Amplification and detection of substrates for protein carboxyl methyltransferases in PC12 cells.
A strategy that facilitates the identification of substrates for protein carboxyl methyltransferases that form "stable" methyl esters, i.e., those that remain largely intact during conventional polyacrylamide gel electrophoresis is described. Rat PC12 cells were cultured in the presence of adenosine dialdehyde (a methylation inhibitor) to promote the accumulation of hypomethylated proteins. Nonidet P-40 cell extracts were then incubated in the presence of S-[methyl-3H]adenosyl-L-methionine to label methyl-accepting sites via endogenous methyltransferases. After labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel slices were incubated in 4 N methanesulfonic acid or 6 N HCl to hydrolyze methyl esters. The resulting [3H]methanol was detected by trapping in liquid scintillation fluid. Seven carboxyl methylated proteins were observed with masses ranging from 18 to 96 kDa. Detection of five of these proteins required prior treatment of cells with adenosine dialdehyde, while methyl incorporation into one protein at 18 kDa was substantially enhanced by the treatment. The use of acidic conditions for methyl ester hydrolysis has an important advantage over assays that utilize alkaline hydrolysis conditions. In PC12 cells, and possibly other cell types where there are significant levels of arginine methylation, the methanol signal becomes obscured by high levels of volatile methylamines generated under the alkaline conditions. Carrying out diffusion assays under acidic conditions eliminates this interference. Adenosine dialdehyde, by virtue of increasing the methyl-accepting capacity of substrates for protein carboxyl methyltransferases, in combination with a more selective assay for carboxyl methylation, should prove useful in the isolation and characterization of new protein carboxyl methyltransferases and their substrates.
Najbauer
J
J
School of Biological Sciences, University of California, Irvine 92717.
Johnson
B A
BA
Aswad
D W
DW
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D066298:In Vitro Techniques; D000432:Methanol / Q000032:analysis; D008745:Methylation; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D012436:S-Adenosylmethionine / Q000378:metabolism
D011506:Proteins; C027579:periodate-oxidized adenosine; D012436:S-Adenosylmethionine; D008780:Methyltransferases; D000241:Adenosine; D000432:Methanol