Role Of Cytochrome B5 Research Articles

Characteristics and roles of cytochrome b5 in cytochrome P450-mediated oxidative reactions in Locusta migratoria

Cytochrome b5 (Cyt-b5) is a small heme protein and known to be involved in a wide range of biochemical transformations, including cytochrome P450 monooxygenase (CYP)-mediated metabolism of endogenous and exogenous compounds. Studies on Cyt-b5 are more concentrated in mammals, but are relatively rare in insects. The characteristics and function of Cyt-b5 from Locusta migratoria have not been described yet. We sequenced the full-length cDNA sequence of Cyt-b5 from L. migratoria (LmCyt-b5) by reverse transcription-PCR (RT-PCR) based on locust transcriptome database. The phylogenetic analysis showed that LmCyt-b5 was closely related to the Cyt-b5 from Blattodea. LmCyt-b5 was highly expressed in ovary, Malpighian tubules, midgut, gastric caeca, and fat bodies. Silencing of LmCyt-b5 had no effect on the susceptibility of L. migratoria to four different insecticides. Suppression of LmCyt-b5 or silencing of both LmCyt-b5 and LmCPR did not significantly change the total CYP activity toward the substrate 7-ethoxycoumarin (7-EC). However, coexpression of LmCYP6FD1 with LmCPR and LmCyt-b5 together in Sf9 cells by using Bac-to-Bac baculovirus expression system significantly increased the catalytic activity of LmCYP6FD1 toward 7-EC as compared with the coexpression of LmCYP6FD1 with cytochrome P450 reductase (LmCPR) or LmCyt-b5 separately. These results suggest that LmCyt-b5 plays an important role in the catalytic reaction of LmCYP6FD1 toward 7-EC in our in vitro experiments. Further study is needed to clarify the role of LmCyt-b5 in CYP-mediated catalytic reactions in L. migratoria.

Role of Cytochrome b5 and α-Tocopherol to Microsomal and Mitochondrial Oxidation

The existence of lipid-radical cycles in membranes as an LPO alternative has been proven. Cytochrome b5 seems to play the key role in the formation of these cycles. The functional role of lipid-radical cycles in cell metabolism, including the positive effect on microsomal and mitochondrial oxidation, is discussed.

Investigations on the role of cytochrome b5 and divalent cations in the maximal nifedipine oxidase activity of human liver.

Native human cytochrome P4503A4 was most active in nifedipine oxidation when incorporated into a binary phospholipid vesicular system with human NADPH-cytochrome P450 reductase. The turnover numbers were estimated to be 17.6 and 19.6 min-1 in the presence of Mg2+ or Ca2+ ions (5 mmol/l) in the test system, respectively. Inclusion of b5 in the vesicular CYP3A4: reductase system results in a slightly lower nifedipine oxidase activity of 16.9 min-1 in the presence of Mg2+ ions. These results demonstrate that b5 is not an essential component in CYP3A4 catalyzed nifedipine oxidation in human liver.

Defining the in Vivo Role for Cytochrome b5 in Cytochrome P450 Function through the Conditional Hepatic Deletion of Microsomal Cytochrome b5

In vitro, cytochrome b5 modulates the rate of cytochrome P450-dependent mono-oxygenation reactions. However, the role of this enzyme in determining drug pharmacokinetics in vivo and the consequential effects on drug absorption distribution, metabolism, excretion, and toxicity are unclear. In order to resolve this issue, we have carried out the conditional deletion of microsomal cytochrome b5 in the liver to create the hepatic microsomal cytochrome b5 null mouse. These mice develop and breed normally and have no overt phenotype. In vitro studies using a range of substrates for different P450 enzymes showed that in hepatic microsomal cytochrome b5 null NADH-mediated metabolism was essentially abolished for most substrates, and the NADPH-dependent metabolism of many substrates was reduced by 50–90%. This reduction in metabolism was also reflected in the in vivo elimination profiles of several drugs, including midazolam, metoprolol, and tolbutamide. In the case of chlorzoxazone, elimination was essentially unchanged. For some drugs, the pharmacokinetics were also markedly altered; for example, when administered orally, the maximum plasma concentration for midazolam was increased by 2.5-fold, and the clearance decreased by 3.6-fold in hepatic microsomal cytochrome b5 null mice. These data indicate that microsomal cytochrome b5 can play a major role in the in vivo metabolism of certain drugs and chemicals but in a P450- and substrate-dependent manner.

