Differences in phosphorylation of the two large subunits of brine shrimp Na, K‐ATPase

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Abstract
Analysis of purified Na,K‐ATPase from brine shrimp nauplii revealed two molecular forms of the α subunit separable by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis [G.L. Peterson, R.D. Ewing, S.R. Hootman, and F.P. Conte (1978) J. Biol. Chem. 253:4762]. The molecular form with lower mobility is designated α1 and the one with higher mobility, α2 in a neutral or alkaline gel system. Differences in Na+‐dependent, K+‐sensitive phosphorylation of these two molecular forms have been investigated by directly measuring the radioactivity present in each phosphoprotein after separation of the two forms by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. In the presence of Na+, Mg2+, and ATP, when the ATP concentration is above 1 μM, both α subunits are phosphorylated, although the phosphoprotein content of α1 is considerably greater than that of α2. Below 1 μM ATP, the phosphoprotein content of α2 is even further reduced. These striking differences in phosphorylation at low ATP concentrations are not due to a greater instability of the α2 phosphoprotein during the long electrophoresis times or during fixation, staining, and destaining. The proportion of total phosphoprotein content in α2, as well as the relationship between phosphoprotein content and ATP concentration, is unchanged when the radioactive analysis is performed on frozen gels that have been electrophoresed for shorter times, even though the actual amount of phosphorylation is 15 times greater than with fixed gels. Since the concentration of α1 and α2 vary during development [G.L. Peterson, L. Churchill, J.A. Fisher, and L.E. Hokin (1982) J. Exp. Zool. 221:295], the differences in phosphorylation may be relevant to differences in Na,K‐ATPase activity during different development stages.