Subcellular localization and kinetic properties of aromatase activity in rat brain.
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 (approximately 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.
Roselli
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CE
Department of Physiology, Oregon Health Sciences University, Portland 97201-3098, USA.
D000679:Amygdala / Q000201:enzymology / Q000648:ultrastructure; D000818:Animals; D001141:Aromatase / Q000032:analysis* / Q000378:metabolism*; D001921:Brain / Q000201:enzymology* / Q000648:ultrastructure*; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D004958:Estradiol / Q000378:metabolism; D007031:Hypothalamus / Q000201:enzymology / Q000648:ultrastructure; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D009919:Orchiectomy; D011301:Preoptic Area / Q000201:enzymology / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000201:enzymology*; D013574:Synaptosomes / Q000201:enzymology; D013739:Testosterone / Q000378:metabolism; D014461:Ultracentrifugation
D013739:Testosterone; D004958:Estradiol; D009249:NADP; D001141:Aromatase