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PubMed1975

Heme biosynthesis in bacterium-protozoon symbioses: enzymic defects in host hemoflagellates and complemental role of their intracellular symbiotes.

Heme biosynthetic activity in the symbiotic association involving crithidial flagellates and intracellular bacteroids was studied by enzymic, nutritional, and isotope incorporation experiments. Component organisms and their complexes in this association were analyzed separately to determine the underlying cause of the hemin requirement of hemoflagellates and the role of symbiotes in sparing this requirement of two crithidial species. Nutritional study of symbiote-free flagellates showed that their growth requires at least 0.1 mug/ml of hemin, which can be substituted by protoporphyrin IX, but not by the porphyrin precursors, delta-amino-levulinic acid or porphobilinogen. These flagellates, in the presence of protoporphyrin IX, incorporated 59Fe into heme, indicating that they possess ferrochelatase (EC 4.99.1.1), the terminal enzyme in the heme biosynthetic pathway, which catalyzes the insertion of iron into protoporphyrin IX. In symbiote-containing flagellates serially cultured in a defined medium free of tetrapyrrole compounds, heme and porphyrins can be detected by a fluorophotometric method, indicative of heme biosynthesis. Study of [14C]glycine incorporation into heme showed that the rate is much higher in symbiote-containing flagellates than in those without symbiotes. Microassay of uroporphyrinogen I synthase [EC 4.3.1.8; porphobilinogen ammonia-lyase (polymerizing)] revealed that the specific activity is high in symbiote-containing flagellates and higher still in isolated symbiotes, but essentially negligible in symbiote-free organisms. It is concluded that the bacterial symbiotes augment a very limited heme biosynthetic capacity of host flagellates by supplying uroporphyrinogen I synthase and perhaps other enzymes preceding ferrochelatase in the heme biosynthetic chain.

Aminolevulinic Acid↗

PubMed1979

Eukaryotic mRNA cap binding protein: purification by affinity chromatography on sepharose-coupled m7GDP.

A 24,000-dalton polypeptide that binds strongly and can be specifically crosslinked to the 5'-terminal cap structure m7GpppN in eukaryotic mRNAs has been detected in protein synthesis initiation factor preparations [Proc. Natl. Acad. Sci. USA (1978) 75, 4843--4847]. This polypeptide has been purified to apparent homogeneity by one chromatographic passage through an affinity resin prepared by coupling the levulinic acid O2',3'-acetal of m7GDP to AH-Sepharose 4B. Translation, in HeLa cell extracts, of capped mRNAs including Sindbis virus, reovirus, and rabbit globin mRNAs was stimulated by the cap-binding protein under conditions that did not increase translation of noncapped RNAs of encephalomyocarditis virus and satellite tobacco necrosis virus.

Animals↗

PubMed1981

Biosynthesis of porphyrins and heme from gamma, delta-dioxovalerate by intact hepatocytes.

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.

Alanine↗

PubMed1996

Identification of a conserved and functional iron-responsive element in the 5'-untranslated region of mammalian mitochondrial aconitase.

Iron-responsive elements (IREs) are RNA stem-loop motifs found in genes of iron metabolism. When cells are iron-depleted, iron regulatory proteins (IRPs) bind to IREs in the transcripts of ferritin, transferrin receptor, and erythroid amino-levulinic acid synthetase. Binding of IRPs to IRE motifs near the 5' end of the transcript results in attenuation of translation while binding to IREs in the 3'-untranslated region of the transferrin receptor results in protection from endonucleolytic cleavage. Iron deprivation results in activation of IRE binding activity, whereas iron replete cells lose IRE binding activation. Here, we report the identification of a conserved IRE in the 5'-untranslated region of the transcript of the citric acid cycle enzyme mitochondrial aconitase from four different mammalian species. The IRE in the transcript of mitochondrial aconitase can mediate in vitro translational repression of mitochondrial aconitase by IRPs. Furthermore, levels of mitochondrial aconitase are decreased in mice maintained on a low iron diet, whereas levels of mRNA remain unchanged. The decrease in levels of mitochondrial aconitase is likely due to activation of IRP binding and consequent attenuation of translation. Thus, expression of the iron-sulfur protein mitochondrial aconitase and function of the citric acid cycle may be regulated by iron levels in cells.

Aconitate Hydratase↗

PubMed1975

Lead. Possible toxicity in urban vs rural rats.

