Knowledge concerning the interaction between protein O-carboxyl methyltransferase (PCM; E.C. 2.1.1.24) and neuronal calmodulin binding proteins may better understanding of the regulation and repair of neuronal proteins during development, aging, and disease. Two hypotheses may explain the potential functions of PCM: PCM recognizes abnormal aspartyl residues on age-damaged proteins or PCM regulates the functional state of calmodulin binding proteins such as calcineurin, a calmodulin-dependent protein phosphatase. We will examine these hypotheses using various approaches. 1) We have developed a biotinylated calmodulin overlay technique which is a rapid and sensitive determination of calmodulin binding proteins after gel electrophoresis (SDS-PAGE) and electroblot transfer to nitrocellulose. Purified calmodulin binding proteins and crude samples from brain will be electroblotted, renatured, and carboxylmethylated directly on the nitrocellulose with purified brain PCM; both the extent of methylation and its effect on subsequent biotinylated calmodulin binding will be determined. 2) Purified calmodulin binding proteins and brain homogenates will be carboxylmethylated in vitro with PCM, immobilized on nitrocellulose slot blots, and calmodulin binding will be quantified and correlated with the carboxylmethylation. 3) Homogenates, slices, and neuroblastoma cell lines from brains of developing, adult, and aged rats will be incubated with methyl donors and carboxylmethylated proteins separated using acidic HPLC reversed-phase methodology or by a rapid HPLC calmodulin affinity technique. Comparison with carboxylmethylated proteins resolved using acidic SDS-gel electrophoresis and HPLC methods, will determine a) whether protein methylation increases nonspecifically with age, b) whether calmodulin binding proteins are preferentially carboxylmethylated regardless of age or c) whether changes in regional levels of PCM activity or content influence the amount and type of carboxylmethylated protein. 4) PCM itself will be biotinylated and used as an overlay probe for specific protein-protein interactions with developing, adult, and aged brain proteins. Examination of age related alterations in PCM binding versus specific, age-independent interactions will further clarify one of the hypotheses. 5) Specific immunologic probes for PCM and several of the calmodulin binding proteins will be used to determine age-related alterations in levels of the target antigens using both immunocytochemistry and solid-phase immunoassay. These studies can illuminate the potential roles of protein methylation in nervous tissues during aging.
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