Skip to main content
SpringerLink
Log in

Qualitative Änderungen in Lebermikrosomen Phenobarbital-behandelter Kaninchen

Änderungen der spektralen Dissoziationskonstanten (K S ) von Anilin und N-Äthylanilin, der scheinbaren Michaelis-Konstanten (K M ) für die Hydroxylierung dieser Substrate und der AffinitÄt der beteiligten Enzyme für Sauerstoff und Kohlenmonoxid

Qualitative changes in hepatic microsomes from phenobarbital-treated rabbits

Changes in spectral dissociation constants (KS) of aniline and N-ethylaniline, in the apparent Michaelis constants (KM) for the hydroxylation of these substrates, and in the affinity of the enzymes involved for oxygen and carbon monoxide

  • Published:
Naunyn-Schmiedebergs Archiv für Pharmakologie Aims and scope Submit manuscript

Summary

Phenobarbital treatment of rabbits was found to cause significant changes in some of the apparent Michaelis (K M) constants for p- and N-hydroxylation of aniline [reactions 1 and 2, resp.] and de-ethylation, p- and N-hydroxylation of N-ethylaniline [reactions 3,4, and 5, resp.], and in the spectral dissociation constants (K s) for aniline, N-ethylaniline and ethylisocyanide in isolated hepatic microsomes.

The “critical” oxygen concentrations were increased in all reactions investigated.

The distribution constants (K G) for CO, determined according to Warburg, decreased in the reactions 2, 3, and 4, and increased in reaction 1. Reaction 5 was not inhibited by CO in microsomes from untreated rabbits and was stimulated by CO after phenobarbital treatment of the animals.

Depending on the reaction, theK M-values for aniline and N-ethylaniline were increased, decreased, or remained unchanged by the phenobarbital treatment of the animals. The most striking changes inK M-values were observed with the reactions 2 and 5 in which cytochrome P-450 is either not involved or is not a limiting factor.

TheK s-value for ethylisocyanide measured after addition of dithionite to microsomes was increased. The correspondingK s-value measured without dithionite under aerobic conditions remained unchanged.

TheK s value for aniline was decreased by the phenobarbital treatment of the animals. Using microsomes from untreated animals theK s value for N-ethylaniline could not be determined exactly. The order of magnitude of this value, however, was greater than that of the corresponding value from phenobarbital-treated animals.

The observed alterations in affinity imply that induction by phenobarbital may be associated withqualitative as well as quantitative changes in the hepatic microsomal enzymes or membranes. The qualitative changes could be important for the accessibility of the reacting groups and/or substrate guiding.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Literatur

  • Alvares, A. P., Schilling, G. R., Kuntzman, R.: Differences in the kinetics of benzpyrene hydroxylation by hepatic drug-metabolizing enzymes from phenobarbital and 3-metnylcholanthrene-treated rats. Biochem. biophys. Res. Commun.30, 588 (1969).

    Google Scholar 

  • Anders, M. W.: Acetone enhancement of microsomal aniline para-hydroxylase activity. Arch. Biochem.126, 269 (1968).

    Google Scholar 

  • —: Stimulation in vitro of microsomal aniline hydroxylation by 2,2′-bipyridine. Biochem. Pharmacol.18, 2561 (1969).

    Google Scholar 

  • —, Mannering, G. J.: Kinetics of the inhibition of the N-demethylation of ethylmorphine by 2-diethylaminoethyl-2,2-diphenylvalerate HCl (SKF 525-A). Molec. Pharmacol.2, 319 (1966).

    Google Scholar 

  • Brodie, B. B., Axelrod, J.: The estimation of acetanilide and its metabolic products: aniline, N-acetyl-p-aminophenol and p-aminophenol (free and total conjugated) in biological fluids and tissues. J. Pharmacol. exp. Ther.94, 22 (1948).

    Google Scholar 

  • —, Maickel, B. P.: Comparative biochemistry of drug metabolism. In: Proc. First Int. Pharmacol. Meeting, Vol. 6, ed. by B. B. Brodie and E. G. Erdös, p. 299. Oxford-London-New York-Paris: Pergamon Press 1962.

    Google Scholar 

  • Conney, A. H.: Pharmacological implications of microsomal enzyme induction. Pharmacol. Rev.19, 317 (1967).

