Cytochrome recombinant human

Together with the site of metabolism, the rate of metabolism is a fundamental factor since this will contribute to the rate of elimination of the compound from the body. The rate of metabolism can be measured using different in vitro systems (cytochrome recombinant, human liver microsome, human hepatocites, etc). Normally, the data are produced using different compounds and the rate values are extracted from complex systems like liver microsomes, or human hepatocites. 

Trubetskoy, O.V., Gibson, J.R., and Marks, B.D. 2005. Highly miniaturized formats for in vitro drug metabolism assays using vivid fluorescent substrates and recombinant human cytochrome P450 enzymes. J. Biomol. Screen. 10 56. 

Newburger, P. E., Dai, Q., Whitney, C. (1991). In vitro regulation of human phagocyte cytochrome b heavy and light chain gene expression by bacterial lipopolysaccharide and recombinant human cytokines. J. Biol. Chem. 266,16171-7. 

Pikuleva lA, Bjorkhem 1, Waterman MR. 1997. Expression, purification, and enzymatic properties of recombinant human cytochrome P450c27 CYP27). Arch Biochem Bio-phys 343 123-130. 

Ghosal A, Hapangama N, Yuan Y, et al. Rapid determination of enzyme activities of recombinant human cytochromes P450, human liver microsomes and hepatocytes. Biopharm Drug Dispos 2003 24 375-384. 

Crewe HK et al (2002) Metabolism of tamoxifen by recombinant human cytochrome P450 enzymes formation of the 4-hydroxy, 4 -hydroxy and N-desmethyl metabolites and isomerization of trans-4-hydroxytamoxifen. Drug Metab Dispos 30 869-874... 

Cytochrome b5 affects the kinetics of drug metabolism by certain CYP enzymes hence, coexpression of this microsomal hemoprotein (together with NADPH-CYP reductase) can affect the catalytic efficiency of certain recombinant CYP enzymes (76,109). For example, the presence of cytochrome b5 tends to increase Fmax for reactions catalyzed by CYP3 A4, whereas it tends to decrease Km for reactions catalyzed by CYP2E1. In both cases, cytochrome b5 increases Vmax/Km, which is a measure of in vitro intrinsic clearance. The fact that some commercially available recombinant CYP enzymes are expressed with cytochrome b5 while others are not complicates the interpretation of results of studies performed with recombinant human CYP enzymes. 

Figure 27 Metabolism of DB289 (antiparasitic prodrug) by a panel of recombinant human CYP enzymes. Abbreviation CYP, cytochrome P450.

In addition to the Mo center shown in Fig. 16, SO contains an N-terminal domain with a h5-type cytochrome (72) that dominates the visible absorption and RR spectra of the holoenzyme. Hence, RR characterization of the Mo center has been confined to studies of the Mo-domain of recombinant human SO. Resonance Raman spectra of the Mo-domain obtained with 488-nm excitation for samples prepared by tryptic cleavage of the overexpressed and purified K108R variant of the holoenzyme (73) and by overexpression and purification of the His-tagged Mo-domain (74), are compared in Fig. 19. Of particular importance is that the bands at 1006, 1161, and 1532 cm-1 in the Mo-domain samples prepared by tryptic cleavage [Fig. 19(a)] are no longer observed in the... 

Bauer E, Guo Z, Ueng YF, Bell LC, Zeldin D, Guengerich FP. 1995. Oxidation of benzo[a]pyrene by recombinant human cytochrome P450 enzymes. Chem. Res. Toxicol. 8 136 42... 

Similar to the techniques used for calculation of chemical disposition parameters, in vivo biotransformation kinetic parameters of a substrate can be estimated from in vitro systems such as microsomes, freshly isolated hepatocytes, fiver slices, and isolated perfused livers (24). Intrinsic clearance or Michaelis-Menten parameters for the whole liver can also be obtained by scaling in vitro parameters based on the cytochrome P450 enzyme content (25-27). These parameters can also be estimated from in vitro data obtained from recombinant human CYP systems, and also through allometric scaling of clearance estimates from animal PBPK models. 

Fig. 3. Quantification of Bcl-2 family activities with the long-format cytochrome c release assay. Results are the average of duplicate measurements in (A) and are averages of triplicate measurements +/-SEM in (B-D) (many error bars in panels [B-D] are obscured by the symbols). In panels (A-D) open circles indicate cytochrome c detected when Triton X-100 was added to mitochondria and open diamonds indicate cytochrome c detected when no Bcl-2 family proteins were added to mitochondria. All incubations of mitochondria except those in (B) were for 30 min. (A) Recombinant human Bid (solid circles) and caspase-8-cleaved human Bid (squares) induce release of cytochrome c from isolated mouse liver mitochondria in a dose- dependent manner. (B) Kinetics of 52 nM (solid squares) and 5.2 nM (solid circles) human cleaved Bid-induced cytochrome c release from isolated mouse liver mitochondria. (C) Bcl-xL inhibition of caspase-8-cleaved human Bid and cleaved mouse Bid induced cytochrome c release. Mitochondria were incubated with 52 nM cleaved human Bid without (solid diamond) or with ( solid squares) the indicated concentrations of mouse Bcl-xL. Mitochondria were also incubated with 17 nM cleaved mouse Bid without (open square) or with (solid circles) the indicated concentrations of mouse Bcl-xL. (D) A synthetic peptide (GQVGRQLAIIGDDINR) corresponding to the amino acid 72-87 BH3 region of Bak prevents Bcl-xL from inhibiting caspase-8-cleaved mouse Bid induction of cytochrome c release. Mitochondria were incubated with 17 nM caspase-8-cleaved mouse Bid without (triangle) or with (open square) 155 nM mouse Bcl-xL and the indicated concentrations of Bak-BH3 (solid circle) or the corresponding Bak peptide (GQVGRQAAIIGDDINR) with a L to A substitution (solid squares).

