Immunologic evidence for structural homology between the release factors of Escherichia coli.

A cell-free chloroplast preparation obtained from greening cucumber cotyledons was tested for its ability to synthesize protoporphyrin IX from compounds previously postulated to be precursors of δ-aminolevulinic acid in plants, namely, glutamate, glutamine, α-ketoglutarate, glycine, and succinate. Of these, only glutamate caused a marked stimulation of protoporphyrin biosynthesis. A mixture of cofactors (ATP, KH₂PO₄, glutathione, coenzyme A, and NAD⁺), which was previously shown to be necessary for the incorporation of δ-aminolevulinic acid into protochlorophyll and for the maintenance of etioplasts in vitro also proved to be necessary for the conversion of glutamate to protoporphyrin IX.     

Formation of Mg-Containing Chlorophyll Precursors from Protoporphyrin IX, delta-Aminolevulinic Acid, and Glutamate in Isolated, Photosynthetically Competent, Developing Chloroplasts

Intact developing chloroplasts isolated from greening cucumber (Cucumis sativus L. var Beit Alpha) cotyledons were found to contain all the enzymes necessary for the synthesis of chlorophyllide. Glutamate was converted to Mg-protoporphyrin IX (monomethyl ester) and protoclorophyllide. δ-Aminolevulinic acid and protoporphyrin IX were converted to Mg-protoporphyrin IX, Mg-protoporphyrin IX monomethyl ester, protochlorophyllide and chlorophyllide a. The conversion of δ-aminolevulinic acid or protoporphyrin IX to Mg-protoporphyrin IX (monomethyl ester) was inhibited by AMP and p-chloromercuribenzene sulfonate. Light stimulated the formation of Mg-protoporphyrin IX from all three substrates. In the case of δ-aminolevulinic acid and protoporphyrin IX, light could be replaced by exogenous ATP. In the case of glutamate, both ATP and reducing power were necessary to replace light. With all three substrates, glutamate, δ-aminolevulinic acid, and protoporphyrin IX, the stimulation of Mg-protoporphyrin IX accumulation in the light was abolished by DCMU, and this DCMU block was overcome by added ATP and reducing power.     

Regulation of protoporphyrin IX biosynthesis by intraplastidic compartmentalization and adenosine triphosphate

Subplastidic preparations from cotyledons of cucumber (Cucumis sativus L.) were tested for their ability to synthesize protoporphyrin IX from the substrate 5-aminolevulinic acid. Envelope or thylakoid membranes failed to synthesize protoporphyrin IX from the substrate 5-aminolevulinic acid. Stromal preparations synthesized a very low amount of protoporphyrin IX. In a reconstitution experiment using stroma + envelope membranes, protoporphyrin IX synthesis from 5-aminolevulinic acid was enhanced by 660% over that of stroma alone. However, when thylakoids were added to the stroma + envelope mixture, protoporphyrin IX synthesis from 5-aminolevulinic acid was completely inhibited. In the reconstituted stroma + envelope membrane mixture, the reducing agent dithiothreitol enhanced the protoporphyrin IX-synthesizing ability and completely abolished the inhibition of protoporphyrin IX synthesis by thylakoids. This suggested that the oxidizing agents usually associated with the thylakoid membranes inhibited protoporphyrin IX biosynthesis and the inhibition was alleviated by the reducing power of dithiothreitol. This study exposes the weakness of in vitro reconstitution experiments in mimicking the in vivo-conditions. Addition of ATP stimulated protoporphyrin IX synthesis by 50% in the supernatant fraction of chloroplast lysate. This ATP-induced stimulation of protoporphyrin IX synthesis was due to the enhancement of the activities of uroporphyrinogen decarboxylase and protoporphyrinogen oxidase, involved in tetrapyrrole biosynthesis. The ATP-induced stimulation of porphyrinogen oxidase activity was an energy-dependent reaction. Received: 21 March 2000 / Accepted: 9 May 2000     

Investigation of the porphyrins accumulation in barley leaves incubated with Mn cations and δ-aminolevulinic acid

