Subcellular localization and kinetic properties of aromatase activity in rat brain

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

The conversion of testosterone to estradiol is catalyzed by cytochrome P450 aromatase. In situ aromatization is required for the full expression of the effects of testosterone in the brain. This study examined the subcellular distribution and reaction kinetics of aromatase in the adult rat brain. Preoptic area, hypothalamus and amygdala were homogenized in isotonic sucrose buffered with potassium phosphate. Tissue homogenates were fractionated by ultracentrifugation. Aromatase activity was measured using a previously validated 3H2O assay. Marker enzymes were measured to identify organelles in the different subcellular fractions. Aromatase activity in all 3 tissues was enriched 10-fold in microsomes, but not in other subcellular fractions. The addition of either a NADPH-generating system or 1 mM NADPH to the reaction mixture stimulated aromatase activity in all subcellular fractions, whereas NADH was only minimally effective. In general, substrate affinity constants were equivalent in all brain areas and subcellular fractions (∼10 nM) suggesting that one predominant catalytic form of the enzyme is present in the rat brain. One week after castration, aromatase activity was significantly reduced in all subcellular fractions of preoptic area and in the whole homogenate and microsomal fraction of the hypothalamus. Castration did not significantly alter aromatase activity in any subcellular compartment of amygdala. To more critically evaluate its subcellular localization, aromatase activity was measured in purified synaptosomes. Aromatase activity was not enriched in these preparations suggesting that it is not substantially associated with nerve terminals in rat brain.

Original languageEnglish (US)
Pages (from-to)469-477
Number of pages9
JournalJournal of Steroid Biochemistry and Molecular Biology
Volume52
Issue number5
DOIs
StatePublished - 1995

Fingerprint

Aromatase
Rats
Brain
Subcellular Fractions
Kinetics
Preoptic Area
Castration
Amygdala
NADP
Hypothalamus
Testosterone
Tissue
Aromatization
Synaptosomes
Ultracentrifugation
Enzymes
Microsomes
Reaction kinetics
Organelles
NAD

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Clinical Biochemistry
  • Endocrinology
  • Molecular Biology
  • Molecular Medicine
  • Endocrinology, Diabetes and Metabolism

Cite this

@article{cc1acb1ff1a8433fbec832e2737d4873,
title = "Subcellular localization and kinetic properties of aromatase activity in rat brain",
abstract = "The conversion of testosterone to estradiol is catalyzed by cytochrome P450 aromatase. In situ aromatization is required for the full expression of the effects of testosterone in the brain. This study examined the subcellular distribution and reaction kinetics of aromatase in the adult rat brain. Preoptic area, hypothalamus and amygdala were homogenized in isotonic sucrose buffered with potassium phosphate. Tissue homogenates were fractionated by ultracentrifugation. Aromatase activity was measured using a previously validated 3H2O assay. Marker enzymes were measured to identify organelles in the different subcellular fractions. Aromatase activity in all 3 tissues was enriched 10-fold in microsomes, but not in other subcellular fractions. The addition of either a NADPH-generating system or 1 mM NADPH to the reaction mixture stimulated aromatase activity in all subcellular fractions, whereas NADH was only minimally effective. In general, substrate affinity constants were equivalent in all brain areas and subcellular fractions (∼10 nM) suggesting that one predominant catalytic form of the enzyme is present in the rat brain. One week after castration, aromatase activity was significantly reduced in all subcellular fractions of preoptic area and in the whole homogenate and microsomal fraction of the hypothalamus. Castration did not significantly alter aromatase activity in any subcellular compartment of amygdala. To more critically evaluate its subcellular localization, aromatase activity was measured in purified synaptosomes. Aromatase activity was not enriched in these preparations suggesting that it is not substantially associated with nerve terminals in rat brain.",
author = "Charles Roselli",
year = "1995",
doi = "10.1016/0960-0760(94)00192-O",
language = "English (US)",
volume = "52",
pages = "469--477",
journal = "Journal of Steroid Biochemistry and Molecular Biology",
issn = "0960-0760",
publisher = "Elsevier Limited",
number = "5",

}

TY - JOUR

T1 - Subcellular localization and kinetic properties of aromatase activity in rat brain

AU - Roselli, Charles

PY - 1995

Y1 - 1995

N2 - The conversion of testosterone to estradiol is catalyzed by cytochrome P450 aromatase. In situ aromatization is required for the full expression of the effects of testosterone in the brain. This study examined the subcellular distribution and reaction kinetics of aromatase in the adult rat brain. Preoptic area, hypothalamus and amygdala were homogenized in isotonic sucrose buffered with potassium phosphate. Tissue homogenates were fractionated by ultracentrifugation. Aromatase activity was measured using a previously validated 3H2O assay. Marker enzymes were measured to identify organelles in the different subcellular fractions. Aromatase activity in all 3 tissues was enriched 10-fold in microsomes, but not in other subcellular fractions. The addition of either a NADPH-generating system or 1 mM NADPH to the reaction mixture stimulated aromatase activity in all subcellular fractions, whereas NADH was only minimally effective. In general, substrate affinity constants were equivalent in all brain areas and subcellular fractions (∼10 nM) suggesting that one predominant catalytic form of the enzyme is present in the rat brain. One week after castration, aromatase activity was significantly reduced in all subcellular fractions of preoptic area and in the whole homogenate and microsomal fraction of the hypothalamus. Castration did not significantly alter aromatase activity in any subcellular compartment of amygdala. To more critically evaluate its subcellular localization, aromatase activity was measured in purified synaptosomes. Aromatase activity was not enriched in these preparations suggesting that it is not substantially associated with nerve terminals in rat brain.

AB - The conversion of testosterone to estradiol is catalyzed by cytochrome P450 aromatase. In situ aromatization is required for the full expression of the effects of testosterone in the brain. This study examined the subcellular distribution and reaction kinetics of aromatase in the adult rat brain. Preoptic area, hypothalamus and amygdala were homogenized in isotonic sucrose buffered with potassium phosphate. Tissue homogenates were fractionated by ultracentrifugation. Aromatase activity was measured using a previously validated 3H2O assay. Marker enzymes were measured to identify organelles in the different subcellular fractions. Aromatase activity in all 3 tissues was enriched 10-fold in microsomes, but not in other subcellular fractions. The addition of either a NADPH-generating system or 1 mM NADPH to the reaction mixture stimulated aromatase activity in all subcellular fractions, whereas NADH was only minimally effective. In general, substrate affinity constants were equivalent in all brain areas and subcellular fractions (∼10 nM) suggesting that one predominant catalytic form of the enzyme is present in the rat brain. One week after castration, aromatase activity was significantly reduced in all subcellular fractions of preoptic area and in the whole homogenate and microsomal fraction of the hypothalamus. Castration did not significantly alter aromatase activity in any subcellular compartment of amygdala. To more critically evaluate its subcellular localization, aromatase activity was measured in purified synaptosomes. Aromatase activity was not enriched in these preparations suggesting that it is not substantially associated with nerve terminals in rat brain.

UR - http://www.scopus.com/inward/record.url?scp=0029018363&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029018363&partnerID=8YFLogxK

U2 - 10.1016/0960-0760(94)00192-O

DO - 10.1016/0960-0760(94)00192-O

M3 - Article

C2 - 7748812

AN - SCOPUS:0029018363

VL - 52

SP - 469

EP - 477

JO - Journal of Steroid Biochemistry and Molecular Biology

JF - Journal of Steroid Biochemistry and Molecular Biology

SN - 0960-0760

IS - 5

ER -