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A Mendelian mutation, r-1, in Chlamydomonas reinhardtii has been isolated which elevates protoporphyrin accumulation of the Mendelian protoporphyrin mutants brS-1 and brC-1 more than 20 fold. This increased protoporphyrin accumulation is shown to result from increased delta-aminolevulinic acid synthesis in the double mutants brS-1 r-1 and brC-1 r-1 over that of brS-1 and brC-1 alone. By itself, the r-1 mutation has no detectable protoporphyrin accumulation and has reduced levels of delta-aminolevulinic acid synthesizing activity, chlorophyll, protoheme, and cytochrome oxidase activity. The low levels of chlorophyll and protoheme in r-1 can be increased by feeding delta-aminolevulinic acid. We hypothesize that r-1 may be a mutation of the gene coding for the delta-aminolevulinic acid synthesizing enzyme which reduces the sensitivity of this enzyme to feedback inhibition by protoporphyrin or heme as well as reducing the overall activity of the enzyme. Evidence is also presented for a single delta-aminolevulinic acid synthesizing enzyme serving both chlorophyll and heme biosynthesis.

In vitro induction of aminoketone synthesis (AKS) by allylisopropylacetamide (AIA) in liver slices from 30 day-old male rats was reversibly inhibited by puromycin (50 micrograms/ml incubation mixture). Normal AKS was initially stimulated and thereafter blocked. Chloramphenicol (350 micrograms/ml) inhibited AIA mediated induction of AKS only partially and only when added 1 h before AIA to the incubation mixture. In vivo puromycin (3 i.p. injections of 15 mg/kg in 1 h intervals) did not influence AKS or AIA mediated AKS induction in newborn rats, whereas chloramphenicol (500 mg/kg s.c. 30 min prior to AIA) inhibited AIA mediated AKS induction in newborn rats. In 40 day-old rats pretreatment with chloramphenicol completely inhibited AKS induction by 200 mg/kg AIA.

Chlorophyll and bacteriochlorophyll formation from 14C-labelled precursors was studied during the illumination of etiolated maize leaves excised from dark-grown seedlings and in cell suspensions of respiring, dark-, aerobically-grown Rhodopseudomonas spheroides adapting to the photosynthetic state in the light under anaerobic conditions. It was found that 1-14C-labelled glutamate and 2-oxoglutarate were incorporated into the tetrapyrrole moieties of chlorophyll and bacteriochlorophyll. This suggests that the C5 pathway of tetrapyrrole biosynthesis operates in both Zea mays and R. spheroides since in the alternative Shemin pathway the label would have been lost as 14CO2 during the formation of succinyl-CoA prior to the condensation with glycine to form 5-aminolaevulinate. It was also found that 5(-14)C-labelled glutamate and 2-oxoglutarate were incorporated into these chlorophylls which is consistent with the operation of both the C5 and Shemin pathways. That the Shemin pathway is also involved was confirmed by the incorporation of [2(-14)C]glycine into both chlorophylls. None of these substrates were incorporated into the phytol moieties of either plant or bacterial chlorophyll or into the carotenoids. However, when [1(-14)C]acetate was added to greening maize leaves not only the tetrapyrrole and phytol moieties were labelled but also the carotenoids: the labelling of these lipids is consistent with their formation from acetate via the isopentenyl pyrophosphate pathway. By comparing the incorporation of [1(-14)C]2-oxoglutarate with that of [5(-14)C]2-oxoglutarate the approximate relative contribution of each pathway to chlorophyll biosynthesis was determined. In maize leaves both pathways contributed almost equally but in R. spheroides the contribution by the Shemin and C5 pathways was 90 and 10%, respectively.

Glutamic acid 1-semialdehyde hydrochloride was synthesized and purified. Its prior structural characterization was extended and confirmed by 1H NMR spectroscopy and chemical analyses. In aqueous solution at pH 1 to 2 glutamic acid 1-semialdehyde exists in a stable hydrated form, but at pH 8.0 it has a half-life of 3 to 4 min. Spontaneous degradation of the material at pH 8.0 generated some undefined condensation products, but coincidentally a significant amount isomerized to 5-aminolevulinate. At pH 6.8 to 7.0, glutamate 1-semialdehyde is sufficiently stable to permit routine and reproducible assay for glutamate 1-semialdehyde aminotransferase activity. Only about 20% of the enzyme extracted from chloroplasts was sensitive to inactivation by gabaculine with no pretreatment. However, when the enzyme was exposed to 5-aminolevulinate, levulinate or 4,5-dioxovalerate in the absence of glutamate 1-semialdehyde, it was completely inactivated by gabaculine; 4,6-dioxoheptanoate had no effect on the enzyme. These results lead to the hypothesis that the aminotransferase exists in the chloroplast in a complex with pyridoxamine phosphate, which must be converted to the pyridoxal form before it can form a stable adduct with gabaculine. We propose that the enzyme catalyzes the conversion of glutamate 1-semialdehyde to 5-aminolevulinate via 4,5-diaminovalerate.

