Our long-range objective is to find out whether the observed age-related changes in brain protein carboxylmethylation represent physiologically significant and possibly diagnostic markers of cellular aging in the brain. As part of Objective 1, we intend to a) determine why the in vitro carboxylmethylation of brain memrane proteins 55K and 20K first becomes detectable at 11-12 months and is no longer detectable at 28-29 months; b) determine if proteins 55K and 20K are neuronal, glial, synaptic, or ubiquitous and compare their carboxylmethylation in vitro and in vivo; c) solubilize proteins 55K and 20K and separate them from other proteins by two dimensional electrophoresis-isoelectric focusing (Ref. 57) at pH 2.4, comparing their properties at 3, 11 and 29 months; d) incorporate 35S-methionine into them and obtain peptide maps from them at 3, 11 and 39 months. As part of Objective 2, we intend to a) determine the D-aspartate content of brain proteins at 3, 11 and 29 months; b) identify the carboxyl-3[H]-methylatable amino acid residue of brain proteins as D-asparartic acid; c) determine the extent of the in situ carboxylmethylation of brain proteins in 3, 11 and 29 month-old rats; d) test brain proteins for the presence of Asn-Gly residues and verify the propensity of this sequence to deamidate and yield L-isoaspartyl residues, as a function of age. As part of Objective 3 we intend to purify and compare the cytosolic and membrane-bound components of brain protein carboxylmethyltransferase II in 3, 11 and 29 month-old rats for age-related differences in substrate recognition, affinity labelling (by 3[H]-methylS-adenosylmethionine), heat stability, and membrane embedment.
| Caamano, C A; Azcurra, J M; Sellinger, O Z et al. (1989) Kinetics of carboxylmethylation of the charge isoforms of myelin basic protein by protein methyltransferase II. J Neurochem 53:1883-8 |
