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Biomedical subjects

S I Beale

Publications and source records attributed to S I Beale.

At least 73 records · Page 4Linked to original sources
PubMed1984

Enzymic Transformation of Biliverdin to Phycocyanobilin by Extracts of the Unicellular Red Alga Cyanidium caldarium.

Cell-free extracts of the unicellular red alga Cyanidium caldarium catalyze the transformation of biliverdin to a product indistinguishable from phycocyanobilin, the free bilin derived from phycocyanin by methanolysis. Crude cell-free extract requires biliverdin as the only substrate, but after removal of low molecular weight components by gel filtration, the reaction shows an additional requirement for a reduced pyridine nucleotide. Boiled extract is enzymically inactive, activity is not sedimented by high-speed centrifugation, and mesobiliverdin cannot serve as a substrate.Incubation of cell extracts with biliverdin yields two products with very similar spectrophotometric properties in acidic methanol, but which are separable by reverse-phase high pressure liquid chromatography. The same two products are formed by methanolysis of protein-bound phycocyanin chromophore, with the late-eluting one predominating. The two products derived from either phycocyanin methanolysis or cell extract incubation with biliverdin are partially interconvertible and they form the same ethylidine-free isomeric derivative, mesobiliverdin. Their absorption spectra correspond to those of the Z- and E-ethylidine isomers of phycocyanobilin. Based on previous work showing that the major methanolysis product has the E-ethylidine configuration, the other product of methanolysis and enzymic biliverdin transformation is therefore the Z-ethylidine isomer. The time course for formation of the two products during incubation suggests that the early-eluting product is the precursor of the late-eluting one. These results suggest that Z-ethylidine phycocyanobilin is the precursor of the E-ethylidine isomer, and that the latter may be a normal cellular precursor to protein-bound phycocyanin chromophore.

Journal Article↗
PubMed1983

Separate physiological roles and subcellular compartments for two tetrapyrrole biosynthetic pathways in Euglena gracilis.

delta-Aminolevulinic acid (ALA), the first committed precursor to the tetrapyrrole components of hemes and chlorophylls, is synthesized by two different routes in the photosynthetic phytoflagellate Euglena gracilis: directly from glutamate, mediated by a 5-carbon pathway, and via condensation of glycine and succinyl-CoA, catalyzed by the enzyme ALA synthase. The physiological roles of the two pathways were determined by administration of specifically 14C-labeled ALA precursors to cultures growing under different physiological conditions. Relative activities of the ALA synthase and 5-carbon pathways were monitored by incorporation of radioactivity from [2-14C] glycine and [1-14C]glutamate into highly purified protoheme, heme a and chlorophyll a derivatives. Wild type cells grown photoautotrophically or photoheterotrophically synthesized chlorophyll and incorporated radioactivity from [1-14C]glutamate into the tetrapyrrole nucleus of the pigment. [2-14C]Glycine was incorporated primarily into the nontetrapyrrole-derived portions of chlorophyll. In the same cultures both [2-14C]glycine and [1-14C]glutamate were efficiently incorporated into protoheme, while only [2-14C] glycine was incorporated into heme a. In dark-grown wild type or light-grown aplastidic cells, no chlorophyll was formed, and both protoheme and heme a were labeled exclusively from [2-14C]glycine. These results indicate: (a) ALA synthase and the 5-carbon pathway operate simultaneously in growing green cells; (b) the 5-carbon pathway provides ALA for chloroplast protoheme and chlorophyll, and is associated with chloroplast development; (c) ALA synthase provides ALA only for nonplastid heme biosynthesis; and (d) the two ALA pathways are separately compartmentalized along with complete sets of enzymes for subsequent tetrapyrrole synthesis from each ALA pool. The protoheme that was synthesized from [1-14C] glutamate had a higher specific radioactivity than chlorophyll synthesized from the same precursor. This result together with calculated specific radioactivities of the products synthesized during the incubation period, suggest that both protoheme and heme a undergo metabolic turnover.

5-Aminolevulinate Synthetase↗
PubMed1983

Biosynthesis of phycocyanobilin from exogenous labeled biliverdin in Cyanidium caldarium.

