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Biomedical subjects

A H Datko

Publications and source records attributed to A H Datko.

At least 37 records · Page 2Linked to original sources
PubMed1984

Responses of Sulfur-Containing Compounds in Lemna paucicostata Hegelm. 6746 to Changes in Availability of Sulfur Sources.

The steady state concentrations of S-containing compounds formed in Lemna paucicostata Hegelm. 6746 in response to variations in source and concentrations of sulfur were measured. Neither growth rates nor protein accumulation were markedly affected by the various growth conditions. Ignoring complications due to possible compartmentation, the results are consistent with internal pools of both SO(4) (2-) and cyst(e)ine (or products of their metabolism), but not methionine, being effectors of regulation of high affinity SO(4) (2-) uptake. As SO(4) (2-) in the growth medium was increased to 10 mm, down-regulation of high affinity SO(4) (2-) uptake was more than compensated for by unregulated uptake via the "non-saturating" uptake system. Tissue inorganic SO(4) (2-) accumulated but formation of reduced sulfur remained constant. Some conversion of l-cystine sulfur to SO(4) (2-) occurred. Presence of l-cystine in the medium (a) down-regulated high affinity SO(4) (2-) uptake and (b) decreased the rate of SO(4) (2-) organification. The net results were decreased (7 mum l-cystine) or normal (14 mum l-cystine) total tissue SO(4) (2-) and dose-dependent accumulation of soluble cyst(e)ine and glutathione, but not of soluble methionine. l-Methionine was not metabolized to cyst(e)ine or its products. Presence of l-methionine in the medium led to increased total tissue sulfur, accounted for almost wholly by manyfold increases in soluble methionine, AdoMet, and S-methylmethionine sulfonium. Soluble cyst(e)ine increased slightly.

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PubMed1984

Threonine Synthase of Lemna paucicostata Hegelm. 6746.

Threonine synthase (TS) was purified approximately 40-fold from Lemna paucicostata, and some of its properties determined by use of a sensitive and specific assay. During the course of its purification, TS was separated from cystathionine gamma-synthase, establishing the separate identity of these enzymes. Compared to cystathionine gamma-synthase, TS is relatively insensitive to irreversible inhibition by propargylglycine (both in vitro and in vivo) and to gabaculine, vinylglycine, or cysteine in vitro. TS is highly specific for O-phospho-l-homoserine (OPH) and water (hydroxyl ion). Nucleophilic attack by hydroxyl ion is restricted to carbon-3 of OPH and proceeds sterospecifically to form threonine rather than allo-threonine. The K(m) for OPH, determined at saturating S-adenosylmethionine (AdoMet), is 2.2 to 6.9 micromolar, two orders of magnitude less than values reported for TS from other plant tissues. AdoMet markedly stimulates the enzyme in a reversible and cooperative manner, consistent with its proposed role in regulation of methionine biosynthesis. Cysteine (1 millimolar) caused a slight (26%) reversible inhibition of the enzyme. Activities of TS isolated from Lemna were inversely related to the methionine nutrition of the plants. Down-regulation of TS by methionine may help to limit the overproduction of threonine that could result from allosteric stimulation of the enzyme by AdoMet.No evidence was obtained for feedback inhibition, repression, or covalent modification of TS by threonine and/or isoleucine.

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PubMed1983

Adaptation of Lemna paucicostata to Sublethal Methionine Deprivation.

