Induction of δ-Aminolevulinic Acid Synthase Activity and Inhibition of Heme Synthesis in Euglena gracilis by N-Methyl Mesoporphyrin IX

Abstract

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...

The effect of N-methylprotoporphyrin IX on the synthesis of photosynthetic pigments in Cyanidium caldarium. Further evidence for the role of haem in the biosynthesis of plant bilins

“…This result implies a requirement for ferrochelatase activity and therefore strongly supports previous conclusions (Brown et al, 1981) that haem is a necessary intermediate in algal bilin synthesis. Recent experiments by Beale (1982) are consistent with these findings. In the dark system, it has been well established (Troxler & Bogorad, 1966) that porphyrins accumulate during the incubation.…”

Ranking the impact of human health disorders on gut metabolism: Systemic lupus erythematosus and obesity as study cases

“…Finally, we further noted that SLE patients accumulated mesoporphyrin IX ( p < 0.0008) and protoporphyrin IX ( p = 0.0004), which were absent in HC subjects ( Supplementary Table 5 ). The fact that mesoporphyrin IX is an inhibitor of heme synthesis and ferrochelatase activity is consistent with the accumulation of protoporphyrin IX due to the presumptive inhibition of HemH proteins 27 . We suggest that SLE most likely decreases the iron uptake capacity of the gut microbiota and may also inhibit heme synthesis.…”

N-Methyl Mesoporphyrin IX Inhibits Phycocyanin, but Not Chlorophyll Synthesis in Cyanidium caldarium

“…NMMP did not cause a detectable decrease in the extractable heme levels in C caldarium (Beale and Chen, unpublished), even though the drug did induce a drop in heme level in E. gracilis (3). This difference might be due to the fact that, whereas heme is the major end product of the heme pathway in Euglena, a relatively minor proportion of the heme becomes an end product in C. caldarium, and most of the heme synthesized is further metabolized to phycobilins.…”

“…They act as competitive inhibitors of the last step of the heme biosynthetic pathway, insertion of ferrous iron into protoporphyrin IX by the enzyme ferrochelatase (7,13). In the phytoflagellate Euglena gracilis, growth in the presence of uM concentrations of NMMP caused induction of &aminolevulinic acid synthase activity, inhibition of 59Fe incorporation into heme, and lowering of the level of extractable heme (3). Importantly, Chl synthesis was not affected by NMMP, indicating that the inhibition was restricted to iron chelation, and that magnesium chelation, which is the first step unique to Chl synthesis, was unaffected under the conditions employed.…”