    Google Scholar 

  • —, Levin, W., Jacobson, M., Kuntzman, R.: Specifity in the regulation of the 6Β-, 7α- and 16α-hydroxylation of testosterone by rat liver microsomes. In: Microsomes and drug oxidations, ed. by J. B. Gillette, A. H. Conney, G. J. Cosmides, R. W. Estabrook, J. R. Fouts, and G. J. Mannering. New Tork-London: Academic Press 1969.

    Google Scholar 

  • Gillette, J. R.: Metabolism of drugs and other foreign compounds by enzymatic mechanisms. Fortschr. Arzneimittel-Forsch.6, 11 (1963).

    Google Scholar 

  • —: Biochemistry of drug oxidation and reduction by enzymes in hepatic endoplasmic retieulum. Advanc. Pharmacol.4, 219 (1966).

    Google Scholar 

  • Gornall, A. G., Bardawill, C. J., David, M. M.: Determination of serum proteins by means of the biuret reaction. J. biol. Chem.177, 751 (1949).

    Google Scholar 

  • Gram, T. E., Rogers, L. A., Fouts, J. R.: Further studies on the metabolism of drugs by subfractions of hepatic microsomes. J. Pharmacol. exp. Ther.155, 479 (1967).

    Google Scholar 

  • — — —: Effect of pretreatment of rabbits with phenobarbital or 3-methylcholanthrene on the distribution of drug-metabolizing enzyme activity in subfraotions of hepatic microsomes. J. Pharmacol. exp. Ther.157, 435 (1967).

    Google Scholar 

  • Guarino, A. M., Gram, T. E., Gigon, P. L., Greene, F. E., Gillette, J. R.: Changes in Michaelis and spectral constants for aniline in hepatic microsomes from phenobarbital-treated rats. Molec. Pharmacol.5, 131 (1969).

    Google Scholar 

  • Herr, F., Kiese, M.: Bestimmung von Nitrosobenzol im Blute. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak.235, 351 (1959).

    Google Scholar 

  • Hlavica, P., Kiese, M.: N-oxygenation of N-alkyl- and N,N-dialkylanilines by rabbit liver microsomes. Biochem. Pharmacol.18, 1501 (1969).

    Google Scholar 

  • — —, Lange, G., Mor, G.: Die Wirkung von Kohlenmonoxid auf die N-Hydroxylierung von Anilin durch Kaninchenlebermikrosomen. Naunyn-Sehmiedebergs Arch. Pharmak. exp. Path.263, 269 (1969).

    Google Scholar 

  • Imai, Y., Sato, R.: Evidence for two forms of P-450 hemoprotein in microsomal membranes. Biochem. biophys. Res. Commun.23, 5 (1966a).

    Google Scholar 

  • — —: Activation and inhibition of microsomal hydroxylation by ethylisocyanide. Biochem. biophys. Bes. Commun.25, 80 (1966b).

    Google Scholar 

  • Jackson, H. L., McKusick, B. C.: In: Organic Synthesis, ed. by T. L. Clairns, Vol. 35, p. 62. New York: John Wiley and Sons, Inc. 1955.

    Google Scholar 

  • Jefcoate, C. R. E., Gaylor, J. L.: Ligand interactions with hemoprotein P-450. II. Influence of phenobarbital and methylcholanthrene induction processes on P-450 spectra. Biochemistry8, 3464 (1969b).

    Google Scholar 

  • — —, Calabrese, R. L.: Ligand interactions with cytochrome P-450. I. Binding of primary amines. Biochemistry8, 3455 (1969a).

    Google Scholar 

  • Jones, P. D., Holloway, P. W., Peluffo, P. O., Wakil, S. J.: A requirement for lipids by the microsomal stearyl coenzyme A desaturase. J. biol. Chem.244, 744 (1969).

    Google Scholar 

  • —, Wakil, S. J.: A requirement for phospholipids by the miorosomal reduced diphosphopyridine nucleotide-cytochrome C reductase. J. biol. Chem.242, 5267 (1967).

    Google Scholar 

  • Kaiser, B.: Chromatographie in der Gasphase. Bibliographisches Institut Mannheim (1960).