A molecule of chlorophyll is synthesized from eight molecules of delta-aminolevulinate (DALA), the universal precursor of porphyrins. The light-regulated conversion of glutamate to delta-aminolevulinate in the stroma of greening plastids involves the reduction of glutamate to glutamate-1-semialdehyde and its subsequent transamination. The components performing this conversion have been isolated from barley and Chlamydomonas and separated into three fractions by serial affinity chromatography on Blue Sepharose and haem- or chlorophyllin-Sepharose. The complete reaction can be performed in vitro in a reconstituted assay by combining all three fractions. An RNA is the essential component of the chlorophyllin-Sepharose-bound fraction. By nucleotide sequence analysis, we have now identified this RNA as a chloroplast glutamate acceptor RNA. Glutamate attached by an aminoacyl bond to the 3'-terminal adenosine of this RNA is a substrate for the enzyme(s) which perform the subsequent reactions. This reaction represents a novel role for transfer RNA: participation in the metabolic conversion of its cognate amino acid into another metabolite of low relative molecular mass which subsequently is not used in peptide bond synthesis.

The purpose of this study was to assess the ability of hepatocytes to synthesize porphyrins and heme from delta-aminolevulinic acid derived from gamma, delta-dioxovalerate and alanine. An alternate pathway for delta-aminolevulinic acid synthesis, in contrast to the condensation of succinate and glycine by delta-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37] has been suggested. This has been supported by the isolation of gamma, delta-dioxovalerate transaminase from liver mitochondria (Varticovski, L., Kushner, J. P. & Burnham, B. F. (1980) J. Biol. Chem. 255, 3742-3747). Gamma, delta-dioxovalerate transaminase catalyzes the formation of delta-aminolevulinic acid by a transamination reaction involving gamma, delta-dioxovalerate and alanine. To assess the significance of this alternate route, we prepared suspensions of respiring rat hepatocytes, which were incubated with optimal concentrations of various additives and then analyzed spectrophotometrically for synthesized porphyrins. No porphyrin synthesis was detected in cell suspensions incubated with succinate(1 mM) and glycine (20 mM). Cell suspensions incubated with gamma, delta-dioxovalerate (0.5-1.0 mM) and alanine (20 mM) synthesized 0.19 nmol of porphyrin per 10(7) cells per 2 hr (SD, 0.057). Cell suspensions incubated with delta-aminolevulinic acid (0.1 mM) synthesized 1.26 nmol of porphyrin per 10(7) cells per 2 hr (range, 1.18-1.32). Incubations with chemically synthesized gamma, delta-dioxo[5-14C]valerate were followed by extraction and purification of porphyrin esters and heme. Liquid scintillation counting revealed radiolabel incorporation into both porphyrins and heme. These studies demonstrate significant tetrapyrrole synthesis by the gamma, delta-dioxovalerate transaminase reaction. That gamma, delta-dioxovalerate is an important precursor of heme in vivo must be considered.

The first committed intermediate of the chlorophyll biosynthetic pathway is delta-aminolevulinic acid (ALA). In plant cells, ALA is formed from glutamate by a pathway not yet clearly defined. One of the proposed pathways involves the reduction of glutamate to glutamate-1-semialdehyde (GSA) via a glutamyl-tRNA intermediate. GSA is then converted to ALA by an aminotransferase. We are studying this pathway using partially purified components from Chlamydomonas reinhardtii in in vitro reactions with [3H]L-glutamate as the substrate and analysis of the radioactive reaction products via HPLC. In reactions either lacking GSA-aminotransferase or containing gabaculine (an inhibitor of aminotransferase), a radioactive intermediate is formed which cochromatographs with synthetic GSA. As observed previously for ALA synthesis, the synthesis of this intermediate has an absolute requirement for RNA, ATP, and active enzymes, while the requirement for NADPH is less stringent. Both the accumulated intermediate and the synthetic GSA can be converted to ALA by the aminotransferase without any additional substrates or cofactors. These results support previous observations that GSA or a very similar compound is an intermediate of ALA synthesis.

