The Regulation of Pyruvate Kinase

Seubert, W.; Schoner, Wilhelm

doi:10.1016/b978-0-12-152803-4.50011-7

Cited by 98 publications

(33 citation statements)

References 71 publications

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“…The malate-labeling curve (Fig. 4) (4,22), is the simplest conclusion consistent with our data. Direct activation of pyruvate kinase in higher plants by ammonia is supported by work with the partially purified enzyme.…”

Section: Resultssupporting

confidence: 91%

Ammonia Regulation of Carbon Metabolism in Photosynthesizing Leaf Discs

1977

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The over-all process of nitrate reduction and ammonia incorporation into amino acids in higher plant leaves (along with the role of light in that process) has been a subject of recent research (3,10,12). Given the production of amino acids as well as sucrose during photosynthesis (16), it is of interest to determine whether leaf regulatory mechanisms involve ammonia. The intracellular NH4+ level could influence leaf carbon metabolism during photosynthesis with respect to sucrose synthesis versus the amino acid synthesis necessary for protein production and leaf growth. Kinetic studies of "4C-labeled compounds formed in the unicellular green alga Chlorella pyrenoidosa during photosynthesis with "4CO2 have indicated that ammonia brings about increased amino acid synthesis in part due to stimulation of pyruvate kinase (8). We have now investigated whether a similar regulatory mechanism is active in the leaves of higher plants. The specific plant chosen for investigation was alfalfa because of its potential as a source of leaf protein for direct human consumption (5).In our experiments we have used the techniques of kinetic tracer analysis of steady-state photosynthesis (2,19 MATERIALS AND METHODS Plant Material. Alfalfa (Medicago sativa L., var. El Unico) seeds were planted in 12 cm of vermiculite. The plants were grown at 3,000 ft-c and 15 C with a 9-hr light period and a 15-hr dark period. Plants were fertilized with modified Hoagland solution. Second through fourth unfolded leaves (numbered from the plant apex; not fully expanded) were excised after 1 hr of light from 6-week-old plants. The leaves were placed on a rubber sheet and 1-cm-diameter leaf discs were cut out with a cork borer.Photosynthetic Carbon Fixation. The closed steady-state gas circulation system described previously was employed (19). Leaf discs were exposed to CO2 in 4.5-cm-diameter flasks made from ground-glass joints and having transparent upper and lower surfaces. Each flask was fitted with gas inlet and outlet tubes, a serum stopper (allowing for addition of reagents during experiments), and five glass leaf disc holders (serving to prevent leaf overlap). Immediately after being cut, the leaf discs were floated on 4 ml of pH 7.4 buffer (0.05 M K2HPO4, pH adjusted with HCl) in the disc exposure flasks. Twelve flasks were utilized; each contained five discs comprising a single sample. The flasks were placed in a temperature-regulated shaking device (previously described for chloroplast experiments) (7) and attached to the closed gas circulation system through manifolds. Gas flow was commenced with 0.04% CO2 in air (20% 02); flask temperature was 27 C. After 12 min of preincubation in the dark, the lights were turned on and ammonium chloride in the pH 7.4 buffer was injected into six of the flasks to give a final concentration of 5 mm NH4Cl. Photosynthesis with 12CO2 was observed for 17 min. The unlabeled CO2 in air was then replaced by 0.038% "4CO2 (30.2 ,uCi/Amol) in air (20% 02). Samples of control and ammoniumtreated leaf discs were remove...

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Section: Resultssupporting

confidence: 91%

Ammonia Regulation of Carbon Metabolism in Photosynthesizing Leaf Discs

1977

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“…The activity of pyruvate kinase in extracts of Candida 107 varied according to the growth conditions, being almost completely repressed in n-alkane-grown cells. Although the properties of pyruvate kinase can vary according to its biological source, it is an important regulatory point in glycolysis (Seubert & Schoner, 1971). Its strong repression in alkane-grown yeast ensures a unidirectional flow of carbon from acetyl-CoA, produced by alkane oxidation, via oxaloacetate and pyruvate to the pentoses and hexoses.…”

