Regeneration of Magnesium-2,4-Divinylpheoporphyrin a5 (Divinyl Protochlorophyllide) in Isolated Developing Chloroplasts

A preparation of developing chloroplasts isolated from greening cucumber (Cucumis sativus L. var Beit Alpha) cotyledons was found capable of synthesizing divinyl protochlorophyllide (magnesium-2,4-divinylpheoporphyrin a(5)) in the presence of glutamate, adenosine triphosphate, reducing power, S-adenosyl-l-methionine, and molecular oxygen. Both adenosine triphosphate and molecular oxygen were absolutely required while each of the other three was strongly promotive. Organelle intactness was essential. The divinyl protochlorophyllide (Pchlide) formed in vitro could be completely phototransformed. Regeneration of Pchlide was not inhibited by 0.3 millimolar chloramphenicol. The initial in vitro rate of Pchlide regeneration was considerably higher than the rate of Pchlide synthesis observed when greened cucumber seedlings were returned to darkness. However, Pchlide synthesis in vitro fell off exponentially with a half-life of approximately 21 minutes, whereas Pchlide synthesis in vivo was linear for at least 100 minutes. It is likely that the leveling off of the in vitro rate is due to the loss of chloroplast integrity during the incubation, because neither adding more cofactors, nor phototransforming the accumulated Pchlide in the middle of the incubation period, restored the high initial rate of Pchlide synthesis.