Light and oxygen regulation of the synthesis of bacteriochlorophylls a and c in Chloroflexus aurantiacus.

Control of the synthesis of bacteriochlorophylls (Bchls) a and c by light and oxygen was studied in Chloroflexus aurantiacus grown in batch or chemostat culture with serine as the growth-limiting substrate. For comparison, inhibition by gabaculine of the formation of selected tetrapyrroles was studied. The inhibitory effect of gabaculine decreased in the following order of tetrapyrrole formation: coproporphyrin greater than Bchl c greater than Bchl a. Not only did addition of 5-aminolevulinate (ALA) reverse the inhibition by gabaculine, it also caused an increase in Bchl c content when the cultures grew at high concentrations of ALA. Inhibition of Bchl a, Bchl c, and coproporphyrin formation by oxygen was similar to inhibition by gabaculine. Addition of ALA to aerated cultures led to significant accumulation of coproporphyrin. These results suggest that oxygen inhibits tetrapyrrole formation at a site before ALA formation. Control by light was studied with chemostat cultures transferred from 5 klx to 25 klx. This resulted in only a transient increase of the protein level of the culture, while specific contents of Bchls c and a and the ratio Bchl c/Bchl a decreased to lower steady states. However, the specific content of coproporphyrin increased. Addition of ALA to chemostat cultures adapted to 50 klx increased specific coproporphyrin and Bchl c contents by factors of about 20 and 4, respectively, while the specific Bchl a content was only slightly increased and protein levels were unaffected. Increasing the serine concentration caused an initial increase in the specific Bchl c content, which returned to the original value as soon as the protein content had attained its maximal level. These results suggest that light does not control ALA formation as strictly as oxygen and that competition of biomass formation and tetrapyrrole synthesis for common precursors may be influenced by light.     

Interaction of rabbit hemoplexin with copro- and uroporphyrins.

Rabbit hemopexin forms equimolar complexes in vitro with the I and III isomers of both coproporphyrin and uroporphyrin. The apparent dissociation constants (Kd) of these complexes are estimated to be 4-10(-7) M for coproporphyrin-hemopexin and 10(-6) M for uroporphyrin-hemopexin by equilibrium dialysis and quenching of protein fluorescence. Results of competitive binding experiments suggest that all four porphyrins bind at the heme-binding site of hemopexin, and that the relative affinity of rabbit hemopexin for these porphyrins is: deuteroheme greater than coproporphyrin I or III greater than uroporphyrin I or III. These findings provide further evidence that hemopexin may function as a transport protein for circulating coproporphyrins as well as for heme.     

Effects of diphenyl ether herbicides on porphyrin accumulation by cultured hepatocytes

Several diphenyl ether herbicides, such as acifluorfen methyl, have been previously shown to cause large accumulations of the heme and chlorophyll precursor, protoporphyrin, in plants. Lightinduced herbicidal damage is mediated by the photoactive porphyrin. Here we investigate whether diphenyl ether herbicides can affect porphyrin synthesis in rat and chick hepatocytes. In rat hepatocyte cultures, protoporphyrin, as well as coproporphyrin, accumulated after treatment with acifluorfen or acifluorfen methyl. Combination of acifluorfen methyl with an esterase inhibitor to prevent the conversion of acifluorfen methyl to acifluorfen resulted in a greater accumulation of porphyrins than caused by acifluorfen methyl or acifluorfen alone. In vitro enzyme studies of hepatic mitochondria isolated from rat and chick embryos demonstrated that protopor-phyrinogen oxidase, the penultimate enzyme of heme biosynthesis, was inhibited by low concentrations of acifluorfen, nitrofen, or acifluorfen methyl with the latter being the most potent inhibitor. These findings indicate that diphenyl ether treatment can cause protoporphyrin accumulation in rat hepatocyte cultures and suggest that this accumulation was associated with the inhibition of protoporphyrinogen oxidase. In cultured chick embryo hepatocytes, treatment with acifluorfen methyl plus an esterase inhibitor caused massive accumulation of uroporphyrin rather than protoporphyrin or coproporphyrin. Specific isozymes of cytochrome P450 were also induced in chick embryo hepatocytes. These effects were not observed in the absence of an esterase inhibitor. These results suggest that diphenyl ether herbicides can cause uroporphyrin accumulation similar to that induced by other cytochrome P450-inducing chemicals such as polyhalogenated aromatic hydrocarbons in the chick hepatocyte system.     