In greening etiolated primary leaves of barley (Hordeum vulgare L.), Mn²⁺ ions have been shown to inhibit chlorophyll (Chl) accumulation in a dose dependent manner and to lead to an accumulation of protoporphyrin IX (Proto) and Mg-protoporphyrin IX monomethyl ester (MgPE). The amount of MgPE that accumulated, was 2 times higher than Proto. In the dark, Proto and MgPE were observed to have accumulated to high levels in seven-day old green and etiolated leaves in the presence of 5 mmol/L Mn²⁺, but only if 5 mmol/L δ-aminolevulinic acid (ALA) was present. The 24 hours of irradiation of the green barley leaves treated in this way, resulted in a photodynamic destruction of Proto and MgPE as well as of Chl and carotenoids (Car). The observed porphyrin accumulation caused by the Mn²⁺ ions was reversed in the presence of active iron (Fe²⁺). This effect was observed when the iron concentration in incubation solutions was half the Mn²⁺ concentration, most effective for porphyrin synthesis, i.e. 5 mmol/L. The action of Mn²⁺ on porphyrin accumulation is also discussed.     

Photodynamic Therapy Using Systemic Administration of 5-Aminolevulinic Acid and a 410-nm Wavelength Light-Emitting Diode for Methicillin-Resistant Staphylococcus aureus-Infected Ulcers in Mice

Bacterial resistance to antibiotics has become a worldwide problem. One potential alternative for bacterial control is photodynamic therapy. 5-aminolevulinic acid is a natural precursor of the photosensitizer protoporphyrin IX. Relatively little is known about the antibacterial efficacy of photodynamic therapy using the systemic administration of 5-aminolevulinic acid; a few reports have shown that 5-aminolevulinic acid exerts photodynamic effects on methicillin-resistant Staphylococcus aureus (MRSA) in vitro. In this study, we evaluated the effectiveness of photodynamic therapy using 5-aminolevulinic acid and a 410-nm wavelength light-emitting diode in vitro and in vivo for the treatment of MRSA. We found that 5-aminolevulinic acid photodynamic therapy with the light-emitting diode had an in-vitro bactericidal effect on MRSA. In vivo, protoporphyrin IX successfully accumulated in MRSA on ulcer surfaces after intraperitoneal administration of 5-aminolevulinic acid to mice. Furthermore, 5-aminolevulinic acid photodynamic therapy accelerated wound healing and decreased bacterial counts on ulcer surfaces; in contrast, vancomycin treatment did not accelerate wound healing. Our findings indicate that 5-aminolevulinic acid photodynamic therapy may be a new treatment option for MRSA-infected wounds.     

PLASTID FUNCTION IN PLANT TISSUE CULTURES. I. PORPHYRIN SYNTHESIS BY DARK-GROWN HAPLOID AND DIPLOID ALBINO CULTURES

Tissue cultures lacking chlorophyll formed porphyrins when fed δ-aminolevulinic acid, a precursor of tetrapyrroles. When grown in the dark tissues from Ginkgo biloba L., Taxus, and Rosa formed protoporphyrin and several unidentified compounds. When grown in the light cultures did not form these pigments. The protoporphyrin was detected in the tissues after 3–6 hours incubation with δ-aminolevulinic acid; it was localized in the plastids by ultraviolet light microscopy and was identified by extraction procedures, chromatography, and absorption spectroscopy. No magnesium protoporphyrins were found, suggesting that chlorophyll synthesis was blocked at this point. Both male and female haploid albino tissues from Ginkgo formed protoporphyrin. The female albino tissue was derived from a chlorophyll-containing tissue culture from the female gametophyte by serially subculturing the green tissue in the dark. Upon exposing the female albino tissue to light, no greening occurred. The treatments used thus far have not caused chloroplasts to develop in the haploid albino tissues, even though the tissues contain many amyloplasts. Concurrent with the loss of chloroplasts, the female tissue loses all capacity to differentiate specialized cells, such as tracheids, resin cells, and chlorenchyma.     

