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Biomedical subjects

N E Tolbert

Publications and source records attributed to N E Tolbert.

At least 127 records · Page 7Linked to original sources
PubMed1970

Excretion of Glycolate, Mesotartrate and Isocitrate Lactone by Synchronized Cultures of Ankistrodesmus braunii.

Fixation of (14)CO(2) by synchronized cultures of Ankistrodesmus braunii was highest for young growing cells, low for mature cells, and lowest for dividing cells. The amount of (14)C excreted during photosynthesis followed the same trend. Cells at the end of the growing phase, after 10 hours of a 16-hour light phase, excreted nearly 35% of the total (14)C fixed as one product, glycolate. Dividing cells from the dark phase, when tested in the light, excreted only 4% as much glycolate-(14)C as the young growing cells. Dividing cells also excreted as much mesotartrate as glycolate and also some isocitrate lactone and an unidentified acid. None of these excreted acids were found inside the cells in significant amounts. Methods for isolation and identification of the excreted acids are present. With (14)C-labeled algae, it was shown that the excretion of glycolate was light-dependent and inhibited by 1,1-dimethyl-3-(p-chlorophenyl) urea. The excretion of labeled mesotartrate, isocitrate lactone, and an unknown acid, but not glycolate, also occurred in the dark. The excreted mesotartrate was predominantly carboxyl-labeled even after long periods of (14)CO(2) fixation. Since glycolate is known to be uniformly labeled, glycolate could not be the precursor of the carboxyl-labeled mesotartrate. The reason for the specific excretion of glycolate, mesotartrate, and isocitrate lactone is not known, but the metabolism of all three acids by the algae may be limited and each can form dilactides or lactones by dehydration. In this context isocitrate lactone was excreted rather than the free acid.

Journal Article↗
PubMed1970

Glycolate pathway in green algae.

By three criteria, the glycolate pathway of metabolism is present in unicellular green algae. Exogenous glycolate-1-(14)C was assimilated and metabolized to glycine-1-(14)C and serine-1-(14)C. During photosynthetic (14)CO(2) fixation the distributions of (14)C in glycolate and glycine were similar enough to suggest a product-precursor relationship. Five enzymes associated with the glycolate pathway were present in algae grown on air. These were P-glycolate phosphatase, glycolate dehydrogenase (glycolate:dichloroindophenol oxidoreductase), l-glutamate:glyoxylate aminotransferase, serine hydroxymethylase, and glycerate dehydrogenase. Properties of glycerate dehydrogenase and the aminotransferase were similar to those from leaf peroxisomes. The specific activity of glycolate dehydrogenase and serine hydroxymethylase in algae was 1/5 to 1/10 that of the other enzymes, and both these enzymes appear ratelimiting for the glycolate pathway.Labeling patterns for products of the glycolate pathway during (14)CO(2) fixation are not the same as those obtained with higher plants. In higher plants glycolate, glycine, and serine are uniformly labeled at shortest time periods. In algae, serine was predominately carboxyl-labeled, similarly to 3-phosphoglycerate. This result, plus the lower specific activity of serine hydroxymethylase, indicates that the glycine-serine interconversin in algae is slower than in plants. Initially (2 to 4 seconds) glycolate and glycine were more C-2 labeled. They rapidly became uniformly labeled, with glycine becoming uniformly labeled first. In the presence of isonicotinylhydrazide, labeled glycolate and glycine accumulated, and only a trace of serine-(14)C was detected. Then glycolate and glycine were initially carboxyl-labeled, and glycolate became uniformly labeled almost immediately and before glycine. These results suggest rapid metabolism of glycolate and glycine, in addition to the glycolate pathway.

Journal Article↗
PubMed1969

A survey of plants for leaf peroxisomes.

