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Biomedical subjects

N E Tolbert

Publications and source records attributed to N E Tolbert.

At least 55 records · Page 3Linked to original sources
PubMed1984

Detection of chemicals inhibiting photorespiratory senescence in a large scale survival chamber.

A large scale survival chamber was developed as a screen for detecting chemical treatments that extend the survival time of illuminated soybean seedlings at CO(2) concentrations below the compensation point. In theory, extended survival should indicate potential for improved crop performance via decreased photorespiration and increased photosynthetic efficiency. An automated control system regulated CO(2) concentrations, temperature and plant watering during a continuous CO(2)-removal photoperiod of 72 hours. An endogenously controlled circadian rhythm of net photosynthesis occurred throughout the continuous light treatment.Spray applications of 3.49 millimolar 2-(4-chlorophenoxy)-2-methylpropanoic acid (CPMP) significantly decreased leaf chlorophyll loss, compared with the control, after 72 hours of subcompensation-point stress. Treatment with CPMP also consistently increased leaf chlorophyll per unit area under nonstress greenhouse conditions. These effects may be due to increases in specific leaf weight produced by CPMP although the compound did not consistently act as a height retardant. The compound, 3-butyl-2-hydroxy-4H-pyrido[1,2-a]pyrimidin-4-one (BHPP), inhibited senescence under low CO(2) conditions but did not decrease leaf light transmission at ambient CO(2) levels. The cytokinin N(6)-benzyladenine (BA) retarded low CO(2) stress senescence although greening effects were not observed. Neither 2-hydroxy-3-butynoic acid (HBA) nor its butyl ester, inhibitors of glycolate oxidase, influenced low CO(2) survival. Cyclohexanecarboxylic acid (CHCA) and sodium naphthenate had no effect upon subcompensation-point senescence.Antisenescence effects of CPMP, BHPP, and BA do not appear to be directly attributable to effects upon the competing carbon paths of photosynthesis and photorespiration. Protection against low CO(2) stress and increased chlorophyll synthesis under nonstress conditions may represent separate effects upon plastids by some of the compounds. This screen will identify compounds which inhibit photorespiratory senescence without decreasing the CO(2) compensation point.

Journal Article↗
PubMed1983

Serine: glyoxylate, alanine:glyoxylate, and glutamate:glyoxylate aminotransferase reactions in peroxisomes from spinach leaves.

Two different aminotransferases, that have glyoxylate as the amino acceptor, have specific activities of 1 to 2 mumol . min-1 . mg of protein-1 in the isolated peroxisomal fraction from spinach leaves. Their properties were evaluated after separation on a hydroxylapatite column. Both enzymes had a Km for glyoxylate of 0.15 mM and an amino acid Km of 2 to 3 mM. Reactions proceeded by a Ping Pong Bi Bi mechanism. Serine:glyoxylate aminotransferase was relatively specific for both substrates and could only be slightly reversed with 100 mM glycine, although the Ki of glycine was 33 mM. The glutamate:glyoxylate amino-transferase protein was equally active in catalyzing an alanine:glyoxylate aminotransferase reaction, but the reverse reactions with 100 mM glycine were hardly measureable, although the Ki (glycine) was 8.7 mM. Protection against hydroxylamine inhibition from reaction with pyridoxal phosphate was used to investigate the specificity of amino acid binding. Substrate amino acids protected at about the same concentration as their Km, while glycine protected at its Ki concentration. Thus, the nearly irreversible catalysis with glycine is not due to a failure to bind glycine. The significance of a peroxisomal alanine:glyoxylate aminotransferase activity has not been incorporated into schemes for the oxidative photosynthetic carbon cycle.

Alanine Transaminase↗
PubMed1983

The lability of an intermediate of the ribulose bisphosphate carboxylase reaction.

Interruption of the catalytic cycle of ribulose-bisphosphate carboxylase by acid denaturation liberated an intermediate with a labile phosphate ester. Addition of fresh, buffered carboxylase enzyme to the acidified carboxylase reaction after 5 s inhibited phosphate release from the intermediate. Therefore, the species with a labile phosphate ester was stable for 5 s in acid and was apparently a substrate for the enzymatic reaction, since the labile intermediate was converted to a stable form by the protein. After acid denaturation, the carboxylated intermediate could be stabilized by reduction after 5 s in acid, but after 1 h no carboxylated intermediate remained. The stoichiometries of phosphate released to enzyme active sites and the carboxylated intermediate trapped to enzyme active sites were approximately 0.04. It was concluded that the labile phosphate species is probably the carboxylated intermediate rather than the enediol(ate) intermediate. The carboxylase and oxygenase reactions were probed for intermediates by the ability of the enzymatic reaction to reduce hexacyanoferrate(III), dichlorophenolindophenol, or nitroblue tetrazolium. Reduction of these reagents and hexacyanoferrate(III)-dependent paracatalytic inactivation were not observed. The copper chelate of lysine, a superoxide dismutase active species, did not selectively inhibit ribulose-bisphosphate oxygenase.