During initial exposure to 40 nanomolar propargylglycine (PAG), Lemna paucicostata colonies undergo abnormal fragmentation and a lag in frond emergence, most severe at 24 to 48 hours. Thereafter, frond emergence resumes and the frond/colony ratio rises. Such ;adapted' plants withstand subculture into the same concentration of PAG without fragmentation or decreases in frond emergence, and display enhanced tolerance to higher concentrations. Adaptation is not dependent upon outgrowth of a few preexisting especially tolerant plants. Exogenous methionine prevents these events and overcomes the PAG-induced lag in frond emergence even after it is underway. These changes in frond emergence are not reflected in the rates of protein and wet weight accumulation which decrease by about 25% during the first 24 hours and continue unchanged thereafter. Cystathionine gamma-synthase activity rapidly decreases to 9% of control during the first 12 hours of exposure to 40 nanomolar PAG but thereafter climbs to 12% of control. Studies of the uptake and internal concentration of PAG during these events are reported.Exposure to a combination of 36 micromolar lysine plus 3 micromolar threonine is an alternative means to bring about sublethal methionine deprivation. Thus exposed, Lemna undergoes an analogous sequence of effects on morphology and growth which are preventable by exogenous methionine and which lead to an adapted state. Cystathionine gamma-synthase specific activity in plants adapted to 36 micromolar lysine plus 3 micromolar threonine is 1.8 times control. However, addition of PAG showed that under these conditions enzyme activity can be decreased to as little as 54% of control without affecting the growth rate. Together these results suggest that adaptation is related to methionine limitation and that the plants adjust, in part, by increasing the steady-state concentrations of cystathionine gamma-synthase and other enzymes in the methionine pathway.

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PubMed1983

In vivo metabolism of 5'-methylthioadenosine in lemna.

Evidence is presented that Lemna converts 5'-methylthioadenosine (MTA) to methionine. The methylthio moiety and four of the ribose carbons of the nucleoside contribute the methylthio and the four-carbon moieties of methionine. Plants grown in the presence of inhibitors which block methionine biosynthesis convert MTA to methionine at a rate sufficient to sustain normal growth (at least 4.4 nanomoles per colony per doubling with a molar yield of at least 65%). The pathway for conversion is shown to be constitutive in plants grown in standard medium and to function at a rate sufficient to dispose of MTA arising as a result of polyamine synthesis, and to explain the observed rate (1.4 nanomoles per colony per doubling) of preferential recycling of methionine sulfur (Giovanelli, Mudd, Datko 1981 Biochem Biophys Res Commun 100: 831-839). Rapid entry of methionine methyl into S-adenosylmethionine and phosphorylcholine was observed for plants grown in standard medium. Adenine generated during this cycle is efficiently salvaged into ADP and ATP.Conversion of MTA to methionine completes the steps in methionine thiomethyl recycling (Giovanelli, Mudd, Datko 1981 Biochem Biophys Res Commun 100: 831-839) in which the sulfur of methionine is retained while the four-carbon moiety is not. The findings further show that the four-carbon moiety of methionine can be derived via the ribose moiety of MTA in addition to the established route from O-phosphohomoserine via transsulfuration. Previous observations (Giovanelli, Mudd, Datko 1980 Biochemistry of Plants pp 453-505) can now be interpreted as establishing that exogenous methionine down-regulates its own net synthesis via the transsulfuration pathway.

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PubMed1982

Methionine biosynthesis in lemna: inhibitor studies.

A search was made for compounds that would inhibit methionine biosynthesis in Lemna paucicostata Hegelm. 6746. dl-Propargylglycine (0.15 micromolar) produced growth inhibition and morphological changes which were prevented by exogenous methionine. Also, dl-propargylglycine inhibits cystathionine gamma-synthase activity. l-Aminoethoxyvinylglycine (0.05 micromolar) produced growth inhibition and morphological changes partially preventable by exogenous methionine. l-Aminoethoxyvinylglycine impairs the cleavage of cystathionine to homocysteine. Lysine and threonine, at concentrations which individually had little effect on growth or morphology of Lemna, together produced growth inhibition and morphological changes preventable by exogenous methionine. The resulting metabolic block prevented conversion of cysteine to cystathionine, presumably secondary to depletion of the supply of O-phosphohomoserine.Inhibition of Lemna growth resulted when the molybdate:sulfate ratio in the medium was increased to 20:1 or more. Such inhibition was prevented by lowering this ratio to 0.3 or less. A non-steady-state experiment (molybdate:sulfate, 20:1) showed that molybdate inhibited sulfate uptake, but it provided no evidence of a further impairment in the organification of sulfate. Molybdate-induced growth inhibition of Lemna was prevented by cystine but not by cystathionine or methionine. Cystathionine is not converted by Lemna to cysteine rapidly enough to sustain growth.