Light control of porphyrin accumulation in acifluorfen-methyl-treated Lemna pausicostata

In Lemna pausicostata Hegelm. 6746, light is required for sufficient acifluorfenmethyl (AFM) stimulation of protoporphyrin IX (Proto IX) accumulation to cause significant herbicidal action. In darkness, AFM causes Proto IX levels to increase for about 2 h, after which Proto IX content is stable at levels significantly lower than those accumulated in light. In darkness, sucrose cannot increase levels of AFM-induced Proto IX. However, addition of δ-aminolevulinic acid (ALA) increases Proto IX levels in AFM-treated plants in darkness, demonstrating that the herbicide blocks the porphyrin pathway in darkness as it does in the light. Thus, Proto IX accumulation in darkness appears to be limited by ALA availability. This is supported by the finding that dioxoheptanoic acid caused more ALA to accumulate in light than in darkness. Heme is a feedback inhibitor of ALA synthesis, and heme synthesis is inhibited by AFM. However, total extractable heme levels were reduced by AFM by about the same amount in both light and darkness. Exogenously supplied hemin reduced AFM-caused Proto IX accumulation and herbicidal damage in the light and also reduced Proto IX accumulation caused by AFM or AFM plus ALA in darkness. AFM-stimulated Proto IX accumulation was inversely proportional to the log of the photon flux density between 5 and 500 μmol in m⁻² s⁻¹. Reduced effects of higher photon fluxes on AFM-stimulated Proto IX accumulation are probably due to both increased photobleaching of Proto IX and reduced porphyrin synthesis because of herbicidal damage. AFM-stimulated Proto IX accumulation in darkness could not be demonstrated to be under phytochrome control, but it appeared to be under the negative influence of protochlorophyllide levels.     

Ambient Light Promotes Selective Subcellular Proteotoxicity after Endogenous and Exogenous Porphyrinogenic Stress

Hepatic accumulation of protoporphyrin-IX (PP-IX) in erythropoietic protoporphyria (EPP) or X-linked-dominant protoporphyria (XLP) cause liver damage. Hepatocyte nuclear lamin aggregation is a sensitive marker for PP-IX-mediated liver injury. We tested the hypothesis that extracellular or intracellular protoporphyria cause damage to different subcellular compartments, in a light-triggered manner. Three hepatoma cell lines (HepG2, Hepa-1, and Huh-7) were treated with exogenous PP-IX (mimicking XLP extrahepatic protoporphyria) or with the iron chelator deferoxamine and the porphyrin precursor 5-aminolevulinic acid (ALA) (mimicking intracellular protoporphyrin accumulation in EPP). Exogenous PP-IX accumulated predominantly in the nuclear fraction and caused nuclear shape deformation and cytoplasmic vacuoles containing electron-dense particles, whereas ALA+deferoxamine treatment resulted in higher PP-IX in the cytoplasmic fraction. Protein aggregation in the nuclear and cytoplasmic fractions paralleled PP-IX levels and, in cell culture, the effects were exclusively ambient light-mediated. PP-IX and ALA caused proteasomal inhibition, whereas endoplasmic reticulum protein aggregation was more prominent in ALA-treated cells. The enhanced ALA-related toxicity is likely due to generation of additional porphyrin intermediates including uroporphyrin and coproporphyrin, based on HPLC analysis of cell lysates and the culture medium, as well as cell-free experiments with uroporphyrin/coproporphyrin. Mouse livers from drug-induced porphyria phenocopied the in vitro findings, and mass spectrometry of liver proteins isolated in light/dark conditions showed diminished (as compared with light-harvested) but detectable aggregation under dark-harvested conditions. Therefore, PP-IX leads to endoplasmic reticulum stress and proteasome inhibition in a manner that depends on the source of porphyrin buildup and light exposure. Porphyrin-mediated selective protein aggregation provides a potential mechanism for porphyria-associated tissue injury.     