The degree of lead poisoning in wild rats from two environments has been studied. Wild rats captured in an urban area had markedly elevated tissue lead compared with values in rural rats. This elevation may have been caused by differences in factors affecting absorption of ingested lead or an elevated respiratory exposure to airborne lead, or both, and lead in precipitated dust. Changes in several biologic indexes (depression of delta-amino levulinic acid dehydratase in kidney and red blood cells, presence of renal intranuclear inclusion bodies, and increased kidney weight) confirmed lead-poisoning in urban rats.

Adult↗

PubMed1998

Clinical multi-colour fluorescence imaging of malignant tumours--initial experience.

PURPOSE: The detection of malignant tumours relies on a variety of diagnostic procedures including X-ray images and, for hollow organs, endoscopy. The purpose of this study was to present a new technique for non-invasive tumour detection based on tissue fluorescence imaging. MATERIAL AND METHODS: A clinically adapted multi-colour fluorescence system was employed in the real-time imaging of malignant tumours of the skin, breast, head and neck region, and urinary bladder. Tumour detection was based on the contrast displayed in fluorescence between normal and malignant tissue, related to the selective uptake of tumour-marking agents, such as haematoporphyrin derivative (HPD) and delta-amino levulinic acid (ALA), and natural chromophore differences between various tissues. In order to demarcate basal cell carcinomas of the skin, ALA was applied topically 4-6 h before the fluorescence investigation. For urinary bladder tumour visualisation (transitional cell carcinoma of different stages including carcinoma in situ), ALA was instilled into the bladder 1-2 h prior to the study. Malignant and premalignant lesions in the head and neck region were imaged after i.v. injection of HPD (Photofrin). Finally, the extent of in situ and invasive carcinomas of the breast was investigated in surgically excised specimens from patients that received a low-dose injection of HPD 24 h prior to the study. The tumour imaging system was coupled to an endoscope. Fluorescence light emission from the tissue surface was induced with 100-ns-long optical pulses at 390 nm, generated from a frequency-doubled alexandrite laser. With the use of special image-splitting optics, the tumour fluorescence, intensified in a micro-channel plate, was imaged in 3 selected wavelength bands. These 3 images were processed together to form a new optimised-contrast image of the tumour. This image, updated at a rate of about 3 frames/s, was mixed with a normal colour video image of the tissue. RESULTS: A clear demarcation from normal surrounding tissue was found during in vivo measurements of superficial bladder carcinoma, basal cell carcinoma of the skin, and leukoplakia with dysplasia of the lip, and in in vitro investigations of resected breast cancer. CONCLUSIONS: The initial clinical experience of using multi-colour fluorescence imaging has shown that the technique has the potential to reveal malignant tumour tissue, including non-invasive early carcinoma and also precancerous tissue. Further investigations are needed to fully develop the method.

Adult↗

PubMed2000

DNA damage formation, DNA repair, and survival after exposure of DNA repair-proficient and nucleotide excision repair-deficient human lymphoblasts to UVA1 and UVB.

PURPOSE: The comet assay has been used to visualize DNA damage in single cells after exposure to UV light. These comets are commonly thought to reflect transient, repair-induced DNA breaks. The goal of the work presented here was to further characterize the nature of UV-induced comets and to further elucidate DNA damage formation by different wavelengths of ultraviolet light. MATERIALS AND METHODS: Detailed dose-response and time-course experiments with comet formation were carried out with normal and nucleotide excision repair (NER)-deficient xeroderma pigmentosum (XP) lymphoblasts. Irradiation was carried out with low, intermediate, or high doses of UVA1 or UVB, comet formation was observed, cell survival and viability were determined, and UV-induced apoptosis was measured. RESULTS: All responses were dose-dependent. With the intermediate dose of UVA1, a pronounced comet formation was observed without subsequent growth inhibition. Raising levels of porphyrins, which act as photosensitizers, by preincubation with 5-amino-levulinic acid increased comet formation with UVA1, but not with UVB. UVA1-sensitivity and comet formation in XP cells was not significantly different from the normal cells. With UVB no comet formation was seen without subsequent apoptotic cell death. XP cells exhibited the known UVB-hypersensitivity, but their comet formation was not significantly different from that of normal cells. CONCLUSIONS: The findings are compatible with the hypothesis that UV-induced comets represent transient repair-induced DNA breaks. Both, the NER of dimers and the base excision repair of oxidative DNA modifications are thought to contribute to comet formation.

Apoptosis↗

PubMed1985

Nitrogen utilization, enzyme activity, glucose intolerance and leukocyte chemotaxis in human experimental zinc depletion.

