Structural Peculiarities of Na+,K+-ATPase Isozymes from the Calf Brain |
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Authors: | N M Vladimirova E N Sautkina T I Murav'eva T V Ovchinnikova N A Potapenko |
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Institution: | (1) Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997, Russia |
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Abstract: | Functionally active preparations of Na+,K+-ATPase isozymes from calf brain that contain catalytic subunits of three types ( 1, 2, and 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 their 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 1 1 type and minor amounts of isozymes of the 2 2( 1) and the 3 1( 2) type. The axolemma contains 2 1 and 3 1 isozymes. A carbohydrate analysis indicated that 1 1 enzyme preparations from the brain grey matter substantially differ from the renal enzymes of the same composition in the glycosylation of the 1 isoform. An enhanced sensitivity of the 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 Y493 was localized (residue numbering is that of the human 3 subunit). This sequence corresponds to one of the regions of the greatest variability in 1-, 2-, 3-, and 4-subunits, but at the same time, it is characteristic of the 3 isoforms of various species. The presence of the 3 isoform of tubulin (cytoskeletal protein) was found for the first time in the high-molecular-mass Na+,K+-ATPase 3 1 isozyme complex isolated from the axolemma of brain stem neurons, and its binding to the 3 catalytic subunit was shown. |
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Keywords: | Na
+ K
+
-ATPase axolemma glycosylation isoforms isozymes proteolysis tubulin |
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