K. Chahed et al., Effects of cytokinins on the polypeptide composition of microsomal membranes from Catharanthus roseus cell suspension cultures, ACT BOT GAL, 146(4), 1999, pp. 375-386
The effects of benzylaminopurine (BAP) on the polypeptide pattern of membra
nes from Catharanthus roseus cells were investigated using two-dimensional
gel electrophoresis. In order to characterize the membrane preparation micr
osomes, issued from cells grown in different hormonal conditions (+2,4-D, -
2,4-D, BAP), microsomes were harvested at different times of cell culture a
nd tested for their ATPases prior to 2-D PAGE analysis. Inhibitory studies
revealed that the total ATPase activity within these membranes was slightly
affected by sodium azide and nitrate (less than 5%) indicating that mitoch
ondrial and tonoplat vesicles were little represented in the crude fraction
s. Membranes contained, however, ATPases that were totally dependent on mag
nesium and strongly inhibited by orthovanadate (90% inhibition). Analysis b
y 2-D PAGE of the protein composition of these membranes revealed that the
overall pattern was conserved after cytokinin treatment. A few polypeptides
appeared, however, to be regulated by BAP. Particularly, the accumulation
of a polypeptide with an apparent molecular mass of 33 kDa (P33) was increa
sed markedly after cytokinin treatment. This stimulation was slightly inhib
ited by 2,4-D, a known inhibitor of alkaloid accumulation. Time course stud
ies indicated that the effect of cytokinin on the accumulation of polypepti
de P33 starts within 24 hours of cytokinin treatment and persists for at le
ast 96 hours.