Pyruvate Kinase, a Possible Regulatory Enzyme in Higher Plants

Duggleby, Ronald G.; Dennis, David T.

doi:10.1104/pp.52.4.312

Cited by 50 publications

(31 citation statements)

References 43 publications

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“…The soluble enzyme is similar to the enzyme from other plant sources (4,5,10,11,15), which is stable at 60 C (10, 15). The proplastid isoenzyme is unstable and cannot be solubilized without high concentrations of 2-mercaptoethanol being present.…”

Section: Discussionmentioning

confidence: 86%

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Isoenzyme of Pyruvate Kinase in Proplastids from Developing Castor Bean Endosperm

Luca

Dennis²

1978

Plant Physiol.

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Section: Discussionmentioning

confidence: 86%

“…Pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) has been partially purified from a variety of plants (4,5,10,11,15). Recently, a pyruvate kinase activity has been found in proplastids from developing castor bean endosperm (2).…”

mentioning

confidence: 99%

Isoenzyme of Pyruvate Kinase in Proplastids from Developing Castor Bean Endosperm

Luca

Dennis²

1978

Plant Physiol.

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“…This behavior might also be related to the distinct pattern of the glycolytic enzymes phosphoenolpyruvate carboxylase, ME, and malate dehydrogenase, associated with pyruvate and downstream malate metabolism, observed throughout the experiment, namely reduced activity after 36 h. This characteristic could result from a cascade of events leading to the degradation of the photosynthetic apparatus (65) and enhanced FA synthesis. Moreover, these changes may be modulated by key enzymes, such as phosphoenolpyruvate carboxylase and pyruvate kinase, which are, in turn, regulated by TCA cycle intermediates (66,67). However, a more comprehensive study would be needed to confirm this hypothesis.…”

Section: Discussionmentioning

confidence: 97%

Metabolite Profiling and Integrative Modeling Reveal Metabolic Constraints for Carbon Partitioning under Nitrogen Starvation in the Green Algae Haematococcus pluvialis

Recht

Töpfer

Batushansky

et al. 2014

Journal of Biological Chemistry

109

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Background:The microalga H. pluvialis shows a distinct pattern of carbon repartitioning upon nitrogen starvation. Results: Data-driven integrative modeling pinpoints metabolic adjustments underlying carbon repartition. Conclusion:In vitro and in silico experiments can dissect the systemic response to nitrogen starvation. Significance: Experimental data in conjunction with integrative modeling enables model-driven hypothesis testing.

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“…Since ADP is required for pyruvate kinase, activity without ADP could not have been due to this enzyme. Previous reports (3,18) have assigned this interference to the activity of phosphatases. However, the colorimetric assay, which is dependent upon the formation of 2,4-dinitrophenylhydrazone derivatives of keto acids, is nonspecific.…”

Section: Resultsmentioning

confidence: 99%

Properties of Pyruvate Kinase from Soybean Nodule Cytosol

Peterson¹,

Evans²

1978

Plant Physiol.

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(4,6,29). Control of pyruvate kinase activity is often exerted through ADP levels and activators like AMP and fructose 1,6-diP and inhibitors such as organic acids and ATP. Posttranslational modification (phosphorylation) may also be important (14). In gluconeogenic animal tissues, the enzyme is thought to be critical in regulating the flow of carbohydrates between the degradative glycolytic and synthetic gluconeogenic pathways (4,29). The same may be true for the plant enzyme (4). Regardless of whether gluconeogenesis is operative in the nodule, the availability of carbon skeletons and cofactors for ammonium assimilation could be influenced by pyruvate kinase activity. In addition, pyruvate kinases from the majority of plant and animal sources examined have been shown to have an absolute requirement for a monovalent cation for activity. Ammonium ion is a strong activator (10). As ammonia is the primary product of N2 fixation, and because of the presumed role of pyruvate kinase in regulation of carbohydrate metabolism, regulatory properties of the soybean nodule cytosol enzyme were examined. (8,21) was employed and reactions were terminated after 10 min. ADP and PEP concentrations were raised to 2.5 and 1.5 mm, respectively, for these assays. In both assays the reaction velocity was proportional to enzyme concentration over the range of concentrations used. Reactions were run at 30 C. In all cases, reaction mixtures without ADP were included in order to correct for PEP carboxylase activity. PEP carboxylase was assayed using a malate dehydrogenase enzyme couple. Assays contained 100 mm imidazole HCI (pH 7.5), 2 mm cyclohexylamine PEP, 5 mm MgCl2, 0; 16 mm NADH, 10 mm KHCO3, and 20 units of beef heart malate dehydrogenase. Coupled assays were performed with a Cary 11 or Carya 118 spectrophotometer. The Cary 118 was used for colorimetric assays and spectral studies. One unit of enzyme activity is defined as I ,umol of product/min at 30 C.Plant Extracts. All isolation steps were performed at 0 to 4 C. In a typical extraction, 18 g of nodules were extracted by grinding with 3 volumes of grinding medium and 6 g of insoluble PVP in a mortar and pestle. Grinding medium consisted of a 3:1 (v/v) mixture of buffer (100 mm imidazole-phosphate [pH 7.51 and 1.33 mM DTT)-glycerol. The extracts were filtered through four layers of cheesecloth and centrifuged 15 min at 37,000g. The supernatant was removed and 30 ml applied to a column (3 x 4 cm) of DEAEcellulose. The DEAE column was prepared by equilibrating with a 3:1 (v/v) mixture of buffer (10 mm imidazole-phosphate [pH 7.51 and 0.13 mm DTT)-glycerol (equilibrating medium). After addition of the enzyme, the column was washed with 2 column volumes of the equilibration medium, 0.075 M with respect to KCI. One column volume of equilibration medium, 0.175 M with respect to KCI, removed the single peak ofpyruvate kinase activity (7.5-10 units) from the column. Column flow rate during the entire procedure was 60 ml/hr. The specific activity of pyruvate kinase was increased 3.5-fo...

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Pyruvate Kinase, a Possible Regulatory Enzyme in Higher Plants

Cited by 50 publications

References 43 publications

Isoenzyme of Pyruvate Kinase in Proplastids from Developing Castor Bean Endosperm

Isoenzyme of Pyruvate Kinase in Proplastids from Developing Castor Bean Endosperm

Metabolite Profiling and Integrative Modeling Reveal Metabolic Constraints for Carbon Partitioning under Nitrogen Starvation in the Green Algae Haematococcus pluvialis

Properties of Pyruvate Kinase from Soybean Nodule Cytosol

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