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PubMed1982

Methionine Biosynthesis in Lemna: STUDIES ON THE REGULATION OF CYSTATHIONINE gamma-SYNTHASE, O-PHOSPHOHOMOSERINE SULFHYDRYLASE, AND O-ACETYLSERINE SULFHYDRYLASE.

Regulation of enzymes of methionine biosynthesis was investigated by measuring the specific activities of O-phosphohomoserine-dependent cystathionine gamma-synthase, O-phosphohomoserine sulfhydrylase, and O-acetylserine sulfhydrylase in Lemna paucicostata Hegelm. 6746 grown under various conditions. For cystathionine gamma-synthase, it was observed that (a) adding external methionine (2 mum) decreased specific activity to 15% of control, (b) blocking methionine synthesis with 0.05 muml-aminoethoxyvinylglycine or with 36 mum lysine plus 4 mum threonine (Datko, Mudd 1981 Plant Physiol 69: 1070-1076) caused a 2- to 3-fold increase in specific activity, and (c) blocking methionine synthesis and adding external methionine led to the decreased specific activity characteristic of methionine addition alone. Activity in extracts from control cultures was unaffected by addition of methionine, lysine, threonine, lysine plus threonine, S-adenosylmethionine, or S-methylmethionine sulfonium to the assay mixture. Parallel studies of O-phosphohomoserine sulfhydrylase and O-acetylserine sulfhydrylase showed that O-phosphohomoserine sulfhydrylase activity responded to growth conditions identically to cystathionine gamma-synthase activity, whereas O-acetylserine sulfhydrylase activity remained unaffected. Lemna extracts did not catalyze lanthionine formation from O-acetylserine and cysteine. Estimates of kinetic constants for the three enzyme activities indicate that O-acetylserine sulfhydrylase has much higher activity and affinity for sulfide than O-phosphohomoserine sulfhydrylase.The results suggest that (a) methionine, or one of its products, regulates the amount of active cystathionine gamma-synthase in Lemna, (b) O-phosphohomoserine sulfhydrylase and cystathionine gamma-synthase are probably activities of one enzyme that has low specificity for its sulfur-containing substrate, and (c) O-acetylserine sulfhydrylase is a separate enzyme. The relatively high activity and affinity for sulfide of O-acetylserine sulfhydrylase provides an explanation in molecular terms for transsulfuration, and not direct sulfhydration, being the dominant pathway for homocysteine biosynthesis.

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PubMed1982

Methionine Synthesis in Lemna: Inhibition of Cystathionine gamma-Synthase by Propargylglycine.

Propargylglycine, vinylglycine, and cysteine each cause irreversible inactivations of cystathionine gamma-synthase (and, in parallel, of O-phosphohomoserine sulfhydrylase) activities in crude extracts of Lemna paucicostata. Inactivation by propargylglycine or vinylglycine is completely prevented by 40 millimolar O-phospho- or O-succinyl-l-homoserine; that by cysteine is only partially prevented. Propargylglycine (PAG), the most potent of these inhibitors, causes rapid and drastic inactivation of both activities in intact Lemna. Studies of plants growing in steady states in the presence of various concentrations (0-150 nanomolar) of PAG showed that 16% of control activity is necessary and sufficient to maintain normal rates of growth and methionine biosynthesis, and that 10% of control activity is essential for viability. Addition of either 2 micromolar methionine or 31 micromolar cystine to growth medium containing 150 nanomolar PAG permits growth at 75 to 100% of control rates when enzyme activity is less than 10% of control. Whereas methionine presumably rescues by directly providing the missing metabolite, cystine may rescue by enhancing substrate accumulation and thereby promoting flux through residual cystathionine gamma-synthase. The results indicate that the down-regulation of cystathionine gamma-synthase to 15% of control which occurs when plants are grown in 2 micromolar methionine (Thompson, Datko, Mudd, Giovanelli Plant Physiol 69: 1077-1083), by itself, is not sufficient to reduce the rate of methionine biosynthesis.