We previously reported significant decreases in plasma, whole blood, urinary, seminal and fecal zinc in six young men consuming a semipurified formula diet providing 0.28 mg zinc and 0.8/kg protein per day for 4-9 weeks. During a one-week baseline period, 15.7 mg of zinc (as ZnSO4) were fed; three of the men were repleted with 6.0, 23.2 or 46.3 mg zinc for 2-5 weeks. Biochemical and functional measures of zinc status other than tissue zinc levels were also monitored. No one parameter appeared to parallel dietary zinc status in all subjects, although significant mean changes were seen in serum and leukocyte alkaline phosphatases. Inconsistent changes were noted in erythrocyte delta-amino levulinic acid dehydratase, plasma alkaline ribonuclease and the serum alkaline phosphatase isoenzymes. Nitrogen balance was unaffected by zinc nutritional status. However, alterations in hair root growth phase and morphology, decreases in lymphocyte counts and in transferrin levels during depletion suggest impairment in protein synthesis. Impaired leukocyte chemotaxis and clinical signs indicative of decreased resistance to infection were also noted.

Adult↗

PubMed1985

Incorporation of heme to microsomal cytochrome P-450 in the absence of protein biosynthesis.

Determination of the heme and protein portions of phenobarbital (PB)-inducible and 3-methylcholanthrene inducible forms of cytochrome P-450, P-450(PB-1), and P-450(MC-1), in the liver microsomes of drug-treated animals indicated the presence of 20-30% of apo-cytochrome P-450 in both cases. Inhibition of protein synthesis by cycloheximide injection to the rats did not significantly inhibit the incorporation of delta-amino[14C]levulinic acid (ALA) into the heme of P-450(PB-1) or P-450(MC-1) in the liver, indicating that the heme incorporation into microsomal cytochrome P-450 is not tightly coupled with the synthesis of the apo-cytochrome. When heme-labeled cytosol prepared from [14C]ALA-injected rats was incubated with non-radioactive microsomes in vitro, a significant amount of labeled heme was incorporated into microsomal P-450(PB-1), whereas the incorporation into P-450(MC-1) was much less. The in vitro transfer of heme from cytosol to microsome-bound cytochrome P-450 was stimulated by the addition of an NADPH-generating system to the incubation mixtures, and inhibited when the microsomes were solubilized with sodium cholate and Emulgen-913. Although the in vitro incubation of heme-labeled microsomes with non-radioactive cytosol resulted in some release of labeled heme from the microsomes, no reversible transfer of heme between cytochrome P-450 molecules bound to separate microsomal vesicles was detected when heme-labeled microsomes were incubated with non-radioactive microsomes in the presence and absence of cytosol.

Aminolevulinic Acid↗

PubMed1995

Nucleotide sequence and expression of the genes for the alpha and beta subunits of phycocyanin in Cyanidium caldarium.

The nucleotide sequence of the plastid-encoded operon containing genes for the alpha (cpcA) and beta (cpcB) subunits of phycocyanin in the unicellular red alga Cyanidium caldarium is described. cpcB is located 5' to cpcA and the two genes are separated by a 102-bp spacer region. The transcription start site of cpcBA was mapped to 80 bp upstream of the ATG initiation codon of cpcB. Promoter-like elements similar to the -10 (TATAAT) and -35 (TTGACA) consensus promoters in bacteria were found 6 and 31 bp upstream of the transcription initiation site. Northern blotting revealed an abundant 1.3-kb cpcBA transcript in illuminated cells, but this transcript was undetectable in dark-grown cells. Expression levels of cpcBA in cells incubated with 10(-6) M heme in the dark were similar to those in cells illuminated for 24 h. Cells illuminated with 150 microM gabaculine (an inhibitor of delta-aminolevulinate synthesis) or 10 mM levulinic acid (an inhibitor of delta-aminolevulinate dehydrase) lacked detectable cpcBA transcripts. In cells illuminated with 200 microM N-methyl-mesoporphyrin IX (an inhibitor of ferrocheletase), inhibition of cpcBA expression and phycocyanin synthesis was similar. These results provide strong evidence that light induction of the cpcBA operon is dependent on synthesis of heme.

Amino Acid Sequence↗

PubMed1971

The sites of photoconversion of protochlorophyllide to chlorophyllide in barley seedlings.

