Characterization of cytochrome P-450-dependent aminopyrine N-demethylase in rat brain: Comparison with hepatic aminopyrine N-demethylation

M. P. Marietta, E. S. Vesell, R. D. Hartman, Judith Weisz, B. H. Dvorchik

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Abstract

This study was designed to characterize cytochrome P-450-dependent aminopyrine N-demethylation using microsomes from rat brain and to compare it with N-demethylation of the drug by hepatic microsomes studied in parallel. The cytochrome P-450 content of the microsomal fraction from brain (0.035 nmol/mg of protein) was 3% that of the liver. Using the dithionite difference technique, a carbon monoxide absorbance peak occurred at 448 to 451 nm for brain microsomal cytochrome P-450. Spectral binding of aminopyrine, hexobarbital, androstenedione and aniline to rat brain cytochrome P-450 could not be detected, apparently because of the low concentration of the hemoprotein. Aminopyrine N-demethylase of brain microsomes required oxygen and NADPH for optimal activity and its activity was inhibited by carbon monoxide (CO) and SKF 525-A in a concentration-dependent manner. However, the enzyme was less sensitive to the inhibitory effects of CO and SKF 525-A than the hepatic N-demethylase. Brain aminopyrine N-demethylation was linear with respect to protein concentrations and incubation times of up to 15 min. Enzymatic activity declined when microsomes were incubated longer than 15 min due apparently to enzyme inactivation. Maximal aminopyrine N-demethylase activity of the brain was 0.5 and 17% that of the liver when expressed per milligram of microsomal protein (261.3 pmol/10 min/mg of protein) and nanomole of cytochrome P-450 (6.98 nmol/10 min/nmol of cytochrome P-450), respectively. In contrast to the biphasic kinetic behavior of hepatic aminopyrine N-demethylase, brain aminopyrine N-demethylation exhibited linear kinetics. The single apparent Km of the brain aminopyrine N-demethylase (3.39 mM) was at least 3-fold higher than that of the liver (0.18 and 1.13 mM). Coupled with data presented on the inhibitory effects of CO and SKF 525-A on brain and hepatic aminopyrine N-demethylases, the kinetic data suggest that brain aminopyrine N-demethylase exhibits behavior different from that of the liver enzyme. Hexobarbital, meperidine and 3,4-benzo(a)pyrene also were metabolized by rat brain microsomes. No appreciable nuclear of mitochondrial contamination was seen in electron micrographs of the brain microsomal pellet. The presence of cytochrome P-450-dependent microsomal enzymes in the brain offers some new implications regarding regulation of the activity of centrally active compounds.

Original languageEnglish (US)
Pages (from-to)271-279
Number of pages9
JournalJournal of Pharmacology and Experimental Therapeutics
Volume208
Issue number2
StatePublished - Sep 26 1979

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Aminopyrine N-Demethylase
Aminopyrine
Cytochrome P-450 Enzyme System
Liver
Brain
Microsomes
Carbon Monoxide
Proadifen
Hexobarbital
Enzymes
Proteins
N Demethylating Oxidoreductases
Dithionite
Meperidine
Androstenedione
Benzo(a)pyrene

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Pharmacology

Cite this

@article{79398290bda146258cdf7a18ffb294f9,
title = "Characterization of cytochrome P-450-dependent aminopyrine N-demethylase in rat brain: Comparison with hepatic aminopyrine N-demethylation",
abstract = "This study was designed to characterize cytochrome P-450-dependent aminopyrine N-demethylation using microsomes from rat brain and to compare it with N-demethylation of the drug by hepatic microsomes studied in parallel. The cytochrome P-450 content of the microsomal fraction from brain (0.035 nmol/mg of protein) was 3{\%} that of the liver. Using the dithionite difference technique, a carbon monoxide absorbance peak occurred at 448 to 451 nm for brain microsomal cytochrome P-450. Spectral binding of aminopyrine, hexobarbital, androstenedione and aniline to rat brain cytochrome P-450 could not be detected, apparently because of the low concentration of the hemoprotein. Aminopyrine N-demethylase of brain microsomes required oxygen and NADPH for optimal activity and its activity was inhibited by carbon monoxide (CO) and SKF 525-A in a concentration-dependent manner. However, the enzyme was less sensitive to the inhibitory effects of CO and SKF 525-A than the hepatic N-demethylase. Brain aminopyrine N-demethylation was linear with respect to protein concentrations and incubation times of up to 15 min. Enzymatic activity declined when microsomes were incubated longer than 15 min due apparently to enzyme inactivation. Maximal aminopyrine N-demethylase activity of the brain was 0.5 and 17{\%} that of the liver when expressed per milligram of microsomal protein (261.3 pmol/10 min/mg of protein) and nanomole of cytochrome P-450 (6.98 nmol/10 min/nmol of cytochrome P-450), respectively. In contrast to the biphasic kinetic behavior of hepatic aminopyrine N-demethylase, brain aminopyrine N-demethylation exhibited linear kinetics. The single apparent Km of the brain aminopyrine N-demethylase (3.39 mM) was at least 3-fold higher than that of the liver (0.18 and 1.13 mM). Coupled with data presented on the inhibitory effects of CO and SKF 525-A on brain and hepatic aminopyrine N-demethylases, the kinetic data suggest that brain aminopyrine N-demethylase exhibits behavior different from that of the liver enzyme. Hexobarbital, meperidine and 3,4-benzo(a)pyrene also were metabolized by rat brain microsomes. No appreciable nuclear of mitochondrial contamination was seen in electron micrographs of the brain microsomal pellet. The presence of cytochrome P-450-dependent microsomal enzymes in the brain offers some new implications regarding regulation of the activity of centrally active compounds.",
author = "Marietta, {M. P.} and Vesell, {E. S.} and Hartman, {R. D.} and Judith Weisz and Dvorchik, {B. H.}",
year = "1979",
month = "9",
day = "26",
language = "English (US)",
volume = "208",
pages = "271--279",
journal = "Journal of Pharmacology and Experimental Therapeutics",
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publisher = "American Society for Pharmacology and Experimental Therapeutics",
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Characterization of cytochrome P-450-dependent aminopyrine N-demethylase in rat brain : Comparison with hepatic aminopyrine N-demethylation. / Marietta, M. P.; Vesell, E. S.; Hartman, R. D.; Weisz, Judith; Dvorchik, B. H.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 208, No. 2, 26.09.1979, p. 271-279.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Characterization of cytochrome P-450-dependent aminopyrine N-demethylase in rat brain