A hemA mutant of Escherichia coli containing a multicopy plasmid which complemented the mutation excreted 5-aminolevulinic acid (ALA) into the medium. [1-14C]glutamate was substantially incorporated into ALA by this strain, whereas [2-14C]glycine was not. Periodate degradation of labeled ALA showed that C-5 of ALA was derived from C-1 of glutamate. The synthesis of ALA by two sonicate fractions which had been processed by gel filtration and dialysis, respectively, was dependent on glutamate, ATP, NADPH, tRNA(Glu), and pyridoxal phosphate. tRNA(Glu) stimulated ALA synthesis in a concentration-dependent manner. Pretreatment with RNase reduced this stimulation. The amino acid sequence of the cloned insert, derived from the nucleotide sequence (J.-M. Li, C. S. Russell, and S. D. Cosloy, J. Cell Biol. 107:617a, 1988), showed no homology with any ALA synthase sequenced to date. These results suggest that E. coli synthesizes ALA by the C5 pathway from the intact five-carbon chain of glutamate.

In the chloroplasts of higher plants and algae, the biosynthesis of the chlorophyll precursor delta-aminolevulinic acid (ALA) involves at least three enzymes and a tRNA species. Here we demonstrate that in cell extracts of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 ALA was formed from glutamate in a series of reactions in which activation of glutamate by glutamyl-tRNAGlu formation was the first step. The activated glutamate was reduced by a dehydrogenase which displayed tRNA sequence specificity. Fractionation of strain 6803 tRNA by reverse-phase chromatography and polyacrylamide gel electrophoresis yielded two pure tRNAGlu species which stimulated ALA synthesis in vitro. These tRNAs had identical primary sequences but differed in the nucleotide modification of their anticodon. The 6803 tRNAGlu was similar to the sequences of tRNAGlu species or tRNAGlu genes from Escherichia coli and from chloroplasts of Euglena gracilis and higher plants. Southern blot analysis revealed at least two tRNAGlu gene copies in the 6803 chromosome. A glutamate-1-semialdehyde aminotransferase, the terminal enzyme in the conversion of glutamate to ALA in chloroplasts, was detected in 6803 cell extracts by the conversion of glutamate-1-semialdehyde to ALA and by the inhibition of this reaction by gabaculin.

The formation of delta-aminolevulinic acid in mammals birds, yeast and some bacteria is known to take place by the ALA-synthetase assisted coupling of glycine with succinylCoA. Plants, however, form the bulk of their delta-aminolevulinate in another way. We present evidence here that the intact, 5-carbon chain of glutamate becomes that of delta-aminolevulinate. Greening barley was fed specifically labelled glutamate and levulinate to create a pool of labelled delta-aminolevulinate. Levulinate, a competitive inhibitor of ALA-dehydratase, prevents the metabolism of delta-aminolevulinate. The carbon chain of the labelled delta-aminolevulinate was broken into formaldehyde and succinate by periodate to determine the position of the label. It was found that the C1, carboxyl carbon of glutamate becomes the amino-bearing (C5) carbon of delta-aminolevulinate and forms the formaldehyde on cleavage. delta-Aminolevulinate formed from C3,4-labelled glutamate bears its label in the succinate cleaved fragment. We conclude that during the light induced development of the plastid in barley the carbon chain of delta-aminolevulinate is formed from the intact chain of glutamate. ALA-synthetase catalyzed the formation of delta-aminolevulinate from glycine and succinylCoA cannot play a quantitively important role in the formation of chlorophyll.

