Structural peculiarities of Na+,K+-ATPase isozymes from the calf brain

• Published in: Bioorganicheskaia khimiia Vol. 29; no. 2; p. 146
• Main Authors: Vladimirova, N M, Sautkina, E N, Murav'eva, T I, Ovchinnikova, T V, Potapenko, N A
• Format: Journal Article
• Language: Russian
• Published: Russia (Federation) 01.03.2003
• Subjects: Amino Acid Sequence; Animals; Biochemistry - methods; Brain - enzymology; Brain Stem - cytology; Brain Stem - enzymology; Catalytic Domain; Cattle; Enzyme Inhibitors - pharmacology; Glycosylation; Isoenzymes - antagonists & inhibitors; Isoenzymes - chemistry; Isoenzymes - metabolism; Kidney - enzymology; Kinetics; Microsomes - enzymology; Molecular Sequence Data; Neuroglia - cytology; Neuroglia - enzymology; Neurons - enzymology; Ouabain - pharmacology; Sequence Homology, Amino Acid; Sodium-Potassium-Exchanging ATPase - antagonists & inhibitors; Sodium-Potassium-Exchanging ATPase - chemistry; Sodium-Potassium-Exchanging ATPase - isolation & purification; Sodium-Potassium-Exchanging ATPase - metabolism; Solubility; Substrate Specificity; Tubulin - metabolism
• ISSN: 0132-3423

Functionally active preparations of Na+,K(+)-ATPase isozymes from calf brain that contain catalytic subunits of three types (alpha 1, alpha 2, and alpha 3) were obtained using two approaches: a selective removal of contaminating proteins by the Jorgensen method and a selective solubilization of the enzyme with subsequent reconstitution of the membrane structure by the Esmann method. The ouabain inhibition constants were determined for the isozymes. The real isozyme composition of the Na+ pump from the grey matter containing glial cells and the brain stem containing neurons was determined. The plasma membranes of glial cells were shown to contain mainly Na+,K(+)-ATPase of the alpha 1 beta 1 type and minor amounts of isozymes of the alpha 2 beta 2 (beta 1) and the alpha 3 beta 1 (beta 2) type. The axolemma contains alpha 2 beta 1- and alpha 3 beta 1 isozymes. A carbohydrate analysis indicated that alpha 1 beta 1 enzyme preparations from the brain grey matter substantially differ from the renal enzymes of the same composition in the glycosylation of the beta 1 isoform. An enhanced sensitivity of the alpha 3 catalytic subunit of Na+,K(+)-ATPase from neurons to endogenous proteolysis was found. A point of specific proteolysis in the amino acid sequence PNDNR492 decreases Y493 was localized (residue numbering is that of the human alpha 3 subunit). This sequence corresponds to one of the regions of the greatest variability in alpha 1, alpha 2, alpha 3, and alpha 4-subunits, but at the same time, it is characteristic of the alpha 3 isoforms of various species. The presence of the beta 3 isoform of tubulin (cytoskeletal protein) was found for the first time in the high-molecular-mass Na+,K(+)-ATPase alpha 3 beta 1 isozyme complex isolated from the axolemma of brain stem neurons, and its binding to the alpha 3 catalytic subunit was shown.