another major metabolite (31%) has yet to be unequivocally identified, but is suggested to be 7 beta-hydroxytestosterone. Examination of the substrate dependence of major and minor isomeric metabolites provides evidence for a single substrate-binding site on RLM2. Regardless of the position hydroxylated, a common Km value was obtained. It is suggested that differences in formation of the isomeric and epimeric products relate to differences in distance from the active oxygen center and the position of attack.

Amino Acid Sequence↗
PubMed1985

Chemical characterization of protein-protein interactions between cytochrome P-450 and cytochrome b5.

Native cytochrome b5 interacts with either RLM5 or LM2 to form tight equimolar complexes (Kd = 250 and 540 nM, respectively) in which the content of high spin cytochrome P-450 was substantially increased. Cytochrome b5 caused 3- and 7-fold increases in the binding affinities of RLM5 and LM2 for benzphetamine, respectively, and benzphetamine decreased the apparent Kd for cytochrome b5 binding. Upon formation of the ternary complex between cytochromes P-450, b5, and benzphetamine the percentage of cytochrome P-450 in the high spin state was increased from 28 to 74 (RLM5) and from 9 to 85 (LM2). Cytochrome b5 caused 13- and 7-fold increases in the rate of RLM5- and LM2-dependent p-nitroanisole demethylation, respectively. Amino-modified (ethyl acetimidate or acetic anhydride) cytochrome b5 produced results similar to those obtained above with native cytochrome b5. In contrast, modification of as few as 5 mol of carboxyl groups/mol of amidinated cytochrome b5 resulted in both a substantial loss of the spectrally observed interactions with either cytochrome P-450 LM2 or cytochrome P-450 RLM5, and in a loss of the cytochrome b5-mediated stimulation of p-nitroanisole demethylation catalyzed by either monooxygenase. In further studies, native and fully acetylated cytochromes b5 reoxidized carbonmonoxy ferrous LM2 at least 20 times faster than amidinated, carboxyl-modified cytochrome b5 derivatives. In contrast, amidination, or acetylation of amino groups, or amidination of amino groups plus methylamidination of the carboxyl groups did not appreciably slow the rate of reduction of the cytochrome b5 by NADPH-cytochrome P-450 reductase. Collectively, the results provide strong evidence for an essential role of cytochrome b5 carboxyl groups in functional interactions with RLM5 and LM2.

Animals↗
PubMed1985

Isolation and comparison of four cytochrome P-450 enzymes from phenobarbital-induced rat liver: three forms possessing identical NH2-terminal sequences.

Phenobarbital pretreatment of male rats induced four microsomal cytochrome P-450 enzymes, at least one of which has previously not been reported. Three of the induced forms of the cytochrome possess identical NH2-terminal amino acid sequence for the first 32 residues (PBRLM5, 6 and 7). This sequence is identical to that shown earlier for PB-4 and PB-5. Apparent values for minimum molecular weight on SDS-PAGE were 52,000 (PBRLM4), 53,000 (PBRLM5), 53,500 (PBRLM6) and 54,000 (PBRLM7). Isomeric metabolite patterns from testosterone and progesterone differed for each enzyme further indicating their unique natures. Studies reveal similarity of PBRLM4 to PB-1, of PBRLM5 to P-450b and PB-4, and PBRLM6 to P-450e and PB-5. PBRLM7, which does not correspond to any reported forms, metabolizes steroids poorly. It preferentially hydroxylates testosterone at the 16 beta-position. It is the largest and least active of the enzymes shown for all of the substrates tested. This study further provided a cautionary note against assuming that chromatographic pools, like a P-450 PB-B fraction, are homogeneous.

Amino Acid Sequence↗
PubMed1984

Reduction kinetics of purified rat liver cytochrome P-450. Evidence for a sequential reaction mechanism dependent on the hemoprotein spin state.

The anaerobic reduction kinetics of purified rat liver ferric cytochrome P-450 from phenobarbital-treated rat liver microsomes, reconstituted with saturating NADPH-cytochrome P-450 reductase, have been investigated and were shown not to be monophasic. From experiments correlating changes in the rate of fast-phase reduction with the spin state of the heme iron existing at preequilibrium, data were obtained consistent with a model for spin-state control of cytochrome P-450 reduction wherein the high-spin form of the hemoprotein is more rapidly reduced than the low-spin form. In addition, the temperature dependence of the reduction process in the presence of the substrate benzphetamine was studied. From the results obtained it is suggested that the endothermic nature of the low- to high-spin transition largely accounts for the apparent activation energy observed for the reduction of high-spin cytochrome P-450 being relatively temperature insensitive when compared to the rate constant for reduction of the membrane-bound form of the hemoprotein.

Anaerobiosis↗
PubMed1983

Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 and by microsomes. Metabolite identification.

Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 in a reconstituted system and by rat liver microsomes was examined. Eleven metabolites were detected. Two of these, found in spots 2 and 4 of a thin layer plate, were only formed by the rat liver microsomes and may represent reductive metabolites of testosterone. A number of monohydroxy metabolites were conclusively identified by gas chromatography-mass spectrometry. These include the 2-, 6 beta-, 7 alpha-, and 16 alpha-hydroxy isomers. Liver microsomes formed the 2 alpha- and 2 beta-epimers in a 1:2 ratio and both co-chromatographed with a third reduced metabolite in thin layer plate spot 4. In contrast with RLM5 about 90% of the 2-hydroxy isomer was the 2 alpha-epimer. RLM3 did not perform the 2-hydroxylation in detectable amounts. The 6 beta-isomer was a major metabolite of RLM3 and microsomes, but a minor product of metabolism by RLM5. In contrast, the 7 alpha-isomer was a minor metabolite of RLM3, was not formed by RLM5, and was a major microsomal metabolite. Hydroxylation at position 16 alpha was a major activity of RLM5 and the heterogeneous microsomal cytochromes, but with RLM3 it was a minor reaction. One new metabolite was found which appeared to be hydroxylated in the D-ring, had a mass spectrum different from both 16 alpha- and 16 beta-hydroxytestosterone, and was tentatively identified as a 15-hydroxy isomer. In agreement with the literature, androstene-3,17-dione was found to be an oxidative metabolite of testosterone by both microsomes and purified cytochrome P-450. It was a major metabolite of RLM5 but was not produced by RLM3. Studies with 18O2 and H218O conclusively show that oxidation of testosterone at C-17 does not involve transient incorporation of an oxygen atom in this position. A mechanism is suggested whereby cytochrome P-450 acts as a peroxidase in the formation of androstenedione.

Animals↗
PubMed1982

Relationship between malondialdehyde production and arachidonate consumption during NADPH-supported microsomal lipid peroxidation.

Fatty acid concentrations and malondialdehyde formation were determined before and during NADPH-supported lipid peroxidation in liver microsomes from rat, mouse, guinea pig and rabbit. In agreement with earlier reports, malondialdehyde production was greatest for rat, followed by mouse, and much less for guinea pig and rabbit. The microsomal content of total unsaturated fatty acids (18:1, 18:2, 18:3, 20:3, 20:4, 20:5, 22:5, 22:6) was approximately the same for rat and mouse and was lower in guinea pig and rabbit. Lipid peroxidation caused a time-dependent decrease in the polyunsaturated fatty acids, particularly 20:4 and 22:6, for all species. These decreases were most pronounced for rat and mouse. Alterations in the dietary regime for rat produced marked changes in microsomal fatty acid content as reported by others, and also caused changes in the rates of malondialdehyde production and polyunsaturated fatty acid consumption during lipid peroxidation. A comparison between the rates of malondialdehyde production and the rates of individual unsaturated fatty acid consumption was performed for each animal species and for rats fed different diets. A linear relationship was found between malondialdehyde production and 20:4 disappearance in individual microsomal preparations and in different species. A similar relationship was seen for the initial microsomal concentration of 20:4 and the initial rate of malondialdehyde formation. Other unsaturated fatty acids did not exhibit linear relationships. Various microsomal mixed-function oxidase variables were measured for the different species. No direct relationship between these values and malondialdehyde production was found.

Animals↗
PubMed1982

Kinetics of hepatic cytochrome P-450 reduction: correlation with spin state of the ferric heme.

The reduction kinetics of cytochrome P-450 are known to be biphasic, with a rapid initial phase and a slower subsequent phase, both of which appear linear in semilogarithmic plots. The present report demonstrates that these biphasic reduction kinetics can be described in terms of a preequilibrium between high- and low-spin ferric states. Computer simulations are used which express the rate and extent of the fast phase or burst as being due to the initial proportion of high-spin cytochrome P-450. According to this simplified sequential model, the slow phase of reduction is controlled by the rate of formation of high-spin cytochrome P-450. The substrate-induced alterations in the reduction kinetics are likewise consistent with the model, which indicates that type I compounds exert their effect by virtue of a decrease in the rate constant controlling the shift from high-spin to low-spin ferric cytochrome. The model is further supported by the influence of temperature on the spin equilibrium and reduction kinetics. Potential influences of other intermediate steps in the reduction and assumptions in the hypothesis are described.

Animals↗
PubMed1982

Purification and characterization of two constitutive forms of rat liver microsomal cytochrome P-450.

Two constitutive forms of cytochrome P-450 isozymes, designated RLM3 and RLM5, have been purified from untreated rat liver microsomes. RLM3 and RLM5 have minimum molecular weights of 50,000 and 51,000, respectively, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Absolute oxidized spectra indicate that RLM3 is essentially all in the low spin state, whereas RLM5 contains some high spin component. The CO-reduced difference spectral maximum of RLM3 is at 449 nm and RLM5 is at 451 nm. RLM3 preferentially hydroxylates testosterone at 6 beta and 7 alpha positions and forms an even higher amount of an as yet unidentified polar metabolite, whereas RLM5 hydroxylates testosterone preferentially at 2 beta and 16 alpha positions, and exhibits 6 beta-hydroxylase activity similar to RLM3. RLM5 metabolizes aminopyrine, ethylmorphine, and benzphetamine all faster than does RLM3. When RLM3 and RLM5 were digested with proteolytic enzymes, peptide maps showed that these two cytochrome P-450 isozymes have different primary structures. Cyanogen bromide digestion breaks both isozymes into fragments too small to compare on polyacrylamide gels. Amino acid analyses indicate that RLM3 and RLM5 have very similar but not identical compositions. NH2-terminal residues were determined by Edman degradation and high pressure liquid chromatography separation of phenylthiohydantoin-derivatives. The first four residues of both enzymes are identical, NH2-Met-Asp-Pro-Val, but the fifth residue of RLM5 is -Leu-while that of RLM3 is -Val-. These sequences differ dramatically from all other P-450 isozymes reported to date.

Amino Acids↗