Quantitation of S-adenosylmethionine decarboxylase protein.
A method for the specific labeling of the active site of S-adenosylmethionine decarboxylase was developed. The method consisted of incubating cell extracts with 3H-decarboxylated S-adenosylmethionine and sodium cyanoborohydride in the presence of a spermidine synthase inhibitor. Under these conditions, S-adenosylmethionine decarboxylase was labeled specifically and stoichiometrically. This procedure was used (a) to establish that the subunit molecular weight of S-adenosylmethionine decarboxylase from rat liver, prostate, and psoas and from mouse SV-3T3 cells was 32 000, (b) to titrate the number of active molecules of S-adenosylmethionine decarboxylase in various cell extracts, and (c) to provide a high specific activity labeled preparation of S-adenosylmethionine decarboxylase for use in radioimmunoassay of this enzyme. Competitive radioimmunoassays using this labeled antigen had a sensitivity such that 3 fmol (0.1 ng) of enzyme protein could be quantitated. The rapid loss of S-adenosylmethionine decarboxylase which occurred when SV-3T3 cells were exposed to exogenous polyamines was shown to be due to a rapid decline in the amount of enzyme protein measured both by titration of the active site and by radioimmunoassay.
D012437:Adenosylmethionine Decarboxylase / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002250:Carbon Radioisotopes; D002262:Carboxy-Lyases / Q000378:metabolism*; D002478:Cells, Cultured; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D051379:Mice; D009132:Muscles / Q000201:enzymology; D011467:Prostate / Q000201:enzymology; D011863:Radioimmunoassay / Q000379:methods; D051381:Rats; D012436:S-Adenosylmethionine / Q000378:metabolism; D014316:Tritium
D002250:Carbon Radioisotopes; D014316:Tritium; D012436:S-Adenosylmethionine; D002262:Carboxy-Lyases; D012437:Adenosylmethionine Decarboxylase