The C-5 of 5-aminolaevulinate, a tetrapyrrole precursor which accumulates when inhibitory laevulinate is present, is derived from either the C-2 of glycine by the 5-aminolaevulinate-synthase-mediated Shemin pathway or the C-1 of 2-oxoglutarate by the C5 pathway. Thin-layer-radiochromatographic procedures are described for determining whether [2-14C]glycine or 2-[1-14C]oxoglutarate labelled the macrocycle of bacteriochlorophyll a, in addition to or rather than the methyl ester or phytyl ester moieties of the side-chains. The method was also used for detecting whether the same substrates label the formaldehyde (C-5) or the succinate (C-1 to C-4) fragments, obtained by periodate cleavage of 5-aminolaevulinate. These methods therefore can readily distinguish between the Shemin and C5 pathways as was demonstrated by using Rhodopseudomonas spheroides and Zea mays (maize), respectively, as examples of each pathway. Both [2-14C]glycine and, to a lesser extent 2-[1-14C]oxoglutarate labelled the macrocycle of bacteriochlorophyll a formed during adaptation of respiring R. spheroides cells to photosynthetic (anaerobic, illuminated) conditions. This and earlier evidence suggested augmentation of the Shemin pathway by a minor C5 pathway contribution. The present studies revealed only Shemin pathway activity: with laevulinate present, [2-14C]glycine formed 5-[5-14C]aminolaevulinate as proved by H14CHO production during periodate cleavage. These methods were sufficiently sensitive also to detect the incorporation of 14CO2, from degradation of either substrate, into 5-aminolaevulinate via the Shemin pathway thus labelling the succinate fragment produced with periodate: this explains bacteriochlorophyll a labelling by 2-[1-14C]oxoglutarate and proves double labelling of 5-aminolaevulinate by [2-14C]glycine. The same techniques were applied to etiolated maize leaves exposed to aerobic illuminated conditions with laevulinate and either 2-[1-14C]oxoglutarate or [2-14C]glycine as substrates. Only the C5 pathway was detected: 2-[1-14C]oxoglutarate was converted to 5-[5-14C]aminolaevulinate, which yielded H14CHO on periodate cleavage. This is not inconsistent with our earlier 13C-NMR studies [Porra, R.J., Klein, O. and Wright, P. E. (1983) Eur. J. Biochem. 130, 509-516] showing that the C5 pathway formed all the 5-aminolaevulinate for chlorophyll biosynthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Two biosynthetic pathways are known for the universal tetrapyrrole precursor, delta-aminolevulinic acid (ALA). In the ALA synthase pathway which was first described in animal and some bacterial cells, the pyridoxal phosphate-dependent enzyme ALA synthase catalyzes condensation of glycine and succinyl-CoA to form ALA with the loss of C-1 of glycine as CO2. In the five-carbon pathway which was first described in plant and algal cells, the carbon skeleton of glutamate is converted intact to ALA in a proposed reaction sequence that requires three enzymes, tRNA(Glu), ATP, Mg2+, NADPH, and pyridoxal phosphate. We have examined the distribution of the two ALA biosynthetic pathways among various genera, using cell-free extracts obtained from representative organisms. Evidence for the operation of the five-carbon pathway was obtained by the measurement of RNase-sensitive label incorporation from glutamate into ALA, using 3,4-[3H]glutamate or 1-[14C]glutamate as substrate. ALA synthase activity was indicated by RNase-insensitive incorporation of label from 2-[14C]glycine into ALA. The distribution of the two pathways among the bacteria tested was in general agreement with their previously established phylogenetic relationships and clearly indicates that the five-carbon pathway is the more ancient process, whereas the pathway utilizing ALA synthase probably evolved much later. The five-carbon pathway is apparently the more widely utilized one among bacteria, while the ALA synthase pathway seems to be limited to the alpha subgroup of purple bacteria.

The 13C-NMR spectra of bacteriochlorophyll a formed in the presence of L-[1-13C]glutamate and [2-13C]glycine in Chromatium vinosum strain D were analyzed. The isotope in the glutamate was specifically incorporated into eight carbon atoms in the tetrapyrrole macrocycle derived from the C-5 of 5-aminolevulinic acid (ALA), and the 13C in glycine was incorporated into the methyl carbon of the methoxycarbonyl group attached to the isocyclic ring of bacteriochlorophyll a. These labeling patterns provide evidence for the exclusive operation of the C5 pathway in ALA biosynthesis in the bacterium. The 13C chemical shifts of two quaternary carbons (C-9 and C-16) of bacteriochlorophyll a were reassigned in the present study.