Cytochromes P450—A Family of Proteins and Scientists–Understanding their Relationships

The unifying thread of this review involves NADPH-cytochrome P450 reductase (CYPOR), the microsomal enzyme responsible for transferring electrons to cytochromes P450, as well as several other monooxygenase systems, a lifelong interest of the corresponding author. The intersection of her research with that of Dr. David Kupfer, their resulting collaboration, and the beginning of a long-standing study of fatty acid- and eicosanoid-metabolizing cytochromes P450 (CYP4A gene subfamily), including the role of cytochrome b5, will be reported. The culmination of this interest now involves purification and characterization of the human mutants of CYPOR that have been implicated in pathologies, such as Antley-Bixler syndrome.

The roles of cytochrome b5 in cytochrome P450 reactions.

Cytochrome b(5), a 17-kDa hemeprotein associated primarily with the endoplasmic reticulum of eukaryotic cells, has long been known to augment some cytochrome P450 monooxygenase reactions, but the mechanism of stimulation has remained controversial. Studies in recent years have clarified this issue by delineating three pathways by which cytochrome b(5) augments P450 reactions: direct electron transfer of both required electrons from NADH-cytochrome b(5) reductase to P450, in a pathway separate and independent of NADPH-cytochrome P450 reductase; transfer of the second electron to oxyferrous P450 from either cytochrome b(5) reductase or cytochrome P450 reductase; and allosteric stimulation of P450 without electron transfer. Evidence now indicates that each of these pathways is likely to operate in vivo.

Role of Cytochrome b5 in the 17,20-Lyase Activity of P450c17

Role of Cytochrome b5 in the 17,20-Lyase Activity of P450c17

Role of cytochrome b5in the oxidative metabolism of polychlorinated biphenyls catalyzed by cytochrome P450

1. The role of cytochrome b5 in the cytochrome P450-dependent hydroxylation of tetrachlorobiphenyl (TCB) isomers was examined using a reconstituted system consisting of CYP2B1 and CYP1A1 and rat liver microsomes. 2. By addition of cytochrome b5 to the reconstituted system containing CYP2B1, the 3-hydroxylation of 2,5,2,'5'- and 2,5,3',4'-TCB was increased about six-fold, but the 3- and 5-hydroxylation of 2,4,3',4'-TCB was decreased by about 50% 3. All hydroxylations of 3 ,4,3',4'-,2,5, 3,4'- and 2,4,3',4'-TCBs were decreased by addition of cytochrome b5 to the reconstituted system containing CYPlA1. 4. In stoichiometry measurements, changes in NADPH oxidation and coupling efficiency by addition of cytochrome b5 was observed and these differed according to the position of chlorine atoms of TCBs and cytochrome P450 isoforms used in the systems.

The Role of Cytochrome b5 in the Biosynthesis of Androgens by Human P450c17

Human cytochrome b5 has a profound effect on the 17,20-lyase activities catalyzed by purified, human cytochrome P450c17. It enhances the conversion of 17α-hydroxypregnenolone to dehydroepiandrosterone by 13-fold and the conversion of 17α-hydroxyprogesterone to androstenedione by at least 10-fold. This latter activity is virtually undetectable in the absence of cytochrome b5. Other activities catalyzed by P450c17 include 17α-hydroxylation of progesterone and pregnenolone and are much less influenced by cytochrome b5. The conversion of pregnenolone to 17α-hydroxypregnenolone is increased by 2-fold, while that of progesterone to 17α-hydroxyprogesterone is unchanged. These studies using purified systems suggest that cytochrome b5 plays a role in regulating the activities of P450c17 to optimize the balance between sex hormone synthesis and glucocorticoid synthesis. In particular, they indicate that in human testes which contains a high b5/P450 ratio, 17α-hydraxyprogesterone can serve as an intermediate in testosterone production, rather than being a dead-end product, or stated another way, because of the relatively high concentration of cytochrome b5 in the human testis, both the Δ4 and the Δ5 steroidogenic pathways can lead to testosterone production.