Leishmania spp.: delta-aminolevulinate-inducible neogenesis of porphyria by genetic complementation of incomplete heme biosynthesis pathway

To further develop the Leishmania model for porphyria based on their deficiencies in heme biosynthesis, three Old World species were doubly transfected as before for Leishmania amazonensis with cDNAs, encoding the 2nd and 3rd enzymes in the pathway. Expression of the transgenes was verified immunologically at the protein level and functionally by uroporphyrin neogenesis that occurs only after exposure of the double-transfectants to delta-aminolevulinate. All species examined were equally deficient in heme biosynthesis, as indicated by the accumulation of uroporphyrin as the sole porphyrin and the production of coproporphyrin upon further transfection of one representative species with the downstream gene. The results obtained thus demonstrate that at least the first five enzymes for heme biosynthesis are absent in all species examined, rendering their transfectants inducible with aminolevulinate to accumulate porphyrins and thus useful as cellular models for human porphyrias.     

Effect of Oxygen on Heme and Porphyrin Accumulation from δ-Aminolevulinic Acid by Suspensions of Anaerobically Grown Staphylococcus epidermidis

The effect of various conditions on the accumulation of porphyrins and heme by resting suspensions of anaerobically grown cells of Staphylococcus epidermidis was examined. Anaerobically grown cells contain 10 to 15% of the amount of protoheme found in cells grown aerobically. Resting suspensions of anaerobically grown cells, when incubated aerobically in buffer with delta-aminolevulinic acid and glucose for 60 min, exhibited a fourfold increase in protoheme content. At high levels of delta-aminolevulinic acid, there was also a significant accumulation of porphyrins with the solubility and chromatographic properties of coproporphyrin and uroporphyrin. Protoporphyrin was not accumulated. When oxygen was excluded from the incubation mixture, accumulation of protoheme was prevented, but accumulation of coproporphyrin and total porphyrin was enhanced. Nitrate served as an electon acceptor as indicated by its reduction to nitrite; however, nitrate did not substitute for oxygen in causing the accumulation of protoheme. These results suggested that oxygen is required for one of the late steps of heme synthesis in S. epidermidis, possibly for the conversion of coproporphyrinogen to protoporphyrin. The inability of nitrate to substitute for oxygen suggests a role for molecular oxygen as a substrate rather than as an electron acceptor for heme synthesis.     

Accumulation of porphyrins and pyrrole pigments by Staphylococcus aureus ssp. anaerobius and its aerobic mutant

Abstract The anaerobic, Gram-positive coccus Staphylococcus aureus ssp. anaerobius and its aerobic mutant MVF-SR, when kept under anaerobic conditions, excreted coproporphyrin (mainly type III) into the medium and enriched uroporphyrin (mainly type I) within the cells.
The rate of porphyrin synthesis stayed practically unaltered when the growth medium was supplemented with 50 μ g/ml 5-aminolevulinic acid (ALA), but was significantly enhanced upon supplementation with hemin (0.5 μ g/ml). When hemin and ALA were given simultaneously, a more than two-fold increase in porphyrin production compared to normal growth medium was observed. These observations indicate a stimulation of porphyrin synthesis in S. aureus by hemin.
An as yet unidentified violet pigment with an intense red-violet fluorescence under UV light ( λ = 366 nm) was found to be present in considerable amounts in cells of S. aureus ssp. anaerobius , whereas the supernatant medium of aerobically grown cells of the mutant MVF-SR contained an equally unidentified blue, non-fluorescing pigment.     

The effect on photohaemolysis of variation in the structure of the porphyrin photosensitizer.

