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Articles

Events Surrounding the Early Development of Euglena Chloroplasts

14. Biosynthesis of Cytochrome c-552 in Wild Type and Mutant Cells ¹

^a Institute for Photobiology of Cells and Organelles, Brandeis University, Waltham, Massachusetts 02154

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 O₁BS, O₂BX, G₁BU and P₁BXL (which make normal sized chloroplasts with abnormal internal structure in the light) have 0.02 to 0.1 x 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 Y₁BXD, Y₃BUD, and W₃₄ZUD 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 Y₂BUL, W₃BUL, W₈BHL, and W₁₀BSmL) 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.

³ Present address: Department of Biology, Wellesley College, Wellesley, Mass. 02181.

Part of this work was carried out under the auspices of the Experimental Marine Botany Program, Marine Biological Laboratory, Woods Hole, Mass. where G. C. H. was a summer postdoctoral research associate of the steps toward independence program.

⁴ Present address: Department of Biology, University of Toledo, Toledo, Ohio 43601.

⁵ Abraham and Etta Goodman Professor of Biology; to whom reprint requests should be directed.

¹ Supported by Grants GM14595 from the National Institutes of Health and PCM76-21486 from the National Science Foundation.