Binding Sites↗
PubMed1983

Isolation and characterization of infected and uninfected cells from soybean nodules : role of uninfected cells in ureide synthesis.

The distribution of organelles and associated enzymes between cells containing bacteroids and uninfected cells from nodules of Glycine max L. Merr. cv Amsoy 71 was investigated by separation of protoplasts on a sucrose step-gradient. Infected protoplasts were much larger, irregular in shape, and more dense than uninfected protoplasts. The peroxisomal enzymes, uricase and catalase, were present at much higher specific activity in the uninfected cell fraction. Allantoinase, an enzyme of the endoplasmic reticulum, had a greater specific activity in the uninfected cell fraction. Several enzymes whose products are required for purine biosynthesis, including phosphoglycerate dehydrogenase, aspartate aminotransferase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase, exhibited a higher specific activity in the uninfected cell fraction. Isozymes of aspartate aminotransferase were separated on native gels and located by an activity stain. The soluble isozyme was predominantly found in the uninfected cell fraction. These data suggest that peroxisomes, containing uricase and catalase for conversion of uric acid to allantoin, are present only in the uninfected cells of soybean nodules. The uninfected cells also appear to be the site of the allantoinase reaction.

Journal Article↗
PubMed1983

Effects of glyoxylate on photosynthesis by intact chloroplasts.

Because glyoxylate inhibits CO(2) fixation by intact chloroplasts and purified ribulose bisphosphate carboxylase/oxygenase, glyoxylate might be expected to exert some regulatory effect on photosynthesis. However, ribulose bisphosphate carboxylase activity and activation in intact chloroplasts from Spinacia oleracea L. leaves were not substantially inhibited by 10 millimolar glyoxylate. In the light, the ribulose bisphosphate pool decreased to half when 10 millimolar glyoxylate was present, whereas this pool doubled in the control. When 10 millimolar glyoxylate or formate was present during photosynthesis, the fructose bisphosphate pool in the chloroplasts doubled. Thus, glyoxylate appeared to inhibit the regeneration of ribulose bisphosphate, but not its utilization.The fixation of CO(2) by intact chloroplasts was inhibited by salts of several weak acids, and the inhibition was more severe at pH 6.0 than at pH 8.0. At pH 6.0, glyoxylate inhibited CO(2) fixation by 50% at 50 micromolar, and glycolate caused 50% inhibition at 150 micromolar. This inhibition of CO(2) fixation seems to be a general effect of salts of weak acids.Radioactive glyoxylate was reduced to glycolate by chloroplasts more rapidly in the light than in the dark. Glyoxylate reductase (NADP(+)) from intact chloroplast preparations had an apparent K(m) (glyoxylate) of 140 micromolar and a V(max) of 3 micromoles per minute per milligram chlorophyll.

Journal Article↗
PubMed1983

Aminooxyacetate stimulation of glycolate formation and excretion by chlamydomonas.

Aminooxyacetate (1 millimolar) did not inhibit photosynthetic (14)CO(2) fixation by Chlamydomonas reinhardtii Dangeard, (-) strain (N.90) but greatly stimulated the biosynthesis and excretion of glycolate. Similar results were obtained from cells grown with 5% CO(2) or low CO(2) (air). After 2 minutes with air-grown cells, [(14)C]glycolate increased from 0.3% of the total (14)C fixed by the control to 11.7% in the presence of aminooxyacetate and after 10 minutes from 3.8% to 41.1%. Ammonium nitrate (0.2 millimolar) in the media blocked the aminooxyacetate stimulation of glycolate excretion. Chromatographic analyses of the labeled products in the cells and supernatant media indicated that aminooxyacetate also completely inhibited the labeling of alanine while some pyruvate accumulated and was excreted. A high percentage (35%) of initial (14)CO(2) fixation was into C(4) acids. Initial products of (14)CO(2) fixation included phosphate esters as well as malate, aspartate, and glutamate in treated or untreated cells. Lactate was also a major early product of photosynthesis, and its labeling was reduced by aminooxyacetate. Inasmuch as lactate was not excreted, glycolate excretion seemed to be specific. When photosynthesis was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, labeled organic and amino acids but not phosphate esters were lost from the cells. Aminooxyacetate did not inhibit the enzymes associated with glycolate synthesis from ribulose bisphosphate.