Influence of photodynamic processes induced by 2,2′-dipyridyl on the enzymatic system of chlorophyll biosynthesis

The influence of 2,2′-dipyridyl (2,2′-DP) on the activity of one of the enzymes at the initial stages of chlorophyll (Chl) biosynthesis, δ-aminolevulinic acid dehydratase (ALAD; δ-aminolevulinate hydro-lyase, EC 4.2.1.24), as well as on δ-aminolevulinic acid (ALA) accumulation was investigated in green barley (Hordeum vulgare L.) leaves. In seven-day-old green leaves treated with 3 mM 2,2′-DP for 17 h in darkness and subsequently irradiated with "white light" (15 W m-2) for 4, 8, and 24 h the ALAD activity was 51 % as compared to that in untreated leaves. At the same time, the ALA forming system was most sensitive to the photodynamic processes caused by 2,2′-DP. After 8 h of irradiation, ALA synthesis was entirely inhibited. After the treatment the leaves accumulated exceptionally high amounts of Chl precursors such as protoporphyrin IX (Proto), Mg-protoporphyrin IX (Mg-Proto), its monomethyl ester, and protochlorophyllide (Pchlide) that are photosensitizers of photodynamic processes in plants. A comparatively low Chl and carotenoid (Car) destruction was registered during the subsequent 4 and 8 h of irradiation. At the same time, the content of Chl precursors was negligible. The low photodestruction of Chl and Car included in pigment-protein complexes, against the background of fast porphyrin disappearance, and fast decrease of enzymatic activities at the initial stages of Chl production could mean that the photodynamic effect induced by porphyrins accumulated in the presence of 2,2′-DP affected first the Chl enzymatic system and did not change the pool of already synthesized photosynthetic pigments.     

An analysis of precursors accumulated by several chlorophyll biosynthetic mutants of maize

Summary Several mutants of maize defective in chlorophyll synthesis are analysed. By feeding shoots of dark-grown seedlings -aminolevulinic acid, the regulatory step in chlorophyll biosynthesis is bypassed and chlorophyll precursors accumulate. In normal plants this results in a buildup of protoporphyrin IX and protochlorophyllide, while mutants accumulate precursors, depending on the site of the mutant-induced lesion. Mutants at three loci, l ^*-Blandy4, 113, and oy, are defective in conversion of protoporphyrin IX to Mg-protoporphyrin. Mutants at the oro and oro2 loci are defective in conversion of Mg-protoporphyrin monomethyl ester to protochlorophyllide. A dominant modifier gene, Orom, which allows oro seedlings to bypass their lesion is also described.Journal Paper No J-9076 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa Project No. 2035     

Ferrochelatase activity inAzospirillum brasilense with reference to the influence of metal cations

Summary Ferrochelatase in membrane preparations fromAzospirillum brasilense displayed an activity of 2.17 mol protoheme formed · h^–1 · mg protein^–1 which is 10-fold greater than previous reports for other bacteria. This ferrochelatase showed an apparentK _m of 20.9 M for Fe²⁺, a pH optimum of 6.0–6.5, and stimulation by oleic or stearic acids. Co²⁺, Cu²⁺ and Zn²⁺ inhibited the incorporation of Fe²⁺ into protoporphyrin IX while Ni² and Mg²⁺ had no effect on protoheme synthesis. Activity with Fe²⁺ and mesoporphyrin IX was less than with protoporphyrin IX but deuteroporphyrin IX produced the highest rate of protoheme synthesis. The membrane fraction containing ferrochelatase activity was found to insert Cu²⁺, Ni²⁺, Zn²⁺ and Co²⁺ enzymatically into protoporphyrin IX to produce metalloporphyrins. Cu²⁺ incorporation into protoporphyrin IX proceeded at a rate greater than with Fe²⁺ and theK _m for Cu²⁺ was 21.9 M.     

Porphyrin Biosynthesis in Cell-free Homogenates from Higher Plants

The porphyrin and phorbin biosynthetic activity of etiolated cucumber (Cucumis sativus, L.) cotyledons was compared to that of cotyledonary homogenates. Etiolated cotyledons incubated with δ-aminolevulinic acid accumulate protoporphyrin, coproporphyrin, small amounts of Mg protoporphyrin monoester, and trace amounts of uroporphyrin. They also incorporate 4-¹⁴C-δ-aminolevulinic acid into free porphyrins, protochlorophyllide, protochlorophyllide phytyl ester, and Mg protoporphyrin monoester. Homogenates incubated with δ-aminolevulinic acid likewise accumulate coproporphyrin, uroporphyrin, Mg coproporphyrin, and trace amounts of protoporphyrin. They also incorporate 4-¹⁴C-δ-aminolevulinic acid into Mg protoporphyrin monoester, Mg coproporphyrin, and free porphyrins. However, the capacity to synthesize protochlorophyllide and protochlorophyllide phytyl ester is lost and the endogenous protochlorophylls gradually disappear. Mg protoporphyrin monoester represents the terminal biosynthetic step in this cell-free system.     

