Francisco Cánovas, Victoriano Valpuesta, Ignacio Núñez De Castro
{"title":"Characterization of tomato leaf glutamine synthetase","authors":"Francisco Cánovas, Victoriano Valpuesta, Ignacio Núñez De Castro","doi":"10.1016/0304-4211(84)90207-4","DOIUrl":null,"url":null,"abstract":"<div><p>Glutamine synthetase was purified to apparent homogeneity from the leaves of light-grown tomato plants. The purification steps involved ammonium sulphate precipitation (40–60%), adsorption by hydroxyapatite, DEAE Sephadex ionic exchange chromatography, and Sephacryl S 300 gel filtration. Only one glutamine synthetase form was found, with <em>K</em><sub><em>m</em></sub>-values of 5.5 mM, 0.8 mM and 0.4 mM for glutamate, ATP and ammonium, respectively. The in vitro activity depended on Mg<sup>2+</sup> or Mn<sup>2+</sup> concentrations with positive cooperativity. Enzyme assay pH optima were 7.2 for synthetase, and 6.0 for transferase. The activation energy of the enzymatic reaction was 33.2 kJ/mol. ADP appeared to be a competitive inhibitor with a <em>K</em><sub><em>i</em></sub>-value of 0.4 mM. Enzyme inhibition was also produced by aspartate, alanine and phosphoserine.</p></div>","PeriodicalId":20221,"journal":{"name":"Plant Science Letters","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"1984-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0304-4211(84)90207-4","citationCount":"33","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Plant Science Letters","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/0304421184902074","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 33
Abstract
Glutamine synthetase was purified to apparent homogeneity from the leaves of light-grown tomato plants. The purification steps involved ammonium sulphate precipitation (40