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PubMed1981

Homocysteine Biosynthesis in Green Plants: Physiological Importance of the Transsulfuration Pathway in Lemna paucicostata.

To permit an assessment of the relative contributions of the transsulfuration and the direct sulfhydration pathways for homocysteine biosynthesis, the time course of incorporation of (35)S from (35)SO(4) (2-) into various sulfur-containing compounds in Lemna paucicostata has been determined. Plants were grown with either low (4.5 micromolar) or ample (1,000 micromolar) sulfate in the medium. At the shortest labeling times, (35)S-cystathionine was the predominant (35)S-containing organic sulfur compound. The flux of sulfur into cystathionine was sufficient to sustain the known rate of methionine biosynthesis. It was calculated that transsulfuration accounted for at least 90 and 85% of the total homocysteine synthesis in low and ample sulfate-grown plants, respectively (and may have accounted for 100%). No marked rise in the (35)S-soluble cysteine:(35)S-homocysteine ratio was observed even at the shortest labeling times, but it is argued that this may be due to (a) the observed compartmentation of soluble cysteine, and (b) the impracticality of using labeling times shorter than 17 seconds. Additional evidence supporting the importance of transsulfuration in Lemna is briefly described.

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PubMed1980

Lemna paucicostata Hegelm. 6746: DEVELOPMENT OF STANDARDIZED GROWTH CONDITIONS SUITABLE FOR BIOCHEMICAL EXPERIMENTATION.

Photoautotrophic and mixotrophic growth of Lemna paucicostata Hegelm. 6746 (formerly Lemna perpusilla Torr. 6746) was investigated to establish standardized conditions for biochemical studies. Optimal temperature for growth was 29 to 30 C. The medium used previously (Datko AH, Mudd SH, Giovanelli J 1977 J Biol Chem 252: 3436-3445) was modified by inclusion of NH(4)Cl, decreasing macronutrient and ethylenediamine tetraacetate concentration, increasing micronutrient concentration, and inclusion of bicarbonate (for photoautotrophic growth) or 2-(N-morpholino)ethanesulfonic acid (for mixotrophic growth) buffers. Varying the sulfate concentration between 14 and 1 millimolar had no effect on growth. For photoautotrophic growth in the new medium (medium 4), the effects of CO(2) concentration, light intensity, and pH were measured. Under the optimal conditions, a multiplication rate (MR) of 300 to 315, equivalent to a doubling time of 23 to 24 hours was obtained. Addition of glutamine or asparagine did not increase this MR. For mixotrophic growth in low light, the effects of sucrose concentration and pH were determined. Under optimal conditions, MR was 210. A concentration of sucrose less than maximal for growth was chosen for the medium for experiments which will include (14)C-labeling of intermediates. MR under these conditions was 184. Growth was equally good in medium 4 and in half-strength Hutner's medium when sulfate was high (0.4 to 1 millimolar), but better in medium 4 when sulfate was low (20 micromolar). Growth rates could be restored to normal in half-strength Hutner's with low sulfate by decreasing the molybdate concentration.By modifying medium 4 to contain very low amounts of sulfate, and by preconditioning medium and plants, it was shown that there was an increment in plant protein of approximately 2.5 micrograms per nanomole of added MgSO(4). Colonies undergoing sulfur limitation exhibited a slow growth rate and a high frond to colony ratio. Molybdate and selenate produced growth inhibition reversible by sulfate. Conditions were developed in which the plants could be maintained indefinitely in the presence of either molybdate or selenate in altered metabolic steady-states with lowered growth rates and protein per frond.

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PubMed1980

Lemna paucicostata Hegelm. 6746: LIFE CYCLE AND CHARACTERIZATION OF THE COLONY TYPES IN A POPULATION.

