CHARACTERIZATION OF A VERY-HIGH-AFFINITY OUABAIN BINDING-SITE IN TERMFETAL GUINEA-PIG BRAIN NA-ATPASE(,K+)

OBJECTIVE: To characterize the very-high-affinity ouabain binding site in fetal brain and determine its sensitivity to hypoxia. METHODS: Stu dies were performed on six normoxic and six hypoxic guinea pig fetuses at term and in six adult guinea pigs. Fetuses were delivered after th e pregnant female had been exposed to 21% or 7% oxygen for 1 hour. Bra in cell membranes were prepared and ouabain binding studies were perfo rmed. Ouabain binding was determined in the presence and absence of er ythrosin B, a known inhibitor of high-affinity ouabain binding sites. RESULTS: Normoxic term fetal brain membrane had a Bmax (receptor numbe r) of 84.2 +/- 13.6 pmol/mg protein, which decreased to 5.9 +/- 3.8 pm ol/mg protein (93.0% decrease, P < .001) in the presence of erythrosin B. Normoxic fetal brain had a dissociation constant (Kd) (receptor af finity) of 24.6 +/- 4.5 nmol/L, which was unchanged in the presence of erythrosin B (Kd = 20.7 +/- 15.4 nmol/L, P = nonsignificant [NS]). Hy poxic term fetal brain had a Bmax of 74.7 +/- 8.3 pmol/mg protein, whi ch decreased to 7.1 +/- 3.9 pmol/mg protein (90.5% decrease, P < .001) in the presence of erythrosin B. Hypoxic fetal brain had a Kd of 22.9 +/-1.9 nmol/L, which was unchanged in the presence of erythrosin B) ( Kd = 24.5 +/- 9.9 nmol/L, P = NS). The adult control guinea pig brain had a Bmax of 104.1 +/- 13.3 pmol/mg protein, which decreased to 44.9 +/- 10.5 pmol/mg protein (P < .001) in the presence of erythrosin B, a nd a Kd of 214.3 +/- 31.3 nmol/L, which remained unchanged in the pres ence of erythrosin B (Kd was 165.4 +/- 36.0 nmol/L, P = NS). CONCLUSIO N: Fetal brain has a unique very-high-affinity ouabain binding site th at is absent in adult brain and is sensitive to erythrosin B and resis tant to hypoxia. We speculate that the presence of a Na+,K+-ATPase mol ecule with a very-high-affinity site may be advantageous to the fetal brain during early maturation as well as during hypoxia or ischemia. C opyright (C) 1998 by the Society for Gynecologic Investigation.