NA+,K+-ATPASE ISOFORMS IN CALF BRAIN

The isozyme composition and the type of the Na+, K+-ATPase functional complex in calf brain membranes were determined. To isolate functionally active enzymes from microsomes of calf gray matter, brainstem and stem axolemma, two methodical approaches - selective removal of contaminating proteins according to Jorgensen and selective enzyme solubilization with the following reformation of the membrane structure by the Esmann method - were applied. The protein components of the isolated preparations were separated by SDS-PAAG electrophoresis, electroblotted on PVDF-membranes and subjected to structural screening. The analysis revealed not only the N-terminal amino acid sequences of the subunits: alpha-1-GRDKYEPAAVS; alpha-2-GREYXPAATT; alpha-3-MGDKKDDKXS; beta-1-ARGKAKEEGSXKKFI; beta-2-VIXKEKKSXXXVV, but also the isoform composition and the type of functional complexes alpha-n-beta-m for the studied microsomes. Na+, K+-ATPase of brain gray matter shows the biphase kinetics of ouabain inhibition (K(i) almost-equal-to 10(-6) M and K(i) almost-equal-to 1,5.10(-8) M), and is represented by a set of isozymes alpha-1-beta-1 and alpha-2-beta-m, alpha-3-beta-m (where m = 1 and/or 2), alpha-1-beta-1 being predominant. Na+, K+-ATPase of brainstem and stem axolemma is a mixture of mainly isozymes alpha-2-beta-1, alpha-3-beta-1 having the same constants for ouabain inhibition (K(i) almost-equal-to 10(-7) M), in axolemma form alpha-3-beta-1 being predominant. The increased sensitivity of catalytic subunit alpha-3 of the native enzyme to endogenic proteolysis was revealed, isolation of the enzyme containing this type of the catalytic subunit is possible only in the presence of proteases inhibitor - diisopropylfluorophosphate (DIPF). Without the inhibitor specific fragmentation of the polypeptide chain yielding rather a stable N-terminal fragment (M almost-equal-to 55 kDa) was observed.