A comparison of the photosensitizing ability of a variety of porphyrins for photohaemolysis gives the following order of activity: protoporphyrin greater than deuteroporphyrin, mesoporphyrin, haematoporphyrin dimethyl ester much greater than haematoporphyrin diacetate, haematoporphyrin greater than haematoporphyrin monoacetate, coproporphyrin III, haematoporphyrin derivative, coproporphyrin III tetramethyl ester greater than uroporphyrin I, meso-tetra-(N-methyl-4-pyridinium)porphyrin tetratoluene-p-sulphonate, meso-tetra-(p-carboxyphenyl)porphyrin, protoporphyrin dimethyl ester, meso-tetra-(p-hydroxy-sulphonylphenyl)porphyrin tetrasodium salt, uroporphyrin III, deuteroporphyrin-3,8-disulphonic acid and protohaemin. The results for the metal-free porphyrins are rationalized in terms of solubility and partition properties, and a model is proposed for the incorporation of amphipathic porphyrins into the membrane lipid bilayer. Experiments with erythrocytes from patients with erythropoeitic protoporphyria and with normal erythrocytes to which porphyrin was added in a deuterium oxide medium do not lead to an increase in the rate of photohaemolysis. A possible explanation for this somewhat surprising observation is outlined.     

Uroporphyrinogen III cosynthase-deficient mutant of Salmonella typhimurium LT2.

A new type of heme-deficient mutant of Salmonella typhimurium LT2 was isolated using neomycin. The mutant, designated as strain SASY74, accumulated uroporphyrin I and coproporphyrin I. Extracts of the mutant converted 5-aminolevulinic acid to uroporphyrin I. Extracts of the mutant SASY74 and of the uroporphyrinogen synthase-deficient mutant SASY32 complemented each other and converted, when incubated together, 5-aminolevulinic acid to protoporphyrin. This finding excludes the possibility that uroporphyrinogen I synthase in strain SASY74 is deficient in its cosynthase-binding ability. Hence, the most probable explanation for the accumulation of uroporphyrin I and coproporphyrin I by the mutant is the lack of the uroporphyrinogen III cosynthase activity. This mutant is the first isolated in bacteria with such deficiency, and the mutation is analogous, as far as porphyrin synthesis is concerned, to human congenital porphyria. Mapping of the corresponding gene (hemD) by conjugation and P22-mediated transduction suggests the following gene order on the chromosome: ilv....hemC, hemD, cya....metE. The hemC and hemD genes are probably adjacent; this is the first case in which two hem genes of Enterobacteriaceae are contiguous on the chromosomal map.     

Chlorinated biphenyls induce cytochrome P450IA2 and uroporphyrin accumulation in cultures of mouse hepatocytes

Previous enzymatic and immunological studies from this laboratory have indicated a critical role for cytochrome P450IA2-catalyzed uroporphyrinogen oxidation in the development of uroporphyria caused by halogenated aromatic hydrocarbons. To extend these studies, we investigated whether primary cultures of mammalian hepatocytes which are inducible for cytochrome P450IA2 are also inducible for chemically mediated uroporphyria. Hepatocytes were isolated from C57BL/6 mice and maintained on Matrigel, an extracellular matrix isolated from a mouse tumor. When these cultures were treated with 3,4,5,3',4',5'-hexachlorobiphenyl (HCB) and 5-aminolevulinic acid (ALA), they accumulated cytochrome P450IA2 as well as uroporphyrin (URO) and heptacarboxyporphyrin for up to 12 days. Cultures treated with ALA alone accumulated no P450IA2 and very little URO. Neither URO accumulation nor the level of P450IA2 was affected by addition of iron as the nitrilotriacetate complex. Other inducers of P450IA2 in vivo (3,4,5,3',4'-pentachlorobiphenyl, 3,4,3',4'-tetrachlorobiphenyl, and 3-methylcholanthrene) also increased P450IA2 in the cultures and caused URO accumulation in the presence of added ALA. The tetrachlorobiphenyl and methylcholanthrene caused these effects only when given repeatedly. Inducers of other forms of P450 failed to cause URO accumulation in the presence of ALA and iron. Cultures of hepatocytes from DBA mice (which are resistant to the uroporphyria in vivo) accumulated much less P450IA2 or URO when treated with HCB and ALA. These primary cultures of mammalian hepatocytes represent a new experimental model to investigate the role of cytochrome P450IA2 in the mechanism of chemically induced uroporphyria.     

