Events Surrounding the Early Development of Euglena Chloroplasts: 14. Biosynthesis of Cytochrome c-552 in Wild Type and Mutant Cells

Lack of a suitable assay has thwarted attempts to measure cytochrome c -552 in dark-grown wild type cells of Euglena gracilis var. bacillaris in mutants and in other situations where the concentrations are low. Purification methods are described based on electrofocusing which provide a cytochrome c -552 preparation homogeneous enough to elicit a single reactive antibody in rabbits; this antibody is then used as a specific and sensitive assay for cytochrome c -552. Dark-grown cells of wild type and of mutants O 1 BS, O 2 BX, G 1 BU and P 1 BXL (which make normal sized chloroplasts with abnormal internal structure in the light) have 0.02 to 0.1 × 10 −11 micromoles of cytochrome c -552 per cell, 10 to 150 times less than light-grown cells. Light-grown cells of these mutants and of wild type show a ratio of chlorophyll to cytochrome of about 300 (mole to mole). Cytochrome c -552 is undetectable in dark-grown Y 1 BXD, Y 3 BUD, and W 34 ZUD which cannot carry plastid development beyond the proplastid in light; the light-grown cells of these mutants have levels of cytochrome similar to or lower than dark-grown wild type cells. Cytochrome c -552 is undetectable in light- and dark-grown mutants in which plastid DNA is undetectable (such as Y 2 BUL, W 3 BUL, W 8 BHL, and W 10 BSmL) consistent with the view, but not proving, that this molecule may be coded, at least in part, in plastid DNA. During light-induced chloroplast development in resting cells, cytochrome c -552 formation behaves in all respects like chlorophyll except that the dark-grown cells contain low amounts of the cytochrome c -552 but lack chlorophyll. Thus, both cytochrome c -552 and chlorophyll show the same lag period even when the length is changed by nutritional manipulation; preillumination largely eliminates the lag in the formation of both molecules, cycloheximide and streptomycin both inhibit the biosynthesis of chlorophyll and cytochrome c -552 in the same manner, and the formation of both during chloroplast development is strictly light-dependent. It is shown that chloroplasts isolated from Euglena by methods thought to give intact organelles, lack 95% of the cytochrome c -552; this and the loss of similar molecules may explain why these isolated chloroplasts are not photosynthetically active.