The present study investigates structural characteristics and hypoxia-induced translational modification of the α 1 and α 3 subunits of the Na+,K+-ATPase in guinea pig brain using 2-D polyacrylamide gel electrophoresis (PAGE). Cerebral cortical synaptosomes were prepared from fetal, juvenile and adult guinea pigs. 13 normoxic: 4 preterm, 1 term, 6 at 8-15 days, 2 adults and 4 hypoxic: 2 preterm, 1 term, 1 adult. Brain tissue hypoxia was documented biochemically by decreased levels of ATP and phosphocreatine. Samples were denatured and 2-D PAGE performed. Silver-stained gels were scanned with a computing densitometer and the images analyzed using a Sun SPARCstation with MELANIE software. Immunoblotting was carried out using specific antibodies for the α1 and α 3 subunits of Na,K-ATPase. Immunoreactivity with anti-α1 antibody occurred in two isoelectric series, each with 4 separate components: one at MW≈54 kDa, pI range 4.85-5.05, and the second at MW 63 kD and pI range 6.05-6.70. Immunoreactivity with the anti-α 3 antibody was present in an isoelectric series of 4 spots at MW≈60kDa and pI range 5.10-5.30. No change in relative abundance of spots corresponding to the a1 and a3 Na,K-ATPase subunits were observed in silver-stained protein maps from hypoxic brain tissues compared to normoxic tissues. Although previous studies using unidimensional PAGE have reported doublet bands corresponding to the subunits of the Na,K-ATPase, the presence of 8 distinct spots for the α 1 subunit and 4 for the α 3 subunit demonstrates a range of structural variability not previously described. We speculate that the isoelectric heterogenicity observed is due to modification of the synaptosomal Na,K-ATPase subunits by post-translational phosphorylation.

(Funded by Sci. Res. Found. Min. of Edu., Italy, NIH#HD-20337)