Journal Article↗
PubMed1983

Factors which affect the amount of inorganic phosphate, phosphorylcholine, and phosphorylethanolamine in xylem exudate of tomato plants.

Phosphate in the xylem exudate of tomato (Lycopersicon esculentum) plants was 70 to 98% inorganic phosphate (Pi), 2 to 30% P-choline, and less than 1% P-ethanolamine. Upon adding (32)Pi to the nutrient, Pi in xylem exudate had the same specific activity within 4 hours. P-choline and P-ethanolamine reached the same specific activity only after 96 hours. The amount of Pi in xylem exudate was dependent on Pi concentration in the nutrient and decreased from 1700 to 170 micromolar when Pi in the nutrient decreased from 50 to 2 micromolar. The flux of 0.4 nmoles organic phosphate per minute per gram fresh weight root into the xylem exudate was not affected by the Pi concentration in the nutrient solution unless it was below 1 micromolar. During 7 days of Pi starvation, Pi in the xylem exudate decreased from 1400 to 130 micromolar while concentrations of the two phosphate esters remained unchanged.The concentration of phosphate esters in the xylem exudate was increased by addition of choline or ethanolamine to the nutrient solution, but Pi remained unchanged. Upon adding [(14)C]choline to the nutrient, 10 times more [(14)C]P-choline than [(14)C]choline was in the xylem exudate and 85 to 90% of the ester phosphate was P-choline. When [(14)C]ethanolamine was added, [(14)C]P-ethanolamine and [(14)C]ethanolamine in the xylem sap were equal in amount. P-choline and P-ethanolamine accumulated in leaves of whole plants at the same time and the same proportion as observed for their flux into the xylem exudate. No relationship between the transport of P-choline and Pi in the xylem was established. Rather, the amount of choline in xylem exudate and its incorporation into phosphatidylcholine in the leaf suggest that the root is a site of synthesis of P-choline and P-ethanolamine for phospholipid synthesis in tomato leaves.

Journal Article↗
PubMed1981

Storage and maintaining activity of ribulose bisphosphate carboxylase/oxygenase.

Purified ribulose-1,5-bisphosphate carboxylase/oxygenase in 50% saturated (NH(4))(2)SO(4) was stable when frozen as small beads in liquid nitrogen and stored at -80 C. When stored as a slurry at 4 C most of the activity was lost within four weeks. This loss was due not only to enzyme polymerization. Activity in old preparations purified from spinach leaves, but not tobacco or tomato leaves, can be restored to the level of newly purified enzyme after storage at 4 C by treatment with 50 to 100 millimolar dithiothreitol for several hours followed by dialysis against buffer and 1 millimolar dithiothreitol before CO(2) and Mg(2+) activation and assay. Some enzyme oligomers that had been formed were not converted back to native enzyme by treatment with 100 millimolar dithiothreitol.The purified enzyme contained about 2 gram-atoms iron per mole enzyme that could not be removed by chelating agents. When the enzyme was incubated with 100 millimolar dithiothreitol and exposed to O(2), a purple dithiothreitol-iron complex was formed which could be removed by dialysis. The activities of ribulose-1,5-bisphosphate carboxylase and oxygenase were not altered by reducing the iron content to 0.7 mole per mole enzyme by treatment with dithiothreitol followed by exhaustive dialysis against iron free buffer.

Journal Article↗
PubMed1981

Localization of enzymes of ureide biosynthesis in peroxisomes and microsomes of nodules.

The intracellular location of enzymes involved in the synthesis of the ureides, allantoin and allantoic acid, was investigated in nodules of Glycine max L. Merr. Cellular organelles were separated on isopycnic sucrose density gradients. Xanthine dehydrogenase activity (270 nanomoles per min per gram fresh weight) was totally soluble, whereas approximately 15% of the total uricase and catalase activities (1 and 2000 micromoles per minute per gram fresh weight, respectively) was in the fraction containing intact peroxisomes. Allantoinase activity (680 nanomoles per minute per gram fresh weight) was associated with the microsomal fraction, which apparently originates from the endoplasmic reticulum.

Journal Article↗