Leaves of 10 plant species, 7 with photorespiration (spinach, sunflower, tobacco, pea, wheat, bean, and Swiss chard) and 3 without photorespiration (corn, sugarcane, and pigweed), were surveyed for peroxisomes. The distribution pattern for glycolate oxidase, glyoxylate reductase, catalase, and part of the malate dehydrogenase indicated that these enzymes exist together in this organelle. The peroxisomes were isolated at the interface between layers of 1.8 to 2.3 m sucrose by isopycnic nonlinear sucrose density gradient centrifugation or in 1.95 m sucrose on a linear gradient. Chloroplasts, located by chlorophyll, and mitochondria by cytochrome c oxidase, were in 1.3 to 1.8 m sucrose. In leaf homogenates from the first 7 species with photorespiration, glycolate oxidase activity ranged from 0.5 to 1.5 mumoles x min(-1) x g(-1) wet weight or a specific activity of 0.02 to 0.05 mumole x min(-1) x mg(-1) protein. Glyoxylate reductase activity was comparable with glycolate oxidase. Catalase activity in the homogenates ranged from 4000 to 12,000 mumoles x min(-1) x g(-1) wet weight or 90 to 300 mumoles x min(-1) x mg(-1) protein. Specific activities of malate dehydrogenase and cytochrome oxidase are also reported. In contrast, homogenates of corn and sugarcane leaves, without photorespiration, had 2 to 5% as much glycolate oxidase, glyoxylate reductase, and catalase activity. These amounts of activity, though lower than in plants with photorespiration, are, nevertheless, substantial. Peroxisomes were detected in leaf homogenates of all plants tested; however, significant yields were obtained only from the first 5 species mentioned above. From spinach and sunflower leaves, a maximum of about 50% of the marker enzyme activities was found to be in these microbodies after homogenization. The specific activity for peroxisomal glycolate oxidase and glyoxylate reductase was about 1 mumole x min(-1) x mg(-1) protein; for catalase. 8000 mumoles x min(-1) x mg(-1) protein, and for malate dehydrogenase, 40 mumoles x min(-1) x mg(-1) protein. Only small to trace amounts of marker enzymes for leaf peroxisomes were recovered on the sucrose gradients from the last 5 species of plants. Bean leaves, with photorespiration, had large amounts of these enzymes (0.57 mumole of glycolate oxidase x min(-1) x g(-1) tissue) in the soluble fraction, but only traces of activity in the peroxisomal fraction. Low peroxisome recovery from certain plants was attributed to particle fragility or loss of protein as well as to small numbers of particles in such plants as corn and sugarcane. Homogenates of pigweed leaves (no photorespiration) contained from one-third to one-half the activity of the glycolate pathway enzymes as found in comparable preparations from spinach leaves which exhibit photorespiration. However, only traces of peroxisomal enzymes were separated by sucrose gradient centrifugation of particles from pigweed. Data from pigweed on the absence of photorespiration yet abundance of enzymes associated with glycolate metabolism is inconsistent with current hypotheses about the mechanism of photorespiration. Most of the catalase and part of the malate dehydrogenase activity was located in the peroxisomes. Contrary to previous reports, the chloroplast fractions from plants with photo-respiration did not contain a concentration of these 2 enzymes, after removal of peroxisomes by isopycnic sucrose gradient centrifugation.

Glycoside Hydrolases↗
PubMed1969

Glycolate stimulation of oxygen evolution during photosynthesis.

Glycolate and glyoxylate stimulated 100% to 300% the rate of oxygen evolution by Scenedesmus in the light in the absence of added carbon dioxide. This stimulation occurred either aerobically or anaerobically, and was sensitive to CMU. Aerobic dark respiration was stimulated 25% to 100% by glycolate. This phenomenon was best demonstrated with synchronized Scenedesmus at the stage of cell division. For glycolate stimulation of oxygen evolution, a dark preincubation of 1 minute or less was necessary. In comparative test with other compounds of metabolism and photosynthesis, the stimulation of oxygen evolution was greatest by glycolate and glyoxylate. In a proposed scheme glyoxylate serves as a terminal hydrogen acceptor from NADPH produced by photosynthesis, and it thereby stimulates oxygen evolution when carbon dioxide is not available. Transformation of glycolate to glyoxylate in these cells would have to occur in the absence of oxygen.

Eukaryota↗
PubMed1969

Glycolate and glyoxylate metabolism by isolated peroxisomes or chloroplasts.

Chloroplasts, mitochondria, and peroxisomes from leaves were separated by isopycnic sucrose density gradient centrifugation. The peroxisomes converted glycolate-(14)C or glyoxylate-(14)C to glycine, and contained a glutamate: glyoxylate aminotransferase as indicated by an investigation of substrate specificity. The pH optimum for the aminotransferase was between 7.0 and 7.5, and the Km for l-glutamate was 3.6 mm and for glyoxylate, 4.4 mm. The reaction of glutamate plus glyoxylate was not reversible. The isolated peroxisomes did not convert glycine to glyoxylate nor glycine to serine.Peroxisomes did not oxidize glycolate or glyoxylate to CO(2). Chloroplasts could very slowly oxidize glyoxylate, but not glycolate, to CO(2). Chloroplast oxidation of glyoxylate was heat labile and widely distributed among plants. Oxidation was stimulated by light and oxygen. but was not inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU).A scheme for the distribution of enzymes associated with glycolate metabolism and photorespiration is presented. Glycolate biosynthesis occurs in the chloroplasts. In the peroxisomes, glycolate is oxidized with O(2) uptake to glyoxylate by glycolate oxidase, and the glyoxylate is converted to glycine by glutamate:glyoxylate aminotransferase. Further metabolism of glycine does not occur in the peroxisomes. It is possible that excess glyoxylate from the peroxisomes could return to the chloroplasts to be reduced to glycolate or oxidized to account for part of the CO(2) loss during photorespiration.

