Identification of a key protein associated with cerebral ischemia

The biochemical effects of permanent focal ischemia following unilateral occlusion of the middle cerebral artery in rats were studied by determining the content of specific proteins of the affected areas in the cerebral hemisphere. Brain proteins were prepared 72 h after the occlusion and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. A significant increase in 66 and 80 kDa components and a paradoxical decrease in 260 kDa protein occurred in the ischemic brain tissues. The 66 and 80 kDa protein bands were identified as albumin and transferrin, respectively. The 260 kDa protein was analyzed by peptide mass fingerprinting (PMF) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The isoelectric point of the 260 kDa protein was 4.65 determined by isoelectric focusing. The data obtained from PMF were used in searching the protein database for homologous components. Three proteins with partial homology were identified. They were the microtubule-associated protein 1A, protein-tyrosine phosphatase zeta precursor (phosphacan), and protein kinase A anchoring protein 6. Polyclonal antibodies against the 260 kDa protein were raised and used to immunolocalize the antigen in various tissues. Positive staining occurred with brain neurons and pyramidal cells, islet cells, podocytes of kidney glomeruli, and endothelial cells of the venous sinuses of the spleen. The localization of 260 kDa protein strongly implies its function in these tissues. Its physiological and pathophysiological significances need to be clarified in future.