The sequence of frond emergence and the intervals required for daughter colony separation have been determined for Lemna paucicostata Hegelm. 6746 growing under standardized conditions. After separation of a new mother colony, the first daughter colony is produced from the left meristematic pocket and separates after approximately 60 hours, the second daughter, produced from the right pocket, separates after a further 30 hours, and the third daughter, again from the left, after a further 40 hours. The pattern of alternating longer and shorter intervals for separation of daughters continues throughout the life of the mother colony.Protein contents of fronds and whole colonies were determined either by chemical methods or by labeling to isotopic equilibrium with (35)SO(4) (2-). The smallest fronds measured were 5 to 6% of the final area they would attain when fully expanded and contained 10% of the protein they would finally contain. Thus, most protein accumulation occurred during the phase of rapid frond expansion rather than in an earlier primordial stage. The specific rates of protein accumulation were 0.027 to 0.030 micrograms per microgram protein per hour during rapid frond expansion and relatively constant thereafter at 0.014 to 0.019 for whole colonies until at least four daughter colonies had separated.A substantial amount of (35)S is transferred directly from the mother frond to its attached daughter fronds. The results are consistent with the transfer of soluble compounds arising through turnover of protein in the mother frond.Tree diagrams and a mathematical treatment were utilized to relate the distribution of colony types and the over-all doubling time in a large population of Lemna colonies to the doubling times and modes of division of the individual colonies within that population. It was shown that a population with the growth properties described increases exponentially and that within a relatively few generations reaches an equilibrium population distribution which is independent of the initial proportions of the members of the population. The measured distribution of colony types producing odd-numbered as compared to those producing even-numbered daughters was the same as that predicted by the theoretical analyses. Once a mass culture of Lemna paucicostata has attained its equilibrium distribution of colony types, under our standard conditions the proportions of colonies in which the indicated daughter will be next to separate will be: 60%, first; 15%, second; 11%, third; 5%, fourth; 8% fifth or higher. Thus, about 91% of colonies will have produced no more than four daughter colonies. For at least this period the specific rate of protein accumulation is relatively constant and senescent changes in mother fronds are rare. In the equilibrium distribution, 39% of colonies will themselves be first daughters, 25% will be second daughters, 14% will be third daughters, 9% fourth, and 13% greater than fourth daughters. These combined results suggest that obtaining representative samples of colonies from such a population for biochemical studies should be relatively simple.

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PubMed1978

Homocysteine biosynthesis in green plants. Physiological importance of the transsulfuration pathway in Chlorella sorokiniana growing under steady state conditions with limiting sulfate.

The physiological roles of the transsulfuration and direct sulfhydration pathways in Chlorella sorokiniana growing under steady state photoautotrophic conditions with limiting sulfate were studied by following the patterns of assimilation of 35SO4(2-) into sulfur amino acids. The labeling patterns expected of each pathway were defined by means of models based on the rates of net synthesis of the terminal pools of GSH, protein cysteine, and protein methionine. The labeling patterns observed are entirely consistent with the transsulfuration pathway and inconsistent with the direct sulfhydration pathway. By analysis of the amounts of radioactivity present in key intermediates at labeling times as short as 1 s, it was demonstrated that direct sulfhydration makes no detectable contribution to homocysteine biosynthesis, and if operative contributes no more than approximately 3% of the total homocysteine biosynthesized. From the combined determinations of the initial rates of labeling and net rates of synthesis of the various sulfur amino acids, a tentative working model is presented that summarizes our best current estimates of the major fluxes of sulfur in the experimental system. The labeling data further showed that soluble cysteine consists of at least two pools. One pool, termed "rapidly turning over" cysteine comprises less than 1% of the total soluble cysteine, and is the precursor of GSH, protein cysteine, and, almost certainly, cystathionine. The other pool, "slowly turning over" cysteine, appears to be in equilibrium with "rapidly turning over" cysteine, but not to be further metabolized.

Chlorella↗
PubMed1978

Phytostat for the growth of lemna in semicontinuous culture with low sulfate.

