Na(+)-K(+)-ATPase isoforms in different areas of calf brain.

Abstract

The isoform composition and type of Na(+)-K+ ATPase functional complexes in a number of calf brain membranes were determined. Functionally active enzymes were obtained from microsomes from calf cerebral cortex grey matter, brain stem, and stem axolemma by two different methods involving (1) the selective removal of contaminating proteins according to Jorgensen (1974) and (2) the selective solubilization of the enzyme with subsequent reformation of the membrane structure according to Esmann (1988). The protein components of the isolated preparations were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, transferred to an immobilon membrane [poly(vinylidene difluoride) membrane] by electroblotting, and subjected to structural analysis. The N-terminal amino acid sequences of the alpha- and beta-subunits (alpha 1, alpha 2, alpha 3, beta 1, beta 2) and the isoform composition and type of alpha n beta m functional complexes present in the different microsome preparations were determined. Brain grey matter Na(+)-K+ ATPase was characterized by biphasic kinetics with respect to ouabain inhibition (Ki approximately 10(-6) M and -1.5 x 10(-8) M) and comprised a set of isozymes with subunit compositions of alpha 1 beta 1, alpha 2 beta m, and alpha 3 beta m (where m = 1 and/or 2), with the alpha 1 beta 1 form clearly predominating. Na(+)-K+ ATPase from brain stem and axolemma consisted mainly of a mixture of the isozymes alpha 2 beta 1 and alpha 3 beta 1, which had identical ouabain inhibition constants (Ki approximately 10(-7) M), but in the axolemma there was a large quantity of the alpha 3 beta 1 isozyme. The catalytic subunit alpha 3 within the untreated enzyme complex had increased sensitivity towards endogenous proteolysis. It was therefore possible to isolate enzyme containing the alpha 3 catalytic subunit only in the presence of the protease inhibitor diisopropyl fluorophosphate (DIPF). In the absence of this inhibitor there was a specific fragmentation of the polypeptide chain, resulting in the formation of an extremely stable N-terminal fragment of molecular mass 55 kDa.