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We find that cycloheximide completely blocks the light-induced appearance of Euglena chloroplastic aminoacyl-tRNA synthetases in dark-grown cells of Euglerva gracilis var. baciliaris. Streptomycin, on the other hand, has no effect on the light-induction of these organellar enzymes. These observations, together with the finding that an aplastidic mutant (strain WsBUL, which has neither significant plastid structure nor detectable chloroplast DNA) contains low levels of the chloroplastic synthetases, indicate that the chloroplastic synthetases are transcriptional products of nuclear genes and are translated on cytoplasmic ribosomes prior to compartmentalization within the chloroplasts.The chloroplastic aminoacyl-tRNA synthetases (EC 6.1.1.X) of Euglena have been shown to be quantitatively under the control of light (1) in that exposure of dark-grown cells to light results in a marked increase in the level of these organelle-specific enzymes. In the present report, we have taken advantage of this light inducibility as well as of the techniques developed by Schiff et al. (2, 3). These techniques permit the study of chloroplast development in the absence of such complicating factors as cell growth and cell division in order to de-termine the site of translation of chloroplastic synthetases. More specifically, we examined the effect of streptomycin (4-6) and cycloheximide (7), which block protein synthesis on organelle (68S) and cytoplasmic (87S) ribosomes, respectively, on the light induction of chloroplastic synthetases in "resting" or nondividing dark-grown Euglena.We observe that, while cycloheximide completely blocks induction, streptomycin has no effect upon induction of chloroplast synthetases. In addition, we find as indicated previously (1), that low levels of the chloroplast synthetases are present in strain W3BUL (3,(8)(9)(10), a mutant strain that lacks both detectable chloroplast DNA and chloroplast structure. Thus we interpret our results to indicate that the chloroplast-specific aminoacyl-tRNA synthetases are translated on cytoplasmic ribosomes from transcriptional products of the nuclear genome, and are subsequently compartmentalized within the organelle, as has also been suggested for the NADP: triose phosphate dehydrogenase and alkaline DNase of Euglena (11)(12)(13).Abbreviation: SynPh, chloroplast phenylalanyl-tRNA synthetase.

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Significant purification of the ubiquitous cytochrome P-450-A and the strain-specific P-450-B from Drosophila melanogaster has been achieved by sequential chromatography on octylamino-agarose, DEAE-cellulose and hydroxyapatite. Preparations of P-450-A (specific contents of 7-9 nmol/mg) were homogeneous as determined by SDS/polyacrylamide-gel electrophoresis (PAGE) analysis. Preparations enriched for P-450-B (specific contents of 4-7 nmol/mg) contained significant amounts of P-450-A but were essentially free of other proteins as judged by SDS/PAGE. Partial reconstitution of 7-ethoxycoumarin de-ethylase activity was achieved using rabbit NADPH: cytochrome P450 reductase and purified preparations containing P450-B.