RESUMEN
In crude extracts of primary leaves from dark grown seedlings of Phaseolus vulgaris L., relatively high hydrolytic activity of chlorophyllase (chlorophyll-chlorophyllido-hydrolase, EC 3.1.1.14) was observed. When plants were exposed to continuous illumination, the enzyme activity in the extracts was doubled within 3 days and both chlorophyll a and b were synthesized. However, when exposed to periodic illumination (1 min light-59 min dark) the enzyme activity was doubled within 1 to 2 days and chlorophyll a was synthesized but the formation of chlorophyll b was suppressed. When plants were transferred from periodic illumination to continuous illumination chlorophyll b was synthesized but the activity of chlorophyllase declined. Chloramphenicol blocked the increase in enzyme activity independent of the light regimes, but cycloheximide inhibited the activity more effectively during growth in the light. The presence of chlorophyllase activity in the leaves is discussed in relation to the chlorophyll-proteins and the membranes known to be present in chloroplasts. It is suggested that the enzyme is synthesized on plastid ribosomes.
RESUMEN
Chlorophyllase (chlorophyll-chlorophyllidohydrolase, EC 3.1.1.14) was isolated and purified from Phaseolus vulgaris L. chloroplasts and etioplasts dissolved in 1% Triton X-100 and 10% glycerol. A 100 and 40-fold purification, respectively, was achieved. Enzyme preparations from both sources had similar affinities for chlorophyll a when assayed in a Triton X-100 medium. When electrophoresed in sodium dodecyl sulphate polyacrylamide gels the major band in both preparations migrated as a peptide of 30,000 daltons. Chlorophyll containing liposomes were also used as a substrate for chlorophyllase. The rate of hydrolysis did not follow Michaelis-Menten kinetics. When chlorophyllide a or methyl chlorophyllide a was incorporated in the liposomes, then in the presence of phytol dissolved in methanol, methylchlorophyllide a and chlorophyll a were shown to be synthesized. Apparently the purified enzyme in the presence of lipids, is endowed with both synthetic and hydrolytic activity.
RESUMEN
During light-induced greening of 10-dayold etiolated bean seedlings a strong increase is observed of ferredoxin (Fd) and of ferredoxin-NADP-oxidoreductase (FNR; E.C. 1.6.99.4) activity, both known to reside in chloroplasts. The production of Fd starts immediately upon illumination and proceeds almost linearly for at least the next 72 h. It is inhibited by chloramphenicol (CAP) but not by cycloheximide (CHI), beit that in the presence of the latter Fd synthesis was impaired after 48 h of illumination. We conclude that for the elaboration of functional Fd in greening chloroplasts protein synthesis on chloroplast ribosomes is required. The increase of FNR activity shows a lag of about 24 h and is blocked by both CAP and CHI. When CAP is applied at 24 h after the illumination started it has no effect. We suggest that the synthesis of FNR on cytoplasmic ribosomes requires prior synthesis of protein(s) on chloroplast ribosomes.The nature of possible interactions between chloroplasts and cytoplasm is discussed.
