Accumulation of substrates for protein L-isoaspartyl methyltransferase in adenosine dialdehyde-treated PC12 cells.

Protein isoaspartyl methyltransferase is implicated in the repair of age-damaged proteins containing atypical, isoaspartyl linkages. To test the prediction that isoaspartyl proteins would accumulate during methyltransferase inhibition, rat PC12 cells were treated with the indirect methylation inhibitor, adenosine dialdehyde. We observed a marked, dose- and time-dependent, reversible accumulation of substrates for the enzyme that closely paralleled the elevation of its competitive inhibitor, S-adenosylhomocysteine. The accumulation of substrates also paralleled a cytostatic action of adenosine dialdehyde; however, 30 microM 3-deazaadenosine, another indirect methylation inhibitor, also caused an accumulation of substrates without affecting cell division, and other cytostatic agents did not affect substrate levels. Acidic gel electrophoresis revealed increased methyl-accepting capacity in a broad spectrum of proteins, with predominant increases in discrete bands of M(r) 46,000 and 110,000. A major substrate (M(r) 17,400) in untreated cells did not increase in methyl-accepting capacity during treatment. Methyl groups at the accumulated sites did not survive conventional electrophoresis, indicating the lability characteristic of isoaspartyl methyl esters in damaged proteins. These results are consistent with an involvement of the methyltransferase in the metabolism of damaged proteins, and they provide a basis for the characterization of physiological substrates for the enzyme.