Hemin feedback inhibition at reticulocyte δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydratase

Addition of hemin (5–200 μM) to a rabbit reticulocyte iron-free incubation medium, resulted in a progressive inhibition of heme synthesis as measured by incorporation of (¹⁴C)-glycine. In contrast when (¹⁴C) δ-aminolevulinic acid incorporation into heme was studied, significant inhibition below that of the (¹⁴C)-glycine control only occurred with hemin concentrations greater than 100 μM. Hemin progressively inhibited cellular and mitochondrialδ-aminolevulinic acid synthetase activity, as well as cellular δ-aminolevulinic acid dehydratase activity. The results indicated that elevated levels of hemin initially control heme synthesis by feedback inhibition at the rate-limiting enzyme of heme synthesis, δ-aminolevulinic acid synthetase. Hemin inhibition of δ-aminolevulinic acid dehydratase is only significant for the entrire heme synthetic pathway when greater than one-third of this enzyme's activity is inhibited.     

Regulation of Magnesium Chelatase Activity during Excessive Accumulation of Porphyrins in Green Barley Leaves

Activity of magnesium chelatase was studied in green barley leaves treated with 5-aminolevulinic acid (ALA). After this treatment, leaves accumulated excessive amounts of porphyrinic precursors of chlorophyll : protoporphyrin IX (PP), magnesium-protoporphyrin IX (MgPP), its monomethyl ester (MgPPE), and protochlorophyllide. The enzyme activity was found to be inversely dependent on the amount of MgPP formed from exogenous ALA. A conclusion was drawn about the existence of a mechanism for the regulation of the enzyme activity in vivo via its inhibition by the reaction product.     

Oxygen Consumption Stimulated by δ-Aminolevulinic Acid in Darkness and during Irradiation of Dark Grown Wheat Leaves

Dark grown wheat leaves (Triticum aestivum L. cv. Starke II Weibull), treated with δ-aminolevulinic acid in darkness, showed an increased oxygen uptake as measured by a Warburg method. The production of CO₂ was also increased in darkness, giving an RQ ? 1. The increased respiration was dependent on the treatment time as well as on the concentration of the δ-aminolevulinic acid. Potassium cyanide suppressed both the normal and the increased respiration. The treatment with δ-aminolevulinic acid caused accumulation of high amounts of protochlorophyllide. Levulinic acid suppressed the increased oxygen uptake as well as the protochlorophyllide accumulation in δ-aminolevulinic acid treated leaves. Irradiation rapidly decreased the protochlorophyllide content with a simultaneous increase in oxygen uptake over the dark value. The peak value of the increase in oxygen uptake was reached after about 5 min. The light induced oxygen uptake was dependent on the amount of PChlide present at the onset of irradiation. Also the CO₂ production was increased during the first minutes of irradiation but soon fell under the buffer control value. Neither potassium cyanide nor heat denaturation affected the oxygen uptake in light in contrast to the effect on the CO₂ production, which was blocked by heat denaturation. The increased oxygen uptake in light initially seems to be a purely photochemical process leading to a release of CO₂, which release is probably an enzymatic process induced by the photo-oxidative decomposition of pigment.     

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

N-Methyl mesoporphyrin IX, an inhibitor of heme synthesis, increases extractable δ-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 × 10⁻⁶ 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.     