Journal Article↗
PubMed1967

Glycolate pathway in algae.

No glycolate oxidase activity could be detected by manometric, isotopic, or spectrophotometric techniques in cell extracts from 5 strains of algae grown in the light with CO(2). However, NADH:glyoxylate reductase, phosphoglycolate phosphatase and isocitrate dehydrogenase were detected in the cell extracts. The serine formed by Chlorella or Chlamydomonas after 12 seconds of photosynthetic (14)CO(2) fixation contained 70 to 80% of its (14)C in the carboxyl carbon. This distribution of label in serine was similar to that in phosphoglycerate from the same experiment. Thus, in algae serine is probably formed directly from phosphoglycerate. These results differ from those of higher plants which form uniformly labeled serine from glycolate in short time periods when phosphoglycerate is still carboxyl labeled. In glycolate formed by algae in 5 and 10 seconds of (14)CO(2) fixation, C(2) was at least twice as radioactive as C(1). A similar skewed labeling in C(2) and C(3) of 3-phosphoglycerate and serine suggests a common precursor for glycolate and 3-phosphoglycerate. Glycine formed by the algae, however, from the same experiments was uniformly labeled. Manganese deficient Chlorella incorporated only 2% of the total (14)CO(2) fixed in 10 minutes into glycolate, while in normal Chlorella 30% of the total (14)C was found in glycolate. Manganese deficient Chlorella also accumulated more (14)C in glycine and serine.Glycolate excretion by Chlorella was maximal in 10 mm bicarbonate and occurred only in the light, and was not influenced by the addition of glycolate. No time dependent uptake of significant amounts of either glycolate or phosphoglycolate was observed. When small amounts of glycolate-2-(14)C were fed to Chlorella or Scenedesmus, only 2 to 3% was metabolized after 30 to 60 minutes. The algae were not capable of significant glycolate metabolism as is the higher plant. The failure to detect glycolate oxidase, the low level glycolate-(14)C metabolism, and the formation of serine from phosphoglycerate rather than from glycolate are consistent with the concept of an incomplete glycolate pathway in algae.

Carbon Isotopes↗
PubMed1967

Changes in Chlorophyll a/b Ratio and Products of CO(2) Fixation by Algae Grown in Blue or Red Light.

Chlamydomonas and Chlorella were grown for 10 days in white light. 955 muw/cm(2) blue light (400-500 mmu) or 685 muw/cm(2) red light (above 600 mmu). Rates of growth in blue or red light were initially slow, but increased over a period of 5 days until normal growth rates were reestablished. During this adaptation period in blue light, total chlorophyll per volume of algae increased 20% while the chlorophyll a/b ratio decreased. In red light no change was observed in the total amount of chlorophyll or in the chlorophyll a/b ratio. After adaptation to growth in blue light and upon exposure to (14)CO(2) with either blue or white light for 3 to 10 minutes, 30 to 36% of the total soluble fixed (14)C accumulated in glycolate-(14)C which was the major product. However, with 1 minute experiments, it was shown that phosphate esters of the photosynthetic carbon cycle were labeled before the glycolate. Glycolate accumulation by algae grown in blue light occurred even at low light intensity. After growth of the algae in red light, (14)C accumulated in malate, aspartate, glutamate and alanine, whereas glycolate contained less than 3% of the soluble (14)C fraction.

Journal Article↗
PubMed1966

Rate of Glycolate Formation During Photosynthesis at High pH.

The products of C(14)O(2) fixation by Chlamydomonas and Chlorella were studied under conditions most favorable for glycolate synthesis. The highest percentage of the C(14) was incorporated into glycolate in the pH range of 8 to 9. After 1 to 2 minutes as much as 40% of the C(14) was found in glycolate products and only a trace of C(14) was present as phosphoglycerate. Below pH 8 the rate of photosynthesis was much faster, but only a small percent of the C(14) was incorporated into glycolate in 1 or 2 minutes, while a high percent of the C(14) accumulated in phosphoglycerate. C(14) labeling of glycolate even at pH 8 or above did not occur at times shorter than 10 seconds. During the first seconds of photosynthesis, nearly all of the C(14) was found in phosphoglycerate and sugar phosphates. Thus glycolate appears to be formed after the phosphate esters of the photosynthetic carbon cycle.Washing Chlamydomonas with water 2 or 3 times resulted in the loss of most of their free phosphate. When a small aliquot of NaHC(14)O(3) was added to washed algae in the absence of this buffering capacity, the pH of the algal medium became 8 or above and much of the fixed C(14) accumulated in glycolate.

Journal Article↗