宽叶吊兰叶绿素生物合成的昼夜节律变化

在被子植物中,从谷氨酰-tRNA到叶绿素的生物合成是由许多酶催化的级联反应,其中间代谢产物具有较强的光反应活性和细胞毒性,因此这一过程在细胞内受到严格的调控。本研究通过检测宽叶吊兰叶片叶绿素生物合成途径的14种中间产物含量随昼夜节律的变化,探讨昼夜节律对宽叶吊兰叶绿素生物合成的影响。结果表明,中间产物ALA(δ-氨基乙酰丙酸)、PBG(胆色素原)、ProtoⅨ(原卟啉Ⅸ)、Heme(血红素)、Mg-ProtoⅨ(镁原卟啉Ⅸ)、Chlide a(叶绿素酸酯a)、Chlide b(叶绿素酸酯b)、Chl a(叶绿素a)、Chl b(叶绿素b)受光诱导,而UrogenⅢ(尿卟啉Ⅲ)、CoprogenⅢ(粪卟啉Ⅲ)和Pchlide(原叶绿素酸脂)受黑暗诱导,尤其是Pchlide在黑暗中的积累量显著增加;Mpe(镁原卟啉Ⅸ单甲酯)和Mpde(镁原卟啉Ⅸ二酯)具有2个积累峰值,分别出现在中午12∶00和夜间24∶00。说明叶绿素生物合成受昼夜节律的调控,但其中间代谢产物含量的变化规律与昼夜节律并不完全一致。     

Relative roles of CYP2E1 and CYP1A2 in mouse uroporphyria caused by acetone

Porphyria cutanea tarda is a liver disease characterized by excess production of uroporphyrin. We previously reported that acetone, an inducer of CYP2E1, enhances hepatic uroporphyrin accumulation in mice treated with iron dextran (Fe) and 5-aminolevulinic acid (ALA). Cyp2e1(-/-) mice treated with Fe and ALA were used to investigate whether CYP2E1 is required for the acetone effect. Hepatic uroporphyrin accumulation was stimulated by acetone in Cyp2e1(-/-) mice to the same extent as in wild-type mice. In the absence of acetone, uroporphyrin accumulated in Cyp2e1(-/-) mice treated with Fe and ALA, but less than in wildtype mice. However, in Cypla2(-/-) mice, uroporphyrin accumulation caused by Fe and ALA, with or without acetone, was completely prevented. Acetone was not an inducer of hepatic CYP1A2 in the wild-type mice. Although acetone is an inducer of CYP2E1, CYP1A2 appears to have the essential role in acetone-enhancement of uroporphyria.     

Effects by heme, insulin, and serum albumin on heme and protein synthesis in chick embryo liver cells cultured in a chemically defined medium, and a spectrofluorometric assay for porphyrin composition.