Chloroplast biogenesis: Biosynthesis and accumulation of Mg-protoporphyrin IX monoester and other metalloporphyrins by isolated etioplasts and developing chloroplasts

Developing chloroplasts isolated from greening cotyledons and isolated etioplasts were capable of synthesizing and accumulating Mg-protoporphyrin IX monoester as well as longer wavelength metalloporphyrins when incubated in the dark, in the presence of air, δ-aminolevulinic acid, and cofactors (coenzyme A, glutathione, adenosine triphosphate, nicotinamide adenine dinucleotide, methyl alcohol, magnesium, potassium, and phosphate). The putative metalloporphyrins exhibited distinct fluorescence emission and excitation properties and were detected by spectrofluorometry in situ and after extraction in organic solvents. The cofactors were previously shown to be required for protochlorophyll, and chlorophyll biosynthesis and grana assembly in vitro. The putative long wavelength metalloporphyrins were suggested earlier to represent intermediates between Mg-protoporphyrin IX monomethyl ester and protochlorophyllide. The isolated plastids were similar in this aspect of their biosynthetic activity to etiolated cotyledons greening in distilled H₂O. In contrast to greening cotyledons, however, the biosynthetic activity of the isolated plastids depended on the addition of exogenous cofactors and δ-aminolevulinic acid. This was interpreted as an indication that the isolated plastids were not capable of generating their own δ-aminolevulinic acid and cofactors under the present incubation conditions. Light was not required for the conversion of added ALA to metalloporphyrins in vitro. The metalloporphyrins synthesized in vitro were more highly fluorescent in situ than those of greening cotyledons. In addition to Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins, isolated etioplasts synthesized and accumulated Zn-protoporphyrin and Zn-protoporphyrin IX monoesterlike compounds.     

Biosynthesis of δ-aminolevulinic acid in Rhodopseudomonas sphaeroides

The biosynthesis of δ-aminolevulinic acid was investigated in three strains of Rhodopseudomonas sphaeroides. A wild-type strain (NCIB 8253) possessed both δ-aminolevulinic acid synthetase and γ,δ-dioxovaleric acid transaminase in the cytoplasmic and membrane cell fractions. δ-Aminolevulinic acid synthetase activities were not detected in extracts of mutant strains H5 and H5D. However, γ,δ-dioxovaleric acid transaminase was found in the cytoplasmic and membrane fractions of these latter two strains. Strain H5 required exogenously added δ-aminolevulinic acid for growth and bacteriochlorophyll synthesis. Strain H5D did not require this compound for growth and bacteriochlorophyll synthesis. γ,δ-Dioxovaleric acid added in the growth medium did not support the growth of H5, although it was actively transported into the cells. Addition of γ,δ-dioxovaleric acid to the growth medium did not enhance the growth of either the wild-type or H5D strains. These results indicate that ALA synthetase is not required for growth and bacteriochlorophyll synthesis in H5D and that γ,δ-dioxovaleric acid is probably not an intermediate in the formation of δ-aminolevulinic acid in the strains of Rhodopseudomonas sphaeroides studied. In strain H5D another pathway may function in the formation of δ-aminolevulinic acid other than that catalyzed by δ-aminolevulinic acid synthetase or γ,δ-dioxovaleric acid transaminase.     

Uptake and release of possible false transmitter amino acids by rat brain tissue

—The uptake of l [¹⁴C]glutamine by a crude isolated nerve ending fraction of rat brain was found to be linear with time for at least 5 min, profoundly temperature-dependent, apparently half-saturated at a substrate concentration of 0·26 mm , partially inhibited by dinitrophenol and ouabain and elevated [K⁺], weakly Na⁺-dependent, poorly inhibited by drugs which block uptake of biogenic amines and more strongly inhibited by glutamic acid (IC₅₀= 0·5mm ) than by aspartic acid, GABA, glycine or methionine. The [¹⁴C]glutamine taken up appeared to be associated with nerve endings and was released by membrane-disruption; about 20 per cent was associated with free mitochondria. Glutamine, δ-aminolevulinic acid and several other amino acids were poor inhibitors of [³H]GABA-uptake; δ-aminolevulinic acid was a poor inhibitor of [³H]glutamine-uptake, whereas glutamine was a moderately effective competitive inhibitor (K_i= 1 mm ). [¹⁴C]glutamine and [³H]GABA were released from brain slices by electrical stimulation or 50 mm K⁺, while labeled δ-aminolevulinic acid, leucine, urea, amphetamine and tyramine were poorly released. [¹⁴C]glutamine was not released by unlabeled glutamate or several aromatic amines. We conclude that the neuropsychiatric features of porphyria are not likely due to a ‘false transmitter’ role for δ-aminolevulinic acid although such a role for glutamine in hepatic encephalopathy or other neuropsychiatric diseases should be considered.     