Phycocyanin is a major light-harvesting pigment in bluegreen, red, and cryptomonad algae. This pigment is composed of phycocyanobilin chromophores covalently attached to protein. Phycocyanobilin is an open-chain tetrapyrrole structurally close to biliverdin. Biliverdin is formed in animals by oxidative ring-opening of protoheme. Recent evidence indicates that protoheme is a precursor of phycocyanobilin in the unicellular rhodophyte, Cyanidium caldarium. To find out if biliverdin is an intermediate in the conversion of protoheme to phycocyanobilin, [14C]biliverdin was administered along with N-methylmesoporphyrin IX (which blocks endogenous protoheme formation) to growing cells of C. caldarium. To avoid phototoxic effects due to the porphyrin, a mutant strain was used that forms large amounts of both chlorophyll and phycocyanin in the dark. After 12 or 24 h in the dark, cells were harvested and exhaustively extracted to remove free pigments. Next, protoheme was extracted. Phycocyanobilin was then cleaved from the apoprotein by methanolysis. Protoheme and phycocyanobilin were purified by solvent partition, DEAE-Sepharose chromatography, and preparative reverse-phase high-pressure liquid chromatography. Absorption was monitored continuously and fractions were collected for radioactivity determination. Negligible amounts of label appeared in the protoheme-containing fractions. A major portion of label in the eluates of the phycocyanobilin-containing samples coincided with the absorption peak at 22 min due to phycocyanobilin. In a control experiment, [14C]biliverdin was added to the cells after incubation and just before the phycocyanobilin-apoprotein cleavage step. The major peak of label then eluted with the absorption peak at 12 min due to biliverdin, indicating that during the isolation biliverdin is not converted to compounds coeluting with phycocyanobilin. It thus appears that exogenous biliverdin can serve as a precursor to phycocyanobilin in C. caldarium, and that the route of incorporation is direct rather than by degradation and reincorporation of 14C through protoheme.

Bilirubin↗
PubMed1983

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

The ability of N-methyl mesoporphyrin IX (NMMP) to block heme synthesis by specifically inhibiting enzymic iron insertion into protoporphyrin IX was exploited to test whether heme is a precursor of the bilin chromophore of phycocyanin (PC). A strain of the unicellular rhodophyte Cyanidium caldarium which forms normal amounts of both chlorophyll (Chl) and PC in the dark was employed to avoid phototoxic effects of exogenous porphyrins. Relative Chl and PC content were assayed spectrophotometrically on whole cell suspensions.When cells were grown in the dark on a glucose-based heterotrophic medium at 42 degrees C, neither division rate nor Chl synthesis was affected by NMMP up to 3.0 micromolar and for as long as 72 hours. NMMP had a dose-dependent inhibitory effect on PC synthesis. PC to Chl absorbance ratios, relative to control cell values, were 100%, 89%, 86%, and 50% in cells grown for 48 hours with 0.3, 1.0, 3.0, and 10.0 micromolar NMMP, respectively. NMMP also caused the accumulation of intracellular protoporphyrin.The ability of NMMP to cause intracellular accumulation of protoporphyrin and to block PC synthesis specifically while allowing normal Chl formation is consistent with its action as a specific inhibitor of enzymic iron chelation, and supports the role of heme as a precursor to the phycobilins.

Journal Article↗
PubMed1982

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 delta-aminolevulinic acid (ALA) synthase activity when administered to growing cultures of Euglena gracilis Klebs strain Z Pringsheim in micromolar concentrations. Wild-type light-grown green cells and white aplastidic cells exhibited 2.8-fold and 1.8-fold increases, respectively, in ALA synthase activity within five to six hours after incubation with 4 x 10(-6) molar N-methyl mesoporphyrin IX. Protoheme levels were decreased and (59)Fe 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.

Journal Article↗
PubMed1982

delta-Aminolevulinic Acid Synthase of Euglena gracilis: Regulation of Activity.

delta-Aminolevulinic acid (ALA), a key precursor of the tetrapyrroles heme and chlorophyll, is capable of being synthesized by two different routes in cells of the unicellular green alga Euglena gracilis: from the intact carbon skeleton of glutamate, and via the condensation of glycine and succinyl CoA, mediated by the enzyme ALA synthase. The regulatory properties of ALA synthase were examined in order to establish its role in Euglena.Partially purified Euglena ALA synthase, unlike the case with the bacterial or animal-derived enzyme, does not exhibit allosteric inhibition by the tetrapyrrole pathway products heme, protoporphyrin IX, and porphobilinogen, at concentrations up to 100 micromolar.In aplastidic mutant cells, extractable ALA synthase activity is constant during exponential growth, and decreases to low levels as the cells reach the stationary state. Rapid exponential decline of ALA synthase (t(1/2) = 55 min) occurs after administration of 43 micromolar cycloheximide, but not 6.2 millimolar chloramphenicol. These results suggest that, as in other eukaryotic cells, ALA synthase is synthesized on cytoplasmic ribosomes and is subject to rapid turnover in vivo.Extractable ALA synthase activity increases 2.5-fold within 6 hours after administration of 100 millimolar ethanol, a stimulator of mitochondrial development, and 4.5-fold within 12 hours after administration of 1 millimolar 4,6-dioxoheptanoic acid, which blocks ALA utilization, suggesting that activity is controlled in vivo by a feedback induction-repression mechanism, coupled with rapid enzyme turnover.In heterotrophically grown wild-type cells, low levels of ALA synthase rapidly increase 4.5-fold within 12 hours after cells are transferred from the light to the dark, and decrease exponentially (t(1/2) = 75 min) when cells are transferred from the dark to light. The dark levels are equal to those in light- or dark-grown aplastidic mutant cells. The low level occurring in light-grown wild-type cells is not altered by the presence of 10 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea, which blocks photosynthetic O(2) production. The decrease that occurs on dark-to-light transfer can be diminished by 12- or 24-hour prior incubation with 6.2 millimolar chloramphenicol, which also retards chlorophyll synthesis after the transfer to light.The positive relationship of ALA synthase activity to degree of mitochondrial expression, and the inverse relationship to plastid development and chlorophyll synthesis, suggests that ALA synthase functions to provide precursors to nonplastid tetrapyrroles in Euglena. In light-grown, wild-type cells, the diminished levels of ALA synthase may be due to the ability of developing plastids to export heme or a heme precursor to other cellular regions, which thereby supplants the necessity for ALA formation via the ALA synthase route.

Journal Article↗
PubMed1982

delta-Aminolevulinic Acid Formation from gamma,delta-Dioxovaleric Acid in Extracts of Euglena gracilis.

gamma,delta-Dioxovaleric acid (DOVA) has been proposed as a precursor to heme and chlorophyll in plants and algae. DOVA transaminase activity was found in extracts of the unicellular green alga Euglena gracilis Klebs strain Z Pringsheim. Optimum conversion of DOVA to delta-aminolevulinic acid (ALA) occurred at pH 6.8. ALA formation was linear with time for at least 30 minutes at 37 degrees C and was proportional to amount of cell extract in the incubation mixture. Boiled cell extract was inactive. DOVA transaminase from either wild-type or aplastidic derivative strain W(14)ZNaIL ran as a single band in agarose gel permeation chromatography, with a calculated molecular weight of 98,000 +/- 3,000. l-Glutamic acid was the most effective amino donor. d-Glutamic acid was inactive. K(m) values for l-glutamic acid and DOVA were 11 and 1.1 millimolar, respectively. Pyridoxal phosphate stimulated activity maximally at 30 micromolar, and (aminooxy)acetate was strongly inhibitory. Glyoxylic acid was a competitive inhibitor with respect to DOVA, with an inhibition constant of 0.62 millimolar. Wild-type and aplastidic cells vielded equal activity, 31 +/- 1 nanomoles ALA per 30 minutes per 10(7) cells, whether grown in light or dark. DOVA transaminase could not be separated from glyoxylate transaminase activity by agarose gel permeation or diethylaminoethyl-cellulose column chromatography. In all fractions, glyoxylate transaminase activity was at least 75 times greater than DOVA transaminase activity. DOVA transamination appears to be catalyzed by glyoxylate transaminase, and not to be of physiological significance with respect to chlorophyll synthesis in Euglena.

Journal Article↗
PubMed1981

delta-Aminolevulinic acid synthase from Euglena gracilis.

delta-Aminolevulinic acid (ALA) synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37] activity was detected in cell extracts of the unicellular green flagellate alga Euglena gracilis. The enzyme was identified by substrate and cofactor requirements, and activity was proportional to number of cells extracted and duration of incubation. The incubation product was spectrophotometrically and chromatographically identical to ALA. ALA synthase activity is present in two wild-type strains, Z and bacillaris, and in nongreening, aplastidic strains derived from them. When grown in the dark, wild-type strains have amounts of ALA synthase activity equal to the amounts in their aplastidic derivative strains. Growth in the light or dark does not affect the level of ALA synthase activity in the aplastidic strains, but the wild-type strains have only one-third as much activity when grown in the light. We propose that ALA synthase is responsible for nonplastid tetrapyrrole biosynthesis in Euglena.

5-Aminolevulinate Synthetase↗
PubMed1979

Diurnal variation in situ of photosynthetic capacity in ulva is caused by a dark reaction.

Ulva lactuca (sea lettuce) undergoes large diurnal oscillations of light-saturated photosynthetic O(2) evolution in situ. Freshly collected samples from Great Harbor, Woods Hole, Massachusetts, had a maximum white light-saturated rate at noon that was 2.5-fold higher than the rate of matched samples collected at midnight. When kept under constant low level illumination, the cycle persisted for at least 36 hours, and after 2 weeks damped out to a constant level that was halfway between the minimum and maximum rates. The cyclic oscillations were apparent whether expressed on a weight or chlorophyll content basis, occurred in both lightly and heavily pigmented samples, and were not attributable to changes in chloroplast shading due to variations in chloroplast orientation within the frond cells. There were no cyclic variations in the initial slopes of the light saturation curves, in photosynthetic unit size, or in relative quantum efficiency. Measurement of the "fast" turnover time of photosynthesis by the delayed dual flash technique revealed no diurnal variations of this parameter. These results indicate that the cyclic variations in photosynthetic activity are modulated by a dark reaction at a step occurring after reduction of plastoquinone by electrons from photosystem II.

Journal Article↗
PubMed1976

The biosynthesis of delta-aminolevulinic acid from the intact carbon skeleton of glutamic acid in greening barley.

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.

Aminolevulinic Acid↗
PubMed1975

Biosynthesis of delta-aminolevulinic acid from the intact carbon skeleton of glutamic acid in greening barley.

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.

Aminolevulinic Acid↗
PubMed1974

The Biosynthesis of delta-Aminolevulinic Acid in Higher Plants: I. Accumulation of delta-Aminolevulinic Acid in Greening Plant Tissues.

delta-Aminolevulinic acid dehydrase activity in cucumber (Cucumis sativus L. var. Alpha green) cotyledons did not change as the tissue was allowed to green for 24 hours. delta-Aminolevulinic acid accumulated in greening cucumber cotyledons, and barley (Hordeum sativum L. var. Numar) and bean (Phaseolus vulgaris L. var. Red Kidney) leaves incubated in the presence of levulinic acid, a specific competitive inhibitor of delta-aminolevulinic acid dehydrase. The rate of delta-aminolevulinic acid accumulation in levulinic acid-treated cucumber cotyledons paralleled the rate of chlorophyll accumulation in the controls, and the quantity of delta-aminolevulinic acid accumulated compensated for the decrease in chlorophyll accumulation. When levulinic acid-treated cucumber cotyledons were returned to darkness, delta-aminolevulinic acid accumulation ceased.delta-Aminolevulinic acid accumulation showed an absolute requirement for oxygen and was inhibited drastically by cyanide and azide, and to a lesser extent by arsenite and malonate. 2,4-Dinitrophenol, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, sodium fluoroacetate, and hydroxylamine hydrochloride showed no effect under the conditions tested. Freezing and thawing of the tissue completely prevented the accumulation of delta-aminolevulinic acid.The findings of this investigation are consistent with the hypothesis that delta-aminolevulinic acid is a chlorophyll precursor in higher plants, and that chlorophyll biosynthesis is regulated at the level of the formation of delta-aminolevulinic acid.

Journal Article↗
PubMed1974

The Biosynthesis of delta-Aminolevulinic Acid in Higher Plants: II. Formation of C-delta-Aminolevulinic Acid from Labeled Precursors in Greening Plant Tissues.

delta-Aminolevulinic acid was accumulated by greening cucumber (Cucumis sativus L. var. Alpha green) cotyledons, barley (Hordeum sativum var. Numar) leaves, and bean (Phaseolus vulgaris L. var. Red Kidney) leaves in the presence of various (14)C-labeled precursors and levulinic acid, a competitive inhibitor of delta-aminolevulinic acid dehydrase. The radioactivity in the accumulated delta-aminolevulinic acid was measured.The most effective labeled precursors were the 5 carbon dicarboxylic compounds glutamate, glutamine, and alpha-ketoglutarate. (14)C-Labeled glycine and succinate were relatively poor. The carboxyl and the methylene carbons of glycine were incorporated into delta-aminolevulinic acid to about equal extent. The carboxyl carbon of glutamate was incorporated almost as well as the internal carbons of the same compound. These results are inconsistent with the succinyl CoA-glycine succinyl transferase (delta-aminolevulinic acid synthetase) mode of delta-aminolevulinic acid production.When the same experiments were performed on turkey blood (which, as avian blood in general, possesses delta-aminolevulinic acid synthetase), delta-aminolevulinic acid was labeled most effectively from glycine-2-(14)C, moderately well from glycine-1-(14)C and glutamate-3,4-(14)C and not at all from glutamate-1-(14)C.It appears probable that greening higher plant tissues possess an alternate route to delta-aminolevulinic acid in which the carbon skeleton of glutamate (and alpha-ketoglutarate) is incorporated intact into the first committed metabolite of the chlorophyll pathway.

Journal Article↗
PubMed1974

The Abolition of the Lag Phase in Greening Cucumber Cotyledons by Exogenous delta-Aminolevulinic Acid.

Etiolated cucumber cotyledons treated with delta-aminolevulinic acid accumulated protochlorophyllide which was phototransformable to chlorophyll (ide). The phototransformation process in the delta-aminolevulinic acid-treated tissue was markedly temperature-dependent, consistent with the view that this protochlorophyllide must combine with the holochrome apoenzyme before phototransformation can occur.The treatment which effects lag phase removal in control tissue did not affect the course of chlorophyll (ide) accumulation in delta-aminolevulinic acid-treated tissue under either continuous or intermittent illumination. It was concluded that the lag phase in etiolated tissues must reflect the gradual development of the ability to synthesize delta-aminolevulinic acid at an appreciable rate.

Journal Article↗