The customary route in animals and bacteria for delta-aminolevulinic acid biosynthesis is from glycine and succinyl CoA, catalyzed by the enzyme delta-aminolevulinic acid synthetase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37]. Attempts to demonstrate this route in plants have been unsuccessful. Evidence is given for a new enzymic route of synthesis of delta-aminolevulinic acid in plants. This route involves the incorporation of the intact five-carbon skeleton of glutamic acid into delta-aminolevulinic acid. Demonstration of the new pathway in plants has been made by feeding specifically labeled [14C]glutamic acid to etiolated barley shoots greening in the light. In the presence of levulinate, a competitive inhibitor of delta-aminolevulinic acid dehydrastase [porphobilinogen synthase; delta-aminolevulinate hydro-lyase (adding delta-aminolevulinate and cyclizing); EC 4.2.1.24], delta-aminolevulinate accumulates. The delta-aminolevulinate formed was chemically degraded by periodate to formaldehyde and succinic acid. The C5 (formaldehyde) fragment was separated, as the 5,5-dimethyl-1,3-cyclohexanedione (dimedone) derivative, from the C1-C4 (succinic acid) fragment. The C5 atom contained radioactivity predominantly derived from C1 of glutamic acid. Conversely, the labeled C3 and C4 atoms of glutamic acid were found primarily in the succinic acid (C1-C4) fragment of delta-aminolevulinate. This labeling pattern for delta-aminolevulinic acid is consistent with a biosynthetic route utilizing the intact five-carbon skeleton of alpha-ketoglutarate, glutamate, or glutamine, and is inconsistent with the delta-aminolevulinic acid synthetase pathway utilizing glycine and succinyl CoA as precursors.

delta-Aminolevulinic acid is the first committed precursor in the biosynthesis of hemes, phycobilins, and chlorophylls. Plants and algae synthesize delta-aminolevulinic acid from glutamate via an RNA-dependent 5-carbon pathway. Previous reports demonstrated that cyanobacteria form delta-aminolevulinic acid from glutamate in vivo. We now report the direct measurement of this activity in vitro. Three oxygenic prokaryotes were examined, the unicellular cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 (Agmenellum quadruplicatum PR-6) and the chlorophyll a- and b-containing filamentous prochlorophyte Prochlorothrix hollandica. delta-Aminolevulinic acid-forming activity was detected in soluble extracts of all three species. delta-Aminolevulinic acid formation by Synechocystis extracts was further characterized. Activity depended upon addition of reduced pyridine nucleotide, ATP, and Mg2+ to the incubation mixture. NADPH was a more effective pyridine nucleotide than NADH at low concentrations, but NADPH inhibited delta-amino-levulinic acid formation above 1 mM, whereas NADH did not. The pH optimum was about 7.6, and the ATP concentration optimum was 0.1 mM. Activity was stimulated by addition of RNA derived from Synechocystis or Chlorella, and abolished by preincubation with RNase A. After RNase inactivation, activity was restored by addition of RNasin to block further RNase action, followed by supplementation with Synechocystis RNA. Activity was inhibited by micromolar concentrations of hemin, as was previously found with plant and algal extracts. Complete dependence on added glutamate could not be achieved. Radioactivity was incorporated into delta-aminolevulinic acid when the incubation mixture contained 1-[14C]glutamate. Activity in the Synechocystis enzyme extract was stimulated by the addition of a partially purified enzyme fraction from Chlorella. It thus appears that prokaryotic oxygenic organisms share with chloroplasts the capacity for biosynthesis of photosynthetic pigments from glutamate via the RNA-dependent 5-carbon pathway.

Cell-free preparations from the unicellular green alga, Chlorella vulgaris, catalyze the conversion of glutamate to delta-aminolevulinate, which is the first committed step in heme and chlorophyll biosynthesis. Most activity remains in the supernatant fraction after centrifugation at 264,000g. Additional activity can be solubilized from the high-speed pellet by treatment with 0.5 M NaCl. After gel filtration through Sephadex G-25, the reaction catalyzed by the high-speed supernatant requires glutamate, ATP, Mg2+, and NADPH. Boiled extract is inactive. The pH optimum is between 7.8 and 7.9 and the temperature optimum is 30 degrees C. Concentrations required for half-maximal activity are 0.05 mM glutamate, 0.4 mM ATP, 6 mM MgCl2, and 0.4 mM NADPH or 0.7 mM NADH. The reaction requires no additional amino donor. Involvement of pyridoxal phosphate in the catalytic mechanism is suggested by sensitivity to pyridoxal antagonists; 50% inhibition is achieved with 5 microM gabaculine or 0.4 mM aminooxyacetate. Involvement of two or more enzymes is suggested by the nonlinear reaction rate dependence on protein concentration. Evidence for the involvement of an activated glutamate intermediate was obtained by product formation after sequential addition and removal of substrates, and by inhibition (80%) with 1 mM hydroxylamine. Protoheme inhibits the activity by 50% at 1.2 microM. Preincubation of the extract with ATP causes stimulation and/or stabilization of the activity compared to preincubation without ATP or no preincubation. In preparations obtained from C. vulgaris strain C-10, which requires light for greening, dark-grown cells yield one-third as much activity as 4-h-greened cells.