  • Kampffmeyer, H., Kiese, M.: The hydroxylation of aniline and N-ethylaniline by microsomal enzymes at low oxygen pressures. Biochem. J.339, 454 (1964).

    Google Scholar 

  • — —: The effect of carbon monoxide on the hydroxylation of aniline and some of its derivatives by liver microsomes. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak.250, 1 (1965).

    Google Scholar 

  • Kato, R., Gillette, J. R.: Effects of starvation on NADPH-dependent enzymes in liver microsomes of male and female rats. J. Pharmacol. exp. Ther.150, 279 (1965).

    Google Scholar 

  • Kiese, M.: Relationship of drug metabolism to methemoglobin formation. Ann. N.Y. Acad. Sci.123, 141 (1965).

    Google Scholar 

  • Kratz, F., Staudinger, Hj.: Spektrale Änderungen von Kaninchenlebermikrosomen durch Cumarin. Hoppe-Seylers Z. physiol. Chem.349, 455 (1968).

    Google Scholar 

  • Kuntzman, R., Jacobson, M.: Effect of drugs on the metabolism of progesterone by liver microsomal enzymes from various animal species. Fed. Proc.24, 152 (1965b).

    Google Scholar 

  • —, Lawrence, D., Conney, A. H.: Michaelis constants for the hydroxylation of steroid hormones and drugs by rat liver microsomes. Molec. Pharmacol.1, 163 (1965a).

    Google Scholar 

  • Lange, G.: über einige Eigenschaften der durch Chlorphenothan oder Phenobarbital induzierten Mikrosomenenzyme aus Kaninchenleber. Naunyn-Schmiedebergs Arch. Pharmak. exp. Path.255, 35 (1966).

    Google Scholar 

  • —: Verschiedene Induktion der mikrosomalen N- und p-Hydroxylierung von Anilin und N-Äthylanilin bei Kaninchen. Naunyn-Schmiedebergs Arch. Pharmak. exp. Path.257, 230 (1967).

    Google Scholar 

  • —: Hydroxylase-AktivitÄt und HÄmoprotein-Gehalt von Mikrosomen aus Kaninchenleber. Naunyn-Schmiedebergs Arch. Pharmak. exp. Path.259, 221 (1968).

    Google Scholar 

  • —, Steubl, Chr.: Änderung der Zusammensetzung mikrosomaler Lipide durch Behandlung von Kaninchen mit Phenobarbital. Naunyn-Schmiedebergs Arch. Pharmak. exp. Path.263, 235 (1969).

    Google Scholar 

  • —, Thun, K.-J.: Verschiedene Induktion einiger mikrosomaler Umbaureaktionen von Progesteron durch die Behandlung von Kaninchen mit Phenobarbital. Naunyn-Sehmiedebergs Arch. Pharmak.264, 266 (1969).

    Google Scholar 

  • - - Hydroxylierung von Progesteron durch Lebermikrosomen von Kaninchen. Änderungen im Metabolitmuster durch Phenobarbital-Behandlung der Tiere. Naunyn-Schmiedebergs Arch. Pharmak. (im Druck) (1970).

  • Leibman, K. C., Hildebrandt, A. G., Estabrook, R. W.: Spectrophotometric studies of interactions between various substrates in their binding to microsomal cytochrome P-450. Biochem. biophys. Res. Commun.36, 789 (1969).

    Google Scholar 

  • —, McAllister, W. J., Jr.: Metabolism of trichlorethylene in liver microsomes. III. Induction of the enzymatic activity and its effect on excretion of metabolites. J. Pharmacol. exp. Ther.157, 574 (1967).

    Google Scholar 

  • Levin, W., Kuntzman, R.: Biphasic decrease of radioactive hemoprotein from liver microsomal CO-binding particles. J. biol. Chem.244, 3671 (1969).

    Google Scholar 

  • Lien, E. J., Hansch, C.: Correlation of ratios of drug metabolism by microsomal subfractions with partition coefficients. J. Pharm. Sci.57, 1027 (1968).

    Google Scholar 

  • Lumper, L., Schnellbacher, E., Staudinger, Hj.: Zur Wirkung von Lysolecithin und (Ω-Acyloxyalkyl)-cholin-phosphaten auf die AktivitÄt mikrosomaler Enzyme. Hoppe-Seylers Z. physiol. Chem.351, 459 (1970).

