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ABSTRACTregions, which thereby supplants the necessity for ALA formation via the ALA synthase route.8-Aminolevulinic acid (ALA), a key precursor of the tetrapyrroles heme and chlorophyll, is capable of being synthesized by two different routes in cells of the unicellular green alga Euglena gracilis: from the intact carbon skeleton of glutamate, and via the condensation of glycine and succinyl CoA, mediated by the enzyme ALA synthase. The regulatory properties of ALA synthase were examined in order to establish its role in Euglena.Partially purified Euglena ALA synthase, unlike the case with the bacterial or animal-derived enzyme, does not exhibit allosteric inhibition by the tetrapyrrole pathway products heme, protoporphyrin IX, and porphobilinogen, at concentrations up to 100 micromolar.In aplastidic mutant cells, extractable ALA synthase activity is constant during exponential growth, and decreases to low levels as the cells reach the stationary state. Rapid exponential decline of ALA synthase (t1/2 = 55 min) occurs after administration of 43 micromolar cycloheximide, but not 6.2 millimolar chloramphenicol. These results suggest that, as in other eukaryotic cells, ALA synthase is synthesized on cytoplasmic ribosomes and is subject to rapid turnover in vivo.Extractable ALA synthase activity increases 2.5-fold within 6 hours after administration of 100 millimolar ethanol, a stimulator of mitochondrial development, and 4.5-fold within 12 hours after administration of I millimolar 4,6-dioxoheptanoic acid, which blocks ALA utilization, suggesting that activity is controlled in vivo by a feedback induction-repression mechanism, coupled with rapid enzyme turnover.In heterotrophically grown wild-type cells, low levels of ALA synthase rapidly increase 4.5-fold within 12 hours after cells are transferred from the light to the dark, and decrease exponentially (t1/2 = 75 min) when cells are transferred from the dark to light. The dark levels are equal to those in light-or dark-grown aplastidic mutant cells. The low level occurring in light-grown wild-type cells is not altered by the presence of 10 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea, which blocks photosynthetic 02 production. The decrease that occurs on dark-to-light transfer can be diminished by 12-or 24-hour prior incubation with 6.2 millimolar chloramphenicol, which also retards chlorophyll synthesis after the transfer to light.The positive relationship of ALA synthase activity to degree of mitochondrial expression, and the inverse relationship to plastid development and chlorophyll synthesis, suggests that ALA synthase functions to provide precursors to nonplastid tetrapyrroles in Euglena. In light-grown, wild-type cells, the diminished levels of ALA synthase may be due to the ability of developing plastids to export heme or a heme precursor to other cellular '

N-Methyl mesoporphyrin IX, an inhibitor of heme synthesis, increases extractable 8-aminolevulinic acid (ALA) synthase activity when administered to growing cultures of Euglena gracilis Klebs strain Z Pringsheim in micromolar concentrations. Wild-type light-grown green cells and white aplastidic cells exhibited 2.8-fold and 1.8-fold increases, respectively, in ALA synthase activity within five to six hours after incubation with 4 x 106 molar N-methyl mesoporphyrin IX. Protoheme levels were decreased and 59Fe incorporation into heme was inhibited by N-methyl mesoporphyrin IX, indicating that, as in animal cells, N-methyl mesoporphyrin IX acts specifically to block iron insertion into protoporphyrin IX. Chlorophyll synthesis in wild-type cells was not affected within the first 6 hours after administration of N-methyl mesoporphyrin IX. In animals, fungi, and bacteria, the first identified step hepatic ALA synthase within 4 h after injection into mice. We now report that N-methyl mesoporphyrin IX increases extractable ALA synthase activity, inhibits incorporation of exogenous 59Feinto heme in vivo, and diminishes heme levels in Euglena, but has no effect on Chl formation within the first 6 h after administration.MATERIALS AND METHODS Cultures of Euglena gracilis Klebs strain Z Pringsheim and an aplastidic mutant derived from this strain, W14ZNa 1 L (23), were kindly provided by H. Lyman (State University of New York, Stony Brook, NY). Cells were grown at 23°C and 32 ltE m-2s-' of cool white and red fluorescent light in a glucose-based heterotrophic medium as previously described (6). Cell population densities were determined with a Coulter Counter (Model ZBI, Coulter Electronics). Chl was extracted into methanol and quantitated by spectrophotometry, using the absorption coefficients of MacKinney (19). ALA synthase was extracted and assayed by the methods previously reported (6), except that cells were disrupted by sonication for four periods using a Sonifier (Model W 185, Heat Systems-Ultrasonics), and ALA was measured by condensation with ethyl acetoacetate at pH 6.8 (18) followed by color development with Ehrlich Hg reagent (26). Spectrophotometry was performed on a Cary Model 219 instrument (Varian).Protoheme was extracted by slight modification of the method of Stillman and Gassman (25). All operations were carried out at ice temperature. Cells were extracted with three 5-ml portions of 90% acetone containing 10-2 M NH40H. These extracts were discarded. The cells were then extracted with 2 ml acetone containing 2% HCI. A second l-ml acetone-HCl extraction supernatant was added to the first, and then 2 ml peroxide-free diethyl ether were added to the combined acetone extracts. After mixing, 6 ml H20 were added, the solution was thoroughly mixed, and briefly centrifuged to separate the phases. The upper ether phase was removed and the lower phase was extracted with I ml ether. The combined heme-containing ether phases were backwashed once with 1 ml H20. After evaporation of the ether, protoheme was de...

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