Porphyrogenic effects and induction of heme oxygenase in vivo by δ-aminolevulinic acid

The effects of single large doses of the porphyrin-heme precursor ?d-aminolevulinic acid on tissue porphyrins and on δ-aminolevulinate synthase and heme oxygenase, the rate-living enzymes of liver heme synthesis and degradation respectively, were studied in the chick embryo in ovo, in the mouse and in the rat. δ-Aminolevulinic acid treatment produced a distinctive pattern characterized by extensive tissue porphyrin accumulation and alterations in these rate-limiting enzymes in the liver. Repression of basal or allylisopropylacetamide-induced liver δ-aminolevulinate synthase was observed and, in the mouse and the rat, induction of liver heme oxygenase after δ-aminolevulinic acid treatment, in a manner similar to the known effects of hemin on these enzymes. In the chick embryo liver in ovo heme oxygenase was substantially higher than in rat and mouse liver, and was not significantly induced by δ-aminolevulinic acid or other compounds, including hemin, CS₂ and CoCl₂. Levulinic acid, an analogue of δ-aminolevulinic acid, did not induce heme oxygenase in mouse liver. δ-Aminolevunilic acid treatment did not impair ferrochelatase activity but was associated with slight and variable decreases in liver cytochrome P-450. Treatment of chick embryos with a small ‘priming’ dose of 1,4-dihydro-3,5-dicarbethoxycollidine, which impairs liver ferrochelatase activity, accentuated porphyrin accumulation after δ-aminolevulinic acid in the liver. These observations indicate that exogenous δ-aminolevulinic acid is metabolized to porphyrins in a number of tissues and, at least in the liver, to a physiologically significant amount of heme, thereby producing an increase in the size of one or more of the heme pools that regulate both heme systhesis and degradation. It is also possible than when δ-aminolevulinic acid is markedly overproduced in vivo it may be transported to many tissues and re-enter the heme pathway and alter porphyrin-heme metabolism in cells and tissues other than those in which its overproduction primarily occurs.     

BIOSYNTHESIS OF SMALL MOLECULES IN CHLOROPLASTS OF HIGHER PLANTS

1. Chloroplasts of higher plants contain enzymes which permit them to synthesize many kinds of small molecules in addition to carbohydrates. 2. Either aqueous or non-aqueous techniques may be used to isolate chloroplasts. Aqueous methods permit the isolation of chloroplasts showing high rates of photosynthesis; the organelles can be purified by means of density gradients. Non-aqueously isolated chloroplasts cannot photosynthesize, but show good retention of low-molecular-weight substances and soluble enzymes. 3. Whole cells photoassimilating ¹⁴CO₂ show considerable formation of ¹⁴C-labelled amino acids and lipids, but isolated chloroplasts exhibit very poor synthesis of amino acids and lipids from ¹⁴CO₂. 4. Chloroplasts play an important rôle in reducing nitrate to ammonia. There is controversy about the presence in chloroplasts of nitrate reductase and about the mechanism of the light-dependent reduction of nitrate to nitrite; however, it is generally agreed that non-cyclic electron transport directly supports reduction of nitrite to ammonia via a chloroplastic nitrite reductase. 5. Chloroplasts actively assimilate inorganic nitrogen into amino acids. The assimilation reaction is either the reductive amination of α-ketoglutarate to glutamate or the ATP-dependent conversion of glutamate to glutamine. The enzyme glutamate synthase has recently been found to be present in chloroplasts and may play an important function in nitrogen assimilation. 6. Numerous transaminases (aminotransferases) are present in chloroplasts. 7. The source of α-keto-acid precursors of chloroplastic amino acids is unknown. It remains to be established whether chloroplasts import the required keto acids or whether some of them might be generated via an incomplete tricarboxylic-acid cycle located in the chloroplast. 8. Chloroplasts contain characteristically high levels of mono and digalactosyl diglycerides, sulpholipid and phosphatidyl glycerol. They also have large amounts of polyunsaturated fatty acids. 9. Fatty acids are synthesized by the concerted action of fatty-acid synthetase, elongases and desaturases. Two pathways have been implicated for the formation of α-linolenic acid. 10. The galactosyldiglycerides are synthesized by successive galactosylation of diglyceride. The enzymes responsible are probably located in the chloroplastic envelope. 11. The other major chloroplastic acyl lipids (sulpholipid, phosphatidylglycerol and phosphatidylcholine) have not been, as yet, synthesized de novo by means of isolated chloroplast fractions. However, indirect evidence indicates that the first two are probably formed there. 12. Chlorophyllide synthesis involves the formation of δ-aminolaevulinic acid (δALA) followed by conversion of δALA to protoporphyrin IX, which is then transformed into protochlorophyll. 13. Recent evidence favours the view that δALA synthesis is not mediated by δALA synthetase but by another pathway in which δALA can be derived from α-ketoglutarate or glutamate. It has not been established whether this pathway is localized in plastids. 14. Conversion of δALA to protoporphyrin IX is mediated by soluble enzymes of the plastid stroma. Membrane-bound enzymes mediate the conversion of protoporphyrin to protochlorophyll. 15. Carotenoids are synthesized from acetyl C_oA via geranylgeranyl-pyrophosphate and phytoene intermediates. Evidence has been obtained for both neurosporene and lycopene as precursors of the cyclic carotenoids. 16. The overall pathway of carotenoid formation is subject to photoregulation, particularly during the development of the chloroplast. 17. Carotenes are precursors of xanthophylls, the inserted oxygen being derived from molecular oxygen. 18. Chloroplasts may synthesize or interconvert gibberellin hormones.     

