Brain cell membrane Na⁺,K⁺-ATPase modification following hypoxia in the guinea pig fetus

The effect of hypoxia in utero on the affinity of the active sites of Na⁺,K⁺-ATPase for Na⁺, K⁺ and ATP of the fetal guinea pig brain was investigated. Brain cell membranes were prepared from normoxic and hypoxic guinea pig fetuses, and a detailed enzyme kinetics analysis was carried out. In the hypoxic fetal brain membranes the K_a 0.5 for Na⁺ and K⁺ increased 104% and 20%, respectively, indicating a decrease in the affinity of the active sites of the enzyme for Na⁺ and K⁺. The affinity of the ATP site increased, as indicated by a decrease in the K_m of 37% in hypoxic brain. The results indicate that changes in the affinity of active sites would affect the phosphorylation and dephosphorylation mechanisms of the Na⁺, K⁺-ATPase reaction. The increased affinity for ATP will favor the phosphorylation step, but will be opposed by the decrease in the Na⁺ affinity. The decreased affinity of the active site for K⁺ would oppose the dephosphorylation of the enzyme-P complex causing the enzyme to be trapped in an inactive phosphorylated state. The results demonstrated the sensitivity of the Na⁺,K⁺-ATPase active sites to hypoxia, and illustrated a selective modification of the enzyme active sites under hypoxic conditions, a key mechanism altering the cell membrane function leading to hypoxic brain damage.