Primary chick embryo liver cells, which had been previously cultured in Eagle's medium containing 10% fetal bovine serum, had the same characteristics (inducibility of delta-aminolevulinic acid synthetase and synthesis of plasma proteins) when cultured in a completely defined Ham F-12 medium containing insulin. Insulin was active in the physiological range; 2 to 3 nM were sufficient to increase the induced delta-aminolevulinic acid synthetase to 50% of the maximum effect obtained with a saturating amount of insulin (30 nM). Serum albumin added to the Ham-insulin medium caused protoporphyrin but not uroporphyrin, generated in the cultured liver cells, to be transferred to the medium. As little as 10 mug of human serum albumin per ml caused the transfer of one-half of the protoporphyrin. Bovine serum albumin was only about 1/30 as effective. A spectrofluorometric method and calculation procedure are described for quantitation, in the nanomolar range, of total porphyrin and the percentage of this that is protoporphyrin or uroporphyrin plus coproporphyrin. The method is satisfactory for the measurement of porphyrins generated by 1 mg wet weight of cells in culture in 20 hours. Heme (0.1 to 0.3 muM), when added to the medium as hemin, human hemoglobin, or chicken hemoglobin, specifically inhibited the induction of delta-aminolevulinic acid synthetase by one-half. This high sensitivity for heme was observed under conditions in which the defined medium was free of serum and where a chelator of iron was added to the medium to diminish the synthesis of endogenous heme. Heme endogenously generated from exogenous delta-aminolevulinic acid also inhibited the induction; chelators of iron prevented this inhibition. The migration of heme from the mitochondria to other portions of the cell is discussed in terms of the affinities of different proteins for heme. A hypothesis of a steady state of liver heme metabolism, controlled by the concentration of "free" heme, is presented. The different effects of heme on the synthesis of a number of proteins are summarized.     

Uroporphyrin- and coproporphyrin I-accumulating mutant of Escherichia coli K12.

A new type of haem-deficient mutant was isolated in Escherichia coli K12 by neomycin selection. The mutant was deficient in uroporphyrinogen III cosynthase activity as indicated by the accumulation of uroporphyrin I and coproporphyrin. The mapping of the corresponding hemD gene by P1-mediated transduction showed that the new gene was located between ilv and cya, at min 83 on the chromosomal map of Escherichia coli K12.     

Yeast mutants deficient in heme biosynthesis and a heme mutant additionally blocked in cyclization of 2,3-oxidosqualene.

Mutants of Saccharomyces cerevisiae were isolated which were blocked in heme biosynthesis and required heme for growth on a nonfermentable carbon source. They were rho+, and grew fermentatively on ergosterol or cholesterol and Tween 80, as a source of oleic acid. Cells grown on ergosterol and Tween 80 lacked cytochromes and catalase which were restored by growth on heme. The mutants comprised five nonoverlapping complementation groups. Tetrad analysis showed that the pleiotropic properties of each of the mutants resulted from a single mutation in one of five unlinked loci (hem1 to hem5) affecting heme biosynthesis. Biochemical studies confirmed that each mutation resulted in loss of a single enzyme activity. hem1 mutants grew on delta-aminolevulinate and lacked delta-aminolevulinate synthase activity, hem2 mutants lacked delta-aminolevulinate dehydratase, and hem3 mutants uroporphyrin I synthase. Mutants in hem1, hem2, and hem3 had an additional requirement for methionine on synthetic medium supplemented with either heme or ergosterol and Tween 80, owing to a lack of sulfite reductase which contains siroheme, a modified uroporphyrin III. Since hem4 and hem5 mutants have sulfite reductase activity under all growth conditions, they are blocked after uroporphyrin III. Cell extracts of a hem4 mutant incubated with delta-aminolevulinate accumulated coproporphyrin III suggesting a block in coproporphyrinogenase, the enzyme which converts coproporphyrinogen III to protoporphyrinogen. Cells and extracts of a hem5 mutant accumulated protoporphyrin IX. Since it was the only mutant that grew on heme but not on protoporphyrin IX, a block in ferrochelatase was suggested for this strain. Mutant strains grown on heme had the sterol composition of wild type cells, whereas without heme only squalene, small amounts of lanosterol, and added sterol was observed. A heme product therefore participates in the transformation of lanosterol to ergosterol. A hem3 mutant was isolated which was also blocked between 2,3-oxidosqualene and lanosterol (erg12). When grown on lanosterol or ergosterol (with Tween 80) it accumulated a compound which was identified as 2,3-oxidosqualene by comparison with the synthetic compound in thin layer and gas-liquid chromatography, and by proton magnetic resonance and mass spectroscopy. Supplementation with heme did not remove the requirement for sterol, but it enabled the mutant to convert lanosterol to ergosterol.     