The photoreduction of protochlorophyllide a to chlorophyllide a in intact 6-day-old seedlings of etiolated barley (Hordeum vulgare) exhibits a small initial phase, followed by an induction period of about 1 hour before a rapid phase of additional chlorophyll formation begins. Cycloheximide, an inhibitor of protein synthesis, has no effect on the initial phase of conversion of preformed protochlorophyllide, but it either abolishes or severely inhibits the subsequent phase of rapid chlorophyll synthesis within 45 minutes of its application to the seedlings. An analysis of the biphasic inhibition process suggests that the lifetime of the enzyme controlling protochlorophyllide synthesis (probably delta-amino-levulinic acid synthetase) is not longer than 10 minutes.The rapid phase of chlorophyll formation can be effected by a series of brief (15 second) pulses of light spaced at least 5 minutes apart. When longer dark intervals are used, no increase is observed in the yield of chlorophyll per pulse. We interpret the findings to indicate that the photoconversion takes place at distinct enzymatic sites whose concentration does not increase during a period of 4 hours following the initial illumination. The sites can be used repeatedly with a turnover time determined by the removal of the product chlorophyllide and the synthesis and placement of a new protochlorophyllide molecule.

Journal Article↗

PubMed1983

5-Aminolevulinate Synthesis in Permeabilized Filaments of the Blue-Green Alga Anabaena variabilis.

Filaments of the blue-green alga Anabaena variabilis permeabilized by dimethylsulfoxide (DMSO) produce increased amounts of 5-aminolevulinate in the presence of levulinic acid. The metabolic activity of the filaments remains unperturbed in the presence of up to 7.5% (v/v) DMSO. Studies utilizing DMSO-permeabilized filaments confirm that 5-aminolevulinate is synthesized preferably from glutamate and, to a lesser extent, from alpha-ketoglutarate in this organism.

Journal Article↗

PubMed1984

Isopentenyl Pyrophosphate cis-1,4-Polyisoprenyl Transferase from Guayule (Parthenium argentatum Gray).

Electron micrographs of the mesophyll cells of guayule Parthenium argentatum Gray leaves show deposits of cis-polyisoprene (rubber) in the cytoplasm in the vicinity of mitochondria and chloroplasts and demonstrate that the rubber-synthesizing enzymes are present in guayule leaves. The terminal step in the synthesis of cis-polyisoprene from isopentenyl pyrophosphate (IPP) catalyzed by isopentenyl pyrophosphate cis-1,4-polyisoprenyl transferase has been demonstrated in crude leaf extracts by the enzymic incorporation of [(14)C]isopentenyl pyrophosphate into the polymer and the recovery of [(14)C]levulinic acid following ozonolysis. The rubber transferase activity in the crude extracts of guayule leaves was 5.8 nanomoles isopentenyl pyrophosphate incorporated per milligram protein per hour. This is the first description of the rubber transferase from a nonlaticiferous plant.The specific activity (in units of nanomoles IPP converted per milligram protein per hour) of the partially purified enzyme following chromatography on diethylaminoethyl-cellulose columns was 41.7 units and contained 0.29 units of IPP isomerase activity and 0.08 units of farnesyl pyrophosphate synthetase activity. The rubber transferase requires reduced glutathione and Mg(2+) for maximal activity. There was no incorporation of IPP into cis-1,4-polyisoprene in the absence of rubber particles as primer, and Langmuir isotherm plots showed that the specific activity of the enzyme was proportional to the concentration of the enzyme on the surface of the rubber particles. For a given rubber particle distribution, enzyme activity was proportional to time, IPP concentration, and rubber concentration. The addition of 0.4 millimolar dimethylallyl pyrophosphate to the rubber transferase reaction resulted in a 2-fold increase in the incorporation of IPP into rubber. A comparison was made of the relative activities of rubber transferase in different species of Parthenium, Ficus, and Euphorbia.

Journal Article↗

PubMed1985

A Low Molecular Weight Polypeptide Which Accumulates upon Inhibition of Porphyrin Biosynthesis in Maize.

Levulinic acid, an inhibitor of porphyrin biosynthesis, causes marked accumulation of a low molecular weight polypeptide in greening maize (Zea mays L.) leaves. Additional compounds which interfere with porphyrin synthesis (e.g. aminooxyacetate, iron-chelators, 4,6-dioxoheptanoic acid) had a similar effect. The polypeptide accumulated in the cytosol and could not be detected in the plastid stroma. Its molecular weight was estimated as 4800 daltons by electrophoresis in sodium dodecyl sulfate-acrylamide gels containing urea and glycerol. The accumulation of the polypeptide did not result from inhibition of chlorophyll or protoheme syntheses. Compounds which caused its accumulation markedly reduced the activity of nitrite reductase. It is suggested that the accumulation is caused by inhibition of siroheme synthesis which interferes with the formation of nitrite or sulfite reductase.

Journal Article↗

PubMed1994

Studies of porphyrin-containing specimens using an optical spectrometer connected to a confocal scanning laser microscope.