The role of cytochrome b5 in adrenal microsomal steroidogenesis

The role of cytochrome b5 in adrenal microsomal steroidogenesis was studied in guinea pig adrenal microsomes and also in the liposomal system containing purified cytochrome P-450s and NADPH-cytochrome P-450 reductase. Preincubation of the microsomes with anti-cytochrome b5 immunoglobulin decreased both 17α- and 21-hydroxylase activity in the microsomes. In liposomes containing NADPH-cytochrome P-450 reductase and P-450C21 or P-45017α,lyase, addition of a small amount of cytochrome b5 stimulated the hydroxylase activity while a large amount cytochrome b5 suppressed the hydroxylase activity. The effect of cytochrome b5 on the rates of the electron transfer to P-450C21 in liposome membranes was determined from stopped flow measurements and that of the second electron transfer was estimated from the oxygenated difference spectra in the steady state. It was indicated that a small amount of cytochrome b5 activated the hydroxylase activity by supplying additional second electrons to oxygenated P-450C21 in the liposomes while a large amount of cytochrome b5 might suppress the activity through the interferences in the interaction between the reductase and P-450C21.

N-demethylation of N-nitrosodimethylamine catalyzed by purified rat hepatic microsomal cytochrome P-450: Isozyme specificity and role of cytochrome b5

Metabolism of the potent hepatocarcinogen N-nitrosodimethylamine (NDMA) was evaluated in reconstituted monooxygenase systems containing each of 11 purified rat hepatic cytochrome P-450 isozymes. The reaction has an absolute requirement for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH, as well as a partial dependence on dilauroylphosphatidylcholine. Of the cytochrome P-450 isozymes evaluated, only cytochrome P-450j, purified from livers of ethanol- or isoniazid-treated rats, had high catalytic activity for the N-demethylation of NDMA. At substrate concentrations of 0.5 and 5 m m, rates of NDMA metabolism to formaldehyde catalyzed by cytochrome P-450j were at least 15-fold greater than the rates obtained with any of the other purified isozymes. At the pH optimum (~6.7) for the reaction, the K m,app and V max were 3.5 m m and 23.9 nmol/min/nmol cytochrome P-450j, respectively. With hepatic microsomes from ethanol-treated rats, which contain induced levels of cytochrome P-450j, the K m,app and V max were 0.35 m m and 3.9 nmol/min/nmol cytochrome P-450, respectively. Inclusion of purified cytochrome b 5 in the reconstituted system containing cytochrome P-450j caused a six-fold decrease in K m,app (0.56 m m) of NDMA demethylation with little or no change in V max (29.9 nmol/min/nmol cytochrome P-450j). Trypsinsolubilized cytochrome b 5, bovine serum albumin, or hemoglobin had no effect on the kinetic parameters of the reconstituted system, indicating a specific effect of intact cytochrome b 5 on the K m,app of the reaction. These results demonstrate high isozyme specificity in the metabolism of NDMA to an ultimate carcinogen and further suggest an important role for cytochrome b 5 in this biotransformation process.

The roles of cytochrome b5 in reconstituted monooxygenase systems containing various forms of hepatic microsomal cytochrome P-450.