5-Aminolevulinic acid stimulation of porphyrin and hemoglobin synthesis by uninduced Friend erythroleukemic cells.

Porphyrin synthesis and iron accumulation was stimulated by exogenous 5-aminolevulinic acid (ALA) in uninduced Friend erythroleukemic cells (FELC). Uroporphyrin and protoporphyrin were the major intermediated precursors produced. All porphyrin types were conjugated to protein insoluble cellular components and could be extracted only by methanol sulfuric acid esterification. Heme content of the uninduced FELC was increased 6-fold in the presence of 5 x 10(-4) M ALA. As a consequence, the synthesis of the minor murine hemoglobin component was preferentially induced, an effect similar to that expressed by exogenous hemin. Addition of exogenous ALA to 0.5% DMSO-induced cells increased total hemoglobin synthesis with a higher efficiency of the minor hemoglobin. The endogenous synthesis of porphyrin from exogenous ALA was markedly reduced by hemin. Uroporphyrin, coproporphyrin, protoporphyrin and heme were equally repressed, indicating an inhibitory effect of hemin on ALA dehydrase and urosynthetase activities. In addition, hemin repressed [3H]leucine incorporation into protein by uninduced cells. Incubation of uninduced cells in culture medium without serum in the presence of hemin blocked their protein synthesis activity, whereas addition of serum exerted a protective effect on living FELC.     

Effect of porphyrins and their derivatives on biosynthesis of vitamin B 12 and the development of Propionibacterium shermanii]

The effect of uroporphyrin, coproporphyrin and their cobalt-containing derivatives on the biosynthesis of vitamin B12 and development of propionibacterium shermanii was studied. The compounds under study stimulated the vitamin synthesis by growing cultures and resting suspensions of these bacteria. Cobalt porphyrins as the sole source of cobalt were used in the vitamin B12 biosynthesis. An addition of cobalt porphyrins to the growing culture of propionic bacteria increased in accumulation of their biomass. Possible mechanisms of porphyrin involvement in the biosynthesis of vitamin B12 and the specific role of cobalt porphyrins in the bacterial activity are discussed.     

Rescuing activity of galactoglycerolipids from cellular lesions induced by 5-aminolevulinic acid

An anti-oxygen radical reagent of a bacterial metabolite, M874 monogalactoglycerolipid (di-O-12-methyl-tetradecanoyl-3-O-beta-D-galactopyranosyl-sn-glycerol ), was tested for its ability to protect two organisms against cellular lesions induced by 5-aminolevulinic acid (ALA) and light. In Corynebacterium flavescens ATCC 10340, extracellular uroporphyrin and coproporphyrin were the main porphyrin products. Although less than 2 mM ALA increased porphyrin synthesis, ALA levels above 3 mM inhibited the synthesis. Depending on the light intensity, the amount of porphyrin decreased and ALA-induced cytotoxicity increased. The lesion was more severe in the case of coproporphyrin than uroporphyrin. The porphyrin lesion produced in low intensity light (300 lx) was considerably reduced by 100 microM M874 glycolipid, although the reduction in intense light (3,000 lx) was restricted to a lower level. Similar results were obtained with radish (Raphanus sativus). The ALA concentration that inhibited porphyrin synthesis and stem growth was similar to that seen with C. flavescens. Although the exogenous addition of M874 glycolipid to the radish did not prevent ALA-induced cellular injury, the co-culture of radish and a glycolipid producing bacterium (Microbacterium sp. M874) resulted in a significant prevention of cellular injury. This was true only under enforced adhesion conditions through the action of a polysaccharide flocculant H12. Some species of monogalactoglycerolipids were found in Corynebacterium and radish that showed prominent oxygen radical-protecting activities similar to that of M874 glycolipid. These monogalactoglycerolipids might function in vivo as agents to prevent ALA-induced cytological lesions, although the concentrations were low in Corynebacterium and radish.