We have characterized the activities of several recombinant human and mouse Bcl-2 family proteins (Bcl-2, Bcl-xL, Bcl-w, Bax, Bid, and caspase-8 cleaved Bid) with the cytochrome c ELISA. Dose responses, kinetics, inhibition, and the effects of protein-protein interactions have been quantified. Conducting the cytochrome c release assay in the well of the ELISA plate decreases the time required to perform the assay from 3 h to 35 min. Similar dose responses for induction of cytochrome c release caused by cleaved Bid were obtained with the 35 min and 3 h release assays, indicating that the short format can detect intermediate levels of cytochrome c release. The short format is applicable to automation for screening million-member libraries. Mitochondria obtained from one mouse liver is sufficient to perform 4000 assays in the 96-well plates and scale-up is performed simply by increasing the amount of starting tissue. 

The Jurkat (human acute T-cell leukemia, clone E6-1) cell line was from ATCC. Cytochrome c ELISA ( MCTC0), active caspase-3 ELISA ( KM300), active caspase-7 ELISA ( KM700), bzVKD-fmk ( FMK011 or included with kits), human Bid ( 846-BD), mouse Bid ( 860-MM), caspase-8-cleaved human Bid ( 882-B8), caspase-8 cleaved mouse Bid ( 883-M8), mouse Bcl-xL ( 878-BC) missing the carboxyl terminal mitochondria targeting sequence, Bak-BH3 synthetic peptide ( 881-BA), Bak L to A synthetic peptide ( 879-BK), and HRP conjugated to streptavidin, recombinant human caspase 2 ( 702-C2), caspase 3 ( 707-C3), caspase 7 ( 823-C7), caspase 8 ( 705-C8), and caspase 10 ( 834-CP) were from R D Systems. Biotinylated standards for the ELISAs were made and assayed as previously... 

Recombinant human CYP2D6 was shown to be selective for the O-demethyla-tion of dextromethorphan at or near the K n for this cytochrome s interaction with this substrate (at a concentration of 3 pM), whereas at higher substrate concentrations CYPs of the cytochrome 2C family contributed significantly to the metabolism of the drug. Similarly, diazepam at a concentration of 100 pM was N-demethylated principally by CYPs 3A4 and 2C19. However, at the lower concentration of 20 pM, the metabolism mediated by CYP3A4 was reduced to 30% that of CYP2C19. 

K. Ohyama, N. Hatanaka, S. Asahi et al. (2002). Roles of NADPH-P450 reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human cytochrome P450s expressed in membranes of Escherichia coli. Protein Expr. Purif. 24, 329-337. 

In early work in this field, this point would have been the demonstration of the reaction of interest with an enzyme purified from tissue. Today P450 proteins are generally produced in recombinant systems and seldom purified from tissue sources. In routine practice in the pharmaceutical industry, new reactions are examined with a battery of the major recombinant human (liver) P450s, many of which are available from commercial sources. Systems used for expression include bacteria, yeast, baculovirus (-infected insect cells), and mammalian cells. The P450s need not be purified for these comparisons hut must have suitable provision for NADPH-P450 reductase in a crude system (and cytochrome... 

Shimada, T., E.M.J. Gillam, T.R. Sutter, P.T. Strickland, F.P. Guengerieh, and H. Yamazaki (1997). Roles of recombinant human cytochrome P450 IBl in the oxidation of xenobiotic chemicals. Drug Metab. Dispos. 25, 617—622. 

I. Bjdrkhem (1998). Activities of recombinant human cytochrome P450c27 (CYP27) which produce intermediates of alternative bile acid biosynthetic pathways. J Biol. Chem. 273,18153-18160. 

K. Takeshige, T. Sakai et al. (1998). Purification and characterization of recombinant human neutrophil leukotriene B (o-hydroxylase (Cytochrome 4F3). Arch. Biochem. Biophys. 355, 201-205. 

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Casimiro, D. R., Wong, L. L., Colon, J. L., Zewert, T. E., Richards, J. H., Chang, I-J., Winkler, J. R. and Gray, H. B, 1993, Electron transfer in ruthenium / zinc porphyrin derivatives of recombinant human myoglobins. Analysis of tunneling pathways in myoglobin and cytochrome c. Journal of American Chemical Society 115, 1485-1489. Chang, Y-C. and Ludescher, R. D, 1994, Local conformation of rabbit skeletal myosin rod filaments probed by intrinsic tryptophan fluorescence. Biochemistry 33, 2313 -2321. 

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