T2 - Comparison with hepatic aminopyrine N-demethylation

AU - Marietta, M. P.

AU - Vesell, E. S.

AU - Hartman, R. D.

AU - Weisz, Judith

AU - Dvorchik, B. H.

PY - 1979/9/26

Y1 - 1979/9/26

N2 - This study was designed to characterize cytochrome P-450-dependent aminopyrine N-demethylation using microsomes from rat brain and to compare it with N-demethylation of the drug by hepatic microsomes studied in parallel. The cytochrome P-450 content of the microsomal fraction from brain (0.035 nmol/mg of protein) was 3% that of the liver. Using the dithionite difference technique, a carbon monoxide absorbance peak occurred at 448 to 451 nm for brain microsomal cytochrome P-450. Spectral binding of aminopyrine, hexobarbital, androstenedione and aniline to rat brain cytochrome P-450 could not be detected, apparently because of the low concentration of the hemoprotein. Aminopyrine N-demethylase of brain microsomes required oxygen and NADPH for optimal activity and its activity was inhibited by carbon monoxide (CO) and SKF 525-A in a concentration-dependent manner. However, the enzyme was less sensitive to the inhibitory effects of CO and SKF 525-A than the hepatic N-demethylase. Brain aminopyrine N-demethylation was linear with respect to protein concentrations and incubation times of up to 15 min. Enzymatic activity declined when microsomes were incubated longer than 15 min due apparently to enzyme inactivation. Maximal aminopyrine N-demethylase activity of the brain was 0.5 and 17% that of the liver when expressed per milligram of microsomal protein (261.3 pmol/10 min/mg of protein) and nanomole of cytochrome P-450 (6.98 nmol/10 min/nmol of cytochrome P-450), respectively. In contrast to the biphasic kinetic behavior of hepatic aminopyrine N-demethylase, brain aminopyrine N-demethylation exhibited linear kinetics. The single apparent Km of the brain aminopyrine N-demethylase (3.39 mM) was at least 3-fold higher than that of the liver (0.18 and 1.13 mM). Coupled with data presented on the inhibitory effects of CO and SKF 525-A on brain and hepatic aminopyrine N-demethylases, the kinetic data suggest that brain aminopyrine N-demethylase exhibits behavior different from that of the liver enzyme. Hexobarbital, meperidine and 3,4-benzo(a)pyrene also were metabolized by rat brain microsomes. No appreciable nuclear of mitochondrial contamination was seen in electron micrographs of the brain microsomal pellet. The presence of cytochrome P-450-dependent microsomal enzymes in the brain offers some new implications regarding regulation of the activity of centrally active compounds.

AB - This study was designed to characterize cytochrome P-450-dependent aminopyrine N-demethylation using microsomes from rat brain and to compare it with N-demethylation of the drug by hepatic microsomes studied in parallel. The cytochrome P-450 content of the microsomal fraction from brain (0.035 nmol/mg of protein) was 3% that of the liver. Using the dithionite difference technique, a carbon monoxide absorbance peak occurred at 448 to 451 nm for brain microsomal cytochrome P-450. Spectral binding of aminopyrine, hexobarbital, androstenedione and aniline to rat brain cytochrome P-450 could not be detected, apparently because of the low concentration of the hemoprotein. Aminopyrine N-demethylase of brain microsomes required oxygen and NADPH for optimal activity and its activity was inhibited by carbon monoxide (CO) and SKF 525-A in a concentration-dependent manner. However, the enzyme was less sensitive to the inhibitory effects of CO and SKF 525-A than the hepatic N-demethylase. Brain aminopyrine N-demethylation was linear with respect to protein concentrations and incubation times of up to 15 min. Enzymatic activity declined when microsomes were incubated longer than 15 min due apparently to enzyme inactivation. Maximal aminopyrine N-demethylase activity of the brain was 0.5 and 17% that of the liver when expressed per milligram of microsomal protein (261.3 pmol/10 min/mg of protein) and nanomole of cytochrome P-450 (6.98 nmol/10 min/nmol of cytochrome P-450), respectively. In contrast to the biphasic kinetic behavior of hepatic aminopyrine N-demethylase, brain aminopyrine N-demethylation exhibited linear kinetics. The single apparent Km of the brain aminopyrine N-demethylase (3.39 mM) was at least 3-fold higher than that of the liver (0.18 and 1.13 mM). Coupled with data presented on the inhibitory effects of CO and SKF 525-A on brain and hepatic aminopyrine N-demethylases, the kinetic data suggest that brain aminopyrine N-demethylase exhibits behavior different from that of the liver enzyme. Hexobarbital, meperidine and 3,4-benzo(a)pyrene also were metabolized by rat brain microsomes. No appreciable nuclear of mitochondrial contamination was seen in electron micrographs of the brain microsomal pellet. The presence of cytochrome P-450-dependent microsomal enzymes in the brain offers some new implications regarding regulation of the activity of centrally active compounds.

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