The hemA mutation of Escherichia coli K-12 confers a requirement for delta-aminolevulinic acid (ALA). Cell extract prepared from the hemA strain SASX41B was incapable of producing ALA from either glutamate or glutamyl-tRNA, whereas extract of the hem+ strain HB101 formed colorimetrically detectable amounts of ALA and transferred label from 1-[14C]glutamate and 3,4-[3H]glutamyl-tRNA to ALA. Extracts of both strains converted glutamate-1-semialdehyde to ALA and were capable of aminoacylating tRNAGlu. Glutamyl-tRNA formed by extracts of both strains could be converted to ALA by the extract of hem+ cells. The extract of hemA cells did not convert glutamyl-tRNA formed by either strain to ALA. However, the hemA cell extract, when supplemented in vitro with glutamyl-tRNA dehydrogenase isolated from Chlorella vulgaris cells, formed about as much ALA as did the unsupplemented hem+ cell extract. We conclude from these observations that the enzyme activity that is lacking in the ALA auxotrophic strain carrying the hemA mutation is that of glutamyl-tRNA dehydrogenase.

L-Alanine:4,5-dioxovalerate aminotransferase, the enzyme that catalyzes the transamination between alanine and 4,5-dioxovalerate to yield delta-aminolevulinate and pyruvate, has been purified from extracts Clostridium tetanomorphum by acetone precipitation and successive tetanomorphum by acetone precipitation and successive chromatography on Sephadex G-150, hydroxyapatite, Octyl-Sepharose, and SP-Sephadex C-50. The enzyme is pure by the criterion of disc gel electrophoresis with varying polyacrylamide concentrations. It is dimeric, and has an apparent molecular weight of 111,000. Each molecule contains 2 molecules of pyridoxal 5-phosphate. The apparent Km values for 4,5-dioxovalerate and L-alanine are 0.26 and 1.96 mM, respectively. In addition to alanine, glutamate also is an effective amino group donor. The enzyme is inhibited by various keto acids as well as by inhibitors of pyridoxal phosphate-containing enzymes. It was possible to show that 4,5-dioxovalerate is formed by cultures of C. tetanomorphum when grown in the presence of 0.2 M levulinate, an inhibitor of 5-aminolevulinate dehydratase.

Chlorophyll and haem synthesis in illuminated Jerusalem artichoke tuber tissues were very efficiently inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid). This inhibition seems to be due specifically to a blockade of the pathway for 5-aminolaevulinate biosynthesis which used glutamate as a substrate (the so-called C5 pathway) since we could not detect any inhibition of protein synthesis in the treated tissues and there was no effect of gabaculine on the glycine-dependent yeast 5-aminolaevulinate synthase used as a model. In dark-aged artichoke tissues, gabaculine also effectively blocked cytochrome P-450 induction, peroxidase activity and 5-aminolaevulinic acid synthesis, thus suggesting the involvement of a C5 pathway in cytoplasmic and microsomal haemoprotein synthesis in this higher plant. Allylglycine and (2-amino-ethyloxyvinyl)glycine, two olefinic glycine analogues which are potential suicide inhibitors of pyridoxal phosphate enzymes, were also demonstrated to be effective blockers of chlorophyll synthesis in artichoke tuber and Euglena cells exposed to light.

When levulinic acid, a competitive inhibitor of delta-aminolevulinic acid dehydratase, was added to growing cultures of blue-green algae (cyanobacteria), delta-aminolevulinic acid was excreted into the medium and cell growth was inhibited.

When Chlamydomonas enzymes that convert glutamate to delta-aminolevulinic acid are separated into three fractions, one of the fractions contains RNA, and the RNA moiety is needed for the enzyme activity.

A cell-free extract of the anaerobic eubacterium, Clostridium thermoaceticum, catalyzes the synthesis of 5-aminolevulinic acid (ALA) from glutamate via the C5 pathway. The enzyme reaction resembles that of higher plants and algae in cofactor requirements and sensitivity to ribonuclease. From the phylogenetic distribution it is proposed that the C5 pathway evolved earlier than the ALA synthase pathway.

Glucagon elicits a twofold increase in delta-aminolevulinic acid synthetase activity in the livers of 18-day-old chick embryos. This rise occurs when RNA synthesis is inhibited, but is prevented when protein synthesis is blocked. Cyclic adenosine monophosphate appears not to be involved.