    Google Scholar 

  • McMahon, R. E., Sullivan, H. R., Craig, J. C., Pereira, W. E., Jr.: The microsomal oxygenation of ethyl benzene: isotopic, stereochemical and induction studies. Arch. Biochem.132, 575 (1969).

    Google Scholar 

  • Netter, K.-J.: Die oxydative N-Demethylierung von N-Monomethyl-p-Nitranilin. Naunyn-Schmiedebergs Arch. Pharmak. exp. Path.255, 151 (1966).

    Google Scholar 

  • —, Seidel, G.: An adaptively stimulated O-demethylating system in rat liver microsomes and its kinetic properties. J. Pharmacol. exp. Ther.146, 61 (1964).

    Google Scholar 

  • Nishibayashi, H., Omura, T., Sato, R.: The binding of the ethyl isocyanide by hepatic microsomal hemoprotein. Biochim. biophys. Acta (Amat.)118, 651 (1966).

    Google Scholar 

  • Remmer, H., Merker, H. J.: Effect of drugs on the formation of smooth endoplasmatic reticulum and drug-metabolizing enzymes. Ann. N. Y. Acad. Sci.123, 79 (1965).

    Google Scholar 

  • Rickert, D. E., Pouts, J. R.: Benzpyrene pretreatment changes the kinetics and pH optimum for aniline hydroxylation in vitro, but not those for benzphatamine demethylation in vitro by rat liver microsomes. Biochem. Pharmacol.19, 381 (1970).

    Google Scholar 

  • Rogers, L. A., Morris, H. P., Fouts, J. R.: The effect of phenobarbital on drug metabolic enzyme activity, ultrastructure and growth of a “minimal deviation” hepatoma (Morris 7800). J. Pharmacol. exp. Ther.157, 227 (1967).

    Google Scholar 

  • Rubin, A., Tephly, T. R., Mannering, G. J.: Kinetics of drug metabolism by hepatic microsomes. Biochem. Pharmacol.13, 1007 (1964).

    Google Scholar 

  • Schulze, H. U., Staudinger, Hj.: Änderungen im Gehalt der Phospholipide von Lebermikrosomen unterschiedlich vorbehandelter Ratten und Meerschweinchen. Hoppe-Seylers Z. physiol. Chem.351, 184 (1970).

    Google Scholar 

  • Shoeman, D. W., Chaplin, M. D., Mannering, G. J.: Induction of drug metabolism. III. Further evidence for the formation of a new P-450 hemoprotein after treatment of rats with 3-methylcholanthrene. Molec. Pharmacol.5, 412 (1969).

    Google Scholar 

  • Sladek, N. E., Mannering, G. J.: Evidence for a new P-450 hemoprotein in hepatic microsomes from 3-methylcholanthrene-treated rats. Biochem. biophys. Res. Commun.24, 668 (1966).

    Google Scholar 

  • Warburg, O.: über die Wirkung von Kohlenmonoxyd und Stickoxyd auf Atmung und GÄrung. Biochem. Z.189, 354 (1972).

    Google Scholar 

  • Ziegler, D. M.: Properties of a mixed function amine oxidase isolated from pork liver microsomes. Vortrag auf dem „International Symposium on Microsomes and Drug Oxidations“, Tübingen, Germany, 1969.

Download references

Author information

Authors and Affiliations

  1. Pharmakologisches Institut der UniversitÄt, München

    F. Kröber, G. Lange, S. Mathes & G. Mor

Authors
  1. F. Kröber
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. G. Lange
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S. Mathes
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. G. Mor
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

FrÄulein Barbara Suermann danken wir für wertvolle technische Mitarbeit bei der Messung der Bindungskonstanten von Anilin und N-Äthylanilin.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kröber, F., Lange, G., Mathes, S. et al. Qualitative Änderungen in Lebermikrosomen Phenobarbital-behandelter Kaninchen. Naunyn-Schmiedebergs Arch. Pharmak. 267, 307–326 (1970). https://doi.org/10.1007/BF00999545

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00999545

Key-words

Search

Navigation