Section: Metabolic Controls In Candida 107mentioning

confidence: 99%

An Analysis of Intermediary Metabolism and its Control in a Fat-synthesizing Yeast (Candida 107) Growing on Glucose or Alkanes

Whitworth¹,

Ratledge²

1975

Journal of General Microbiology

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Enzymes of glycolysis, pentose phosphate pathway, gluconeogenesis, tricarboxylate acid cycle, glyoxylate by-pass and fatty-acid biosynthesis were assayed in extracts from Candida 107 grown continuously on glucose under carbon limitation, nitrogen limitation and on n-alkanes. The yeast was therefore either in a lipogenic or lipolytic state. Phosphofructokinase was absent under all conditions whereas enzymes of gluconeogenesis, including fructose I ,6-bisphosphatase and the pentose phosphate cycle, were all present. Glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were specific for NADP+ and were inhibited in a non-competitive manner by NADPH and NADH. Phosphoenolpyruvate, citrate, ATP and acetyl CoA had no inhibitory effects. Thus glucose metabolism appears to be by the pentose phosphate pathway which will rapidly produce NADPH. This can readily be consumed during fatty-acid biosynthesis and, as there appears to be no inhibition of the flow of carbon from glucose to acetyl CoA, fatty-acid synthesis can continue for as long as there is a supply of glucose. These results help to explain the probable causes of fat build-up to high concentrations (about 40 7; of the cell dry weight) in this and other organisms. In alkane-grown cells, lipogenesis is repressed and carbon is able to flow from the alkanes via acetyl CoA, oxaloacetate and pyruvate into pentoses and hexoses in a unidirectional manner, because of the strong repression of pyruvate kinase and the increased activities of phosphoenolpyruvate kinase and fructose I ,6-bisphosphatase under these conditions. Although there was little change in the total activity of the TCA cycle enzymes under the various growth conditions, isocitrate lyase was induced under lipolytic conditions.

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“…Pyruvate kinase (pyruvate-ATP phosphotransferase, EC 2.7.1.40) from a variety of tissues (2,7,18,19,34,36) and organisms (10,17,21,32,36,38,41) appears to be a regulatory enzyme. It usually shows a sigmoid saturation curve towards PEP3 and is activated by FDP in eucaryotes and by AMP in procaryotes.…”

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confidence: 99%

Pyruvate Kinase, a Possible Regulatory Enzyme in Higher Plants

Duggleby¹,

Dennis²

1973

Plant Physiol.

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A number of plant species were examined for the presence of pyruvate kinase (pyruvate-ATP phosphotransferase, EC 2.7.1.40), and of a phosphatase activity which hydrolyzes phosphoenolpyruvate. Of those examined, only cotton (Gossypium sp. L.) seeds were found to be sufficiently free of the phosphatase to permit a kinetic study of pyruvate kinase. (2,7,18,19,34,36) and organisms (10,17,21,32,36,38,41) appears to be a regulatory enzyme. It usually shows a sigmoid saturation curve towards PEP3 and is activated by FDP in eucaryotes and by AMP in procaryotes. However, some workers have described pyruvate kinases which do not fit these generalizations (3,6,30,42).The kinetic properties of pyruvate kinase from higher plants have not been examined in detail. The activation of the enzyme by mono-and divalent cations in relation to plant nutrition has been studied (14,(26)(27)(28)39), but the work to date (26,28,31) indicates that the higher plant enzyme does not show sigmoid kinetics towards PEP and is not activated by FDP. However, since neither of these properties is common to all pyruvate kinases, it was decided to re-examine the higher plant enzyme.

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The Regulation of Pyruvate Kinase

Cited by 98 publications

References 71 publications

Ammonia Regulation of Carbon Metabolism in Photosynthesizing Leaf Discs

Ammonia Regulation of Carbon Metabolism in Photosynthesizing Leaf Discs

An Analysis of Intermediary Metabolism and its Control in a Fat-synthesizing Yeast (Candida 107) Growing on Glucose or Alkanes

Pyruvate Kinase, a Possible Regulatory Enzyme in Higher Plants

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