A spectrometer has been developed for use with a confocal scanning laser microscope. With this unit, spectral information from a single point or a user-defined region within the microscope specimen can be recorded. A glass prism is used to disperse the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit keeps the detector at 277 K, thereby allowing integration times of up to 60 s. The spectral resolving power, lambda/delta lambda, ranges from 350 at lambda = 400 nm to 100 at lambda = 700 nm. Since the entrance aperture of the spectrometer has the same size as the detector pinhole used during normal confocal scanning, the three-dimensional spatial resolution is equivalent to that of normal confocal scanning. Light from the specimen is deflected to the spectrometer by a solenoid controlled mirror, allowing fast and easy switching between normal confocal scanning and spectrometer readings. With this equipment, studies of rodent liver specimens containing porphyrins have been made. The subcellular localization is of interest for the mechanisms of photodynamic therapy (PDT) of malignant tumours. Spectroscopic detection is necessary to distinguish the porphyrin signal from other fluorescent components in the specimen. Two different substances were administered to the tissue, Photofrin, a haematoporphyrin derivative (HPD) and delta-amino levulinic acid (ALA), a precursor to protoporphyrin IX and haem in the haem cycle. Both are substances under clinical trials for PDT of malignant tumours. Following administration of these compounds to the tissue, the potent photosensitizer and fluorescent compound Photofrin, or protoporphyrin IX, respectively, is accumulated.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminolevulinic Acid↗

PubMed1976

UDP-glucuronyltransferase in perfused rat liver and in microsomes. Glucuronidation of bilirubin.

In perfused rat liver and in fistula rats the formation of bilirubin conjugates was studied after labeling with [14C]bilirubin,5-amino [14C]levulinic acid and [14C]hemin. The latter two compounds were used to study heme degradation to bilirubin from intrahepatic and extrahepatic sources, respectively. Bilirubin glucuronides were the major conjugates in fistula bile. In liver perfusion bile the proportion of non-glucuronide conjugates was increased. After a high dose of hemin (2.5 mumol) bilirubin glucuronides were decreased compared with other bilirubin conjugates both in fistula bile and in liver perfusion bile. In addition green pigments were formed. These alterations were reversed in chronically hemin-treated rats in which heme oxygenase had been induced. The interference of UDP-glucose and UDP-glucuronic acid with bilirubin glucuronidation and glucosidation was studied in liver microsomes. UDP-glucose did not affect bilirubin glucuronidation in native microsomes in which UDP-glucuronyltransferase activity is constrained. When this constraint was released by various treatments altering membrane structure UDP-glucose markedly inhibited bilirubin glucuronidation. However, under these conditions bilirubin glucosidation was unaffected by UDP-glucuronic acid. The results suggest that the release of the constraint of UDP-glucuronyltransferase in vivo may lead to a decrease of the proportion of bilirubin glucuronides to other bilirubin conjugates in bile.

Aminolevulinic Acid↗

PubMed1980

Affinity chromatography on immobilised nucleotides. The synthesis, specificity and applications of immobilised inosine 5'-monophosphate.

The synthesis and characterisation of two IMP analogues, 8-(6-aminohexyl)-ionosine 5'-monophosphate, Ahx8IMP, and inosine 2',3'-O-[1-(6-aminohexyl)-levulinic acid amide]-acetyl 5'-monophosphate, (AhxLvn)2',3'IMP, is described. These analogues were attached to CNBr-activated agarose through the terminal amino group of the spacer molecule. The immobilised-IMP analogues displayed specificity for the inosine-nucleotide-dependent enzyme, IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.2.1.14) but not for the NAD+-dependent enzymes, L-alanine and L-acetate dehydrogenases. Escherichia coli IMP dehydrogenase could be eluted biospecifically from immobilised 8-substituted and ribose-substituted IMP adsorbents with IMP, XMP and GMP. Multiple peaks of enzyme activity in the elution profiles were interpreted in terms of aggregation of the enzyme. A protocol for the large-scale purification of E. coli IMP dehydrogenase is proposed. Homogeneous enzyme of specific activity 9.1 units/mg was obtained in 50% overall yield, representing 14 mg pure protein from a 20-1 culture of E. coli. The two IMP analogues were inactive as substrates in the IMP dehydrogenase reaction.

Animals↗

PubMed1986

Labelling of chlorophylls and precursors by [2-14C]glycine and 2-[1-14C]oxoglutarate in Rhodopseudomonas spheroides and Zea mays. Resolution of the C5 and Shemin pathways of 5-aminolaevulinate biosynthesis by thin-layer radiochromatography.

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)

Aminolevulinic Acid↗