delta-Aminolevulinic acid synthesis in a Cyanidium caldarium mutant unable to make chlorophyll a and phycobiliproteins.

Wild-type cells of the unicellular rhodophyte, Cyanidium caldarium, synthesize chlorophyll a, phycobiliproteins, and heme from δ-aminolevulinic acid during light-dependent chloroplast development but are unable to make photosynthetic pigments in the dark. C. caldarium, mutant GGB-Y, is an obligate heterotroph which, in the light, produces a chloroplast devoid of photosynthetic pigments. The present investigation has shown that δ-aminolevulinic acid is synthesized in cells of mutant GGB-Y incubated with levulinic acid, a competitive inhibitor of δ-aminolevulinic acid dehydrase (the second enzyme in the porphyrin biosynthetic pathway). In vivo, cells of mutant GGB-Y preferentially incorporated C₁ of glutamate and α-ketoglutarate into the C₅ fragment (formaldehyde) of δ-aminolevulinic acid after alkaline periodate degradation. This suggested that δ-aminolevulinic acid arises directly from the carbon skeleton of glutamate and α-ketoglutaric acid. The pattern of incorporation of C₃, C₄, and C₅ of α-ketoglutarate into the C₁–C₄ (succinic acid) fragment of δ-aminolevulinic acid after alkaline periodate degradation was consistent with the origin of δ-aminolevulinic acid from a five-carbon precursor. C₁ and C₂ of glycine and C₂ and C₃ of succinate were incorporated into both the formaldehyde and succinate fragments of δ-aminolevulinic acid in a manner inconsistent with condensation of glycine and succinyl CoA by δ-aminolevulinic acid synthetase, the rate-limiting enzyme in the porphyrin pathway in animals and bacteria. Extracts of the soluble protein from cells of mutant GGB-Y displayed a Soret band at 410 nm indicating the presence of hemoproteins. This shows that mutant GGB-Y cells synthesize heme. The respiration of radiolabeled glutamate, α-ketoglutarate, and glycine to ¹⁴CO₂ is consistent with the existence of mitochondrial cytochromes in cells of mutant GGB-Y and with the ability of the mutant to synthesize δ-aminolevulinic acid. The present results suggest that δ-aminolevulinic acid is synthesized directly from glutamate or α-ketoglutarate and that this is the only process by which the rate-limiting intermediate in the porphyrin pathway is synthesized in C. caldarium. If correct, the rate-limiting, regulative enzyme in the biosynthetic pathway for synthesis of chlorophyll a, bile pigment (phycocyanobilin), and heme must have been completely different in the evolutionary antecedents of modern-day plants and animals.