Previous studies have shown that the high affinity Ca++-ATPase enzyme located on the outer nuclear membrane is responsible for ATP-dependent calcium entry into the nucleus. The present study investigates the development of high affinity Ca++-ATPase activity as a function of gestational age. Studies were performed on 18 guinea pig fetuses at 45 days (preterm) and 60 days (term) gestation. The fetuses were delivered by cesarean section, and the fetal brains were harvested and placed in buffer. Fetal cerebral cortical nuclei were isolated by density gradient centrifugation. The activity of the Mg++-dependent and Mg++-independent high affinity Ca++-ATPase was determined. The activity was determined in a 1 ml assay medium containing 20 mM HEPES buffer (pH 7.0), 100 mM KCl, 95 mM CaCl2, 0 or 250 mM MgCl2, 1 mM ouabain, 1 mM ATP, and 150 mg nuclear membrane protein. The reaction was carried out for 30 min at 37°C, a period during which the rate of reaction was linear. The reaction was stopped by the addition of 0.5 ml 12.5% trichloroacetic acid; the samples were centrifuged, and the supernatant analyzed for the inorganic phosphate. The activity of the enzyme was expressed as nmoles Pi/mg protein/hr. In 45 day fetal nuclei, the activity of total, Mg++-dependent and Mg++-independent Ca++-ATPase activity was 485.3 ± 52.2, 333.7 ± 50.6 and 151.7 ± 30.6 nmoles/mg protein/hr, respectively. Activity in 60 day fetal nuclei was 753.0 ± 30.0, 614.7 ± 99.7 and 136.3 ± 72.6 nmoles/mg protein/hr, respectively, indicating a significant increase in the total and Mg++-dependent high affinity Ca++-ATPase activity in term fetal cerebral cortical nuclei compared to the preterm. Since intranuclear calcium controls nuclear functions including gene transcription, we speculate that the increased activity of high affinity Ca++-ATPase will potentially increase intranuclear calcium and influence apoptotic events in the developing brain at term.
