Light dependent incorporation of carbon 14 di oxide into protein by mesophyll protoplasts and chloroplasts isolated from pisum sativum
Huber, S.C.; Hall, T.C.; Edwards, G.E.
Zeitschrift fuer Pflanzenphysiologie 85(2): 153-164
Mesophyll protoplasts and chloroplasts isolated from protoplasts of P. sativum were found to incorporate 14CO2 into protein during light dependent C assimilation. Isolated chloroplasts from young developing leaves incorporated [3H] leucine into protein in the light within 30 min while mesophyll protoplasts did not. After 1 h of 14CO2 fixation by mesophyll protoplasts, approximately 1% of the 14C assimilated was found in protein. Upon fractionation of the protoplasts about 70% of the 14C labeled protein was found in the chloroplasts. The percentage of 14C assimilated into protein with isolated chloroplasts was 1/10 that with protoplasts, even though the CO2 fixation rates were similar (50-70 .mu.mol .cntdot. mg chl-1 .cntdot. h-1). Two methods gave equivalent results for determining the incorporation of 14CO2 into protein: loss of trichloroacetic acid-precipitable radioactivity by protease treatment and trichloroacetic acid precipitable radioactivity after digestion with a mixture of amyloglucosidase, RNase, DNase and cellulase in the presence of 1% bovine serum albumin. Polypeptides of labeled soluble chloroplast proteins were resolved by gel electrophoresis under dissociating conditions. When chloroplasts were incubated with 14CO2 for 30 min, a pair of radioactive peaks were observed with a mobility similar to that of the large subunit of fraction 1 protein. When mesophyll protoplasts were incubated with 14CO2 and the soluble chloroplast proteins resolved under dissociating conditions, polypeptides were labeled throughout the gel including areas corresponding to both the large and small subunit of fraction 1 protein.