The roles of cytochrome b5 in NADPH-dependent monooxygenase reactions catalyzed by reconstituted systems containing four forms of hepatic microsomal cytochrome P-450, i.e. P-450(1), P-450(2), P-448(1), and P-448(2), were examined. Various substrates were metabolized actively in the absence of cytochrome b5 by the system containing P-450(1), but the monooxygenase reactions were accompanied by oxidation of NADPH uncoupled to the product formation. When cytochrome b5 was included in the system, the product formation increased to various extents, depending on the substrates used, while NADPH oxidation changed much less, resulting in an improvement of the coupling efficiency. The increase was large when a substrate metabolized at a low velocity was employed. Evidence is presented that the second of two electrons required for the monooxygenase reactions could be introduced into P-450(1) via cytochrome b5. On the other hand, the rate of P-450(1) reduction was not affected by the addition of cytochrome b5 to the system and that of cytochrome b5 reduction by NADPH-cytochrome P-450 reductase was sufficient to support electron flow to cytochrome P-450 via cytochrome b5 as the second electron. The stimulatory effect of cytochrome b5 on the P-450(1)-catalyzed monooxygenase reactions can be explained by assuming that the rate of the second electron supply to the oxygenated P-450(1)-substrate complex from cytochrome b5 is higher than that directly from NADPH-cytochrome P-450 reductase. An electron flow from NADH via cytochrome b5 can be utilized as the second electron for the O-deethylase reaction of 7-ethoxycoumarin catalyzed by reconstituted systems containing P-450(2) and P-448(2) when both NADH-cytochrome b5 reductase and cytochrome b5 are included in the system, although the cytochrome has no stimulatory effect at all on the deethylase activity of these two cytochrome P-450's. It has been shown that the second electron for P-448(1) can also be supplied from NADH via cytochrome b5 in a reconstituted acetanilide p-hydroxylase system containing P-448(1), cytochrome b5, and the two reductases.

Role of Cytochrome b5 in Hydroxylation by a Reconstituted Cytochrome P-450-containing System

1. The role of cytochrome b5 in the oxidative metabolism of benzphetamine and 3,4-benzpyrene in the presence of NADPH was studied with the reconstituted hydroxylation system isolated from rat liver microsomes whose components were cytochrome P-450 or P-448, NADPH-cytochrome c reductase, and lipid. In the absence of cytochrome b5, the turnover number for benzphetamine and 3,4-benzpyrene (nanomoles per min per nmole of P-450 or P-448) in the reconstituted system was at least as good as that in liver microsomes, indicating that cytochrome b5 is not an obligatory component in the NADPH-dependent benzphetamine or 3,4-benzpyrene hydroxylation system. 2. The addition of cytochrome b5 at a ratio of cytochrome P-450 to b5 normally found in microsomes inhibited NADPH-dependent benzphetamine N-demethylation by the three components (cytochrome P-450, NADPH-cytochrome c reductase, and lipid). This inhibition was shown to be due to competition between cytochrome b5 and cytochrome P-450 for NADPH-cytochrome c reductase. The addition of NADH and cytochrome b5 reductase to the reconstituted system reversed the inhibition of NADPH-dependent benzphetamine N-demethylation by cytochrome b5. Thus, the NADH synergistic effect observed in liver microsomes with benzphetamine as the substrate and NADPH as electron donor could be due to NADH and cytochrome b5 reductase preventing the inhibition by cytochrome b5. 3. The addition of cytochrome b5 at a ratio of cytochrome P-448 to b5 normally found in microsomes did not inhibit the NADPH-dependent hydroxylation of 3,4-benzpyrene by the three components (cytochrome P-448, NADPH-cytochrome c reductase, and lipid). In the presence of NADH, NADPH, cytochrome b5, cytochrome b5 reductase, and the three components, the rate of 3,4-benzpyrene hydroxylation was significantly increased. This increased rate was due to a NADH-dependent hydroxylation pathway via cytochrome b5 and cytochrome b5 reductase. 4. It is concluded that cytochrome b5 is not an obligatory component in the NADPH-dependent benzphetamine or 3,4-benzpyrene hydroxylation systems. However, this cytochrome plays an important role in regulating NADPH-dependent hydroxylation of these substrates and is also an essential component of the NADH-dependent hydroxylation pathway.

On functional role of cytochrome b5. I. NADH-linked cytochrome c reductase in microsomes.

On functional role of cytochrome b5. I. NADH-linked cytochrome c reductase in microsomes.