The carboxylmethylation of cerebral membrane-bound proteins increases with age

Recently, we have characterized a membrane-bound (mb) component of brain protein carboxylmethyltransferase II (PCMT) which effectively carboxylmethylates endogenous mb methyl-accepting proteins (MAPs). ( Neurochem. Int., 10 (1987) 155). We have also shown that exposing mb-MAPs to mild alkali leads to a marked increase in their recognition by PCMT. Since one of the likely consequences of the alkaline treatment appears to be the deamidation of selected protein-bound asparagines or aspartates, followed by the formation, in their place, of d-or l-isoaspartates, it is reasonable to assume that mb-MAPs constitute unique targets for the mb-PCMT because they contain such unnatural aspartate residues. Testing the relevance of this notion to the aging of cerebral mb-MAPs we focus in this report on age-related changes involving mb-MAPs. When two-or six-times washed (in 50 mM NaPO 4 buffer, pH 6.5) 17 500 g, 30-min membranes or Percoll-gradient purified synaptic membranes were prepared from young (3–4 months) and old (11–12 months) rat brains and were incubated with 20 μM [ 3H]methyl S- adenosyl- l- methionine at pH 6.0, mb-MAP carboxyl[ 3H]methylation was significantly more intense in the old than in the young membranes, no additional increase being noted at 28–35 months. Mb-MAP carboxylmethylation increases were confirmed over a wide range of membrane protein concentrations and incubation times and are taken to reflect age-related modifications of the primary structure of susceptible mb-MAPs. To investigate these, we incubated young and old membranes, as well as their Lubrol-P x(1%) extracts (30 min, 0°C), with 0.05 M NH 4OH for 90 min at 37°C, a treatment which left PCMT activity largely unaffected. Our findings reveal