Localization of aromatase in synaptosomal and microsomal subfractions of quail (Coturnix coturnix japonica) brain.
The subcellular distribution patterns of aromatase, 5 alpha- and 5 beta-reductase in the hypothalamus/preoptic area of Japanese quail were studied using standard methods of centrifugation, and fractional constituents were identified by marker enzymes. Aromatase was concentrated 8-fold in the 100,000 g pellet (P3) along with a 3-fold enrichment in the microsomal marker NADPH-cytochrome c reductase (NCR) a result consistent with glandular tissues. In addition, aromatase was enriched 2-fold in the 11,000 g pellet (P2) and, owing to its large size, this fraction accounted for more total activity than P3. Although P2 contained the mitochondrial marker succinate dehydrogenase (SDH), treatment with Triton X-100 to solubilize membranes and release occluded enzymes increased measured NCR and the cytosol marker lactate dehydrogenase (LDH) 2- and 4-fold, respectively--evidence that this fraction was composed of mitochondria plus synaptosomes (pinched-off nerve terminals). To further explore the location of aromatase in the 11,000 g fraction, P2 was exposed to hypotonic buffer, a treatment known to cause lysis of synaptosomes, and then separated into three fractions P2a (11,000 g pellet), P2b (100,000 g pellet) and P2s (100,000 g supernatant). Aromatase colocalized with the microsomal marker NCR (13- and 4-fold increase, respectively) in the 100,000 g (P2b) pellet which was, however, devoid of mitochondrial enzyme activity. We infer from this that a significant portion of aromatase in brain is associated with smooth membranes present inside synaptosomes. 5 beta-Reductase in quail brain subfractions was enriched 6-fold in the 100,000 g supernatant together with a 4-fold enrichment in the cytosolic marker LDH.(ABSTRACT TRUNCATED AT 250 WORDS)
Schlinger
B A
BA
Department of Biological Sciences, Boston University, Mass.
Callard
G V
GV
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000032:analysis; D000818:Animals; D001141:Aromatase / Q000032:analysis*; D003370:Coturnix / Q000378:metabolism*; D007031:Hypothalamus / Q000201:enzymology*; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D010088:Oxidoreductases / Q000032:analysis; D011301:Preoptic Area / Q000201:enzymology; D011784:Quail / Q000378:metabolism*; D013574:Synaptosomes / Q000201:enzymology*; D014018:Tissue Distribution
D010088:Oxidoreductases; D001141:Aromatase; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase; C021936:3-oxo-5 beta-steroid delta 4-dehydrogenase