Purification of mouse brain (Na+ + K+)-ATPase catalytic unit, characterization of antiserum, and immunocytochemical localization in cerebellum, choroid plexus, and kidney

• Published in: The journal of histochemistry and cytochemistry 1984-12, Vol.32 (12), p.1309-1318
• Main Authors: Siegel, GJ, Holm, C, Schreiber, JH, Desmond, T, Ernst, SA
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cerebellum - enzymology; Choroid Plexus - enzymology; Cross Reactions; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Histocytochemistry; Hydrolases; Immune Sera; Intracellular Membranes - enzymology; Kidney Medulla - enzymology; Kidney Tubules - enzymology; Macromolecular Substances; Mice; Microsomes - enzymology; Molecular Weight; Sheep; Sodium-Potassium-Exchanging ATPase - analysis; Sodium-Potassium-Exchanging ATPase - isolation & purification
• ISSN: 0022-1554; 1551-5044
• DOI: 10.1177/32.12.6094658

The denatured catalytic polypeptide of mouse brain (Na+ + K+)-adenosine triphosphatase(ATPase) was separated from microsomal membranes on polyacrylamide gels and used as an immunogen. The antiserum, characterized by immunoblots, recognizes the polypeptide corresponding to the catalytic unit in various fractions of mouse brain and cross-reacts with the catalytic unit from lamb kidney, duck salt gland, and electroplax. The same polypeptide in brain and salt gland is recognized by antiserum raised against purified lamb kidney enzyme. Light microscopy was performed with the peroxidase-conjugated second antibody method. In mouse cerebellum, immunochemical staining outlines Purkinje cell and granule cell perikarya. Intense activity is associated with regions of high synaptic content including the pericellular basket meshes and preaxonal regions of Purkinje cells and the glomeruli in the granular layer. In the molecular layer, the neuropil is diffusely reactive with distinct vertically oriented processes evident. White matter exhibits light stain deposition. Choroid plexus presents abundant reaction product only at ependymal apical surfaces, while the ependymal lining of the fourth ventricle displays little or no immunoreactivity. Specificity of the antiserum was demonstrated further in mouse kidney where staining conforms to the well-characterized localization of the enzyme along basolateral surfaces of cortical and medullary tubules. The biochemical and immunocytochemical data show the efficacy of generating antisera to brain (Na+ + K+)-ATPase using catalytic polypeptide as an immunogen.