Abstract
Treatment of tobacco liquid suspension cultures with methylglyoxal bis(guanylhydrazone) (MGBG) an inhibitor of S-adenosylmethionine decarboxylase, resulted in a dramatic overproduction of a 35-kDa peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Malmberg, R.L., and McIndoo, J. (1983) Nature 305, 623-625). MGBG treatment also resulted in a 20-fold increase in the activity of S-adenosylmethionine decarboxylase. Purification of S-adenosylmethionine decarboxylase from MGBG-treated cultures revealed that the overproduced 35-kDa peptide and S-adenosylmethionine decarboxylase are identical. Precursor incorporation experiments using [3H] methionine and [35S]methionine revealed that MGBG does not induce any increased synthesis of S-adenosylmethionine decarboxylase but rather stabilizes the protein to proteolytic degradation. The half-life of the enzyme activity was increased when MGBG was present in the growth medium. In addition to stabilizing S-adenosylmethionine decarboxylase, MGBG also resulted in the rapid and specific loss of arginine decarboxylase activity with little effect ornithine decarboxylase. The kinetics of this effect suggest that arginine decarboxylase synthesis was rapidly inhibited by MGBG. Exogenously added polyamines had little effect on ornithine decarboxylase, whereas S-adenosylmethionine and arginine decarboxylase activities rapidly diminished with added spermidine or spermine. Finally, inhibition of ornithine decarboxylase was lethal to the cultures, whereas inhibition of arginine decarboxylase was only lethal during initiation of growth in suspension culture.
Highlights
Treatment of tobacco liquid suspensioncultures with decarboxylase, which, in parallel with ornithine decarboxylmethylglyoxalbis(guany1hydrazone) (MGBG) anin- ase, results in the production of putrescine [7]
In addition to stabilizingS- spermine aswell as low ornithine decarboxylase and S-adenadenosylmethioninedecarboxylase, MGBG re- osylmethionine decarboxylase activities [12]. ts4 has never sulted in the rapid ansdpecific loss of arginine decar- regenerated beyond the stageof small shoots
Added polyaminehsad little effect occurring in ts4 was observed in wild-type cultures exposed onornithinedecarboxylase,whereas S-adenosylme- to sublethal levels of MGBG [13].A revertant of ts4 which thionine and arginine decarboxylase activities rapidlycontained low S-adenosylmethionine decarboxylase activity diminishedwithaddedspermidine or spermine.inhibition of ornithine decarboxylase was lethal to the cultures, whereas inhibition of arginine decarboxylase was only lethal during initiatoifongrowth in suspension culture
Summary
Ylase, spermidine results in rapid loss of S-adenosylmethionine decarboxylase activity. Nizer at a ratio of 1 ml Tris/putrescine buffer (100 mM Tris-C1, 100 Adenosylmethionine decarboxylaseand arginine decarboxyimM EDTA, 10 mM dithiothreitol, 0.04 mM pyridoxal phosphate, 0.5 ase activities increased 10-20-fold 2 days after dilution, and mM putrescine, pH 8.0)/gram of cells. Each fraction was precipitated ity chromatography (Fig. 1).Stationary phase cultures were with 60% ammonium sulfate, resuspended in dialysis buffer, and split 4-fold into MS1-N medium c o n ~ i n i n g1mM MGBG and dialyzed overnight against dialysis buffer containing 0.3 M NaCI. Suspension cultures to be assayed for In A, st.ationaryphase suspension cultures were diluted 4-fold into enzymatic activity which contained MGBG werehomogenized and new medium and an aliquot was harvested every 24 h by collection centrifuged as above, but the supernatant was first spun dialyzed on Whatman filters. The growth rate of liquid suspension cultures was measured as previously described [14]
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