An apparatus is described by means of which Lemna perpusilla 6746 was grown photoautotrophically at a series of constant concentrations of inorganic sulfate, as low as 0.26 mum. Theoretical considerations relevant to this system are discussed and examples of the operation of the apparatus are presented. The data obtained were used to calculate sulfate uptake by the plant colonies as a function of sulfate concentration. The apparatus should be useful for the production of relatively large quantities of Lemna, grown under highly uniform conditions, with or without limitation of one or more nutrient(s). Other small vegetatively reproducing aquatic plants could be similarly studied. Uptake studies could be carried out on a variety of plant materials.

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PubMed1978

Sulfur-containing Compounds in Lemna perpusilla 6746 Grown at a Range of Sulfate Concentrations.

Lemna perpusilla 6746, grown photoautotrophically at a series of sulfate concentrations ranging from 0.32 to 1,000 mum, was labeled to radioisotopic equilibrium with (35)SO(4) (2-). Sulfur-containing compounds were isolated and purified from the colonies. Radioactivity in each compound was a measure of the amount of that compound present in the tissue. The following compounds were identified and quantitated: inorganic sulfate, glutathione, homocyst(e)ine, cyst(e)ine, methionine, S-methylmethionine sulfonium, S-adenosylmethionine, S-adenosylhomocysteine, cystathionine, chloroformsoluble (presumed to be sulfolipid), protein cyst(e)ine, and protein methionine. gamma-Glutamylcyst(e)ine, erythro- and threo-thiothreonine, and S-methylcysteine were not detected. No volatile (35)S compounds were formed during plant growth at 1,000 mum sulfate, nor were significant amounts of (35)S compounds excreted into the medium.The amount of each component present in colonies grown over the 3,000-fold range of medium sulfate was relatively constant except for inorganic sulfate. This increased about 30-fold from the lowest to the highest medium sulfate concentration. The total soluble sulfur amino acids increased about 1.5- to 2-fold, due primarily to an increased amount of glutathione. Protein cyst(e)ine and protein methionine were the major organic sulfur compounds in Lemna, and the amounts of these compounds remained virtually constant despite the variation in external sulfate concentration.Procedures for the analysis of S-adenosylmethionine, S-methylmethionine sulfonium, and S-adenosylhomocysteine are presented.

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PubMed1974

Homoserine esterification in green plants.

EXTRACTS OF PHYLOGENETICALLY DIVERSE PLANS WERE SURVEYED FOR THEIR ABILITY TO SYNTHESIZE THE FOLLOWING HOMOSERINE ESTERS WHICH ARE POTENTIAL PRECURSORS FOR METHIONINE AND THREONINE SYNTHESIS IN GREEN PLANTS: O-acetyl-, O-oxalyl-, O-succinyl-, O-malonyl-, and O-phosphohomoserine. Synthesis of O-acylhomoserine esters was detected only in Pisum sativum L. and Lathyrus sativus L. Extracts of P. sativum, a plant known to accumulate O-acetylhomoserine, catalyzed the specific synthesis of this ester from homoserine and acetyl-CoA. Extracts of L. sativus, a plant known to accumulate O-oxalylhomoserine, catalyzed the specific synthesis of this ester from homoserine and oxalyl-CoA. None of the other plants surveyed, including representatives of the green algae, horsetails, gymnosperms, and angiosperms, catalyzed the synthesis of any of the O-acylhomoserine esters studied. In contrast, synthesis of O-phosphohomoserine by the reaction catalyzed by homoserine kinase was demonstrated in extracts of all plants examined, including the two exceptional legumes.These results suggest that, among the five homoserine esters studied, O-phosphohomoserine is the major activated homoserine derivative in plants. Direct confirmation of the dominant physiological role of O-phosphohomoserine in the synthesis of cystathionine in the transsulfuration pathway of methionine biosynthesis in plants has recently been provided (Datko, A. H., Giovanelli, J., and Mudd, S. H. 1974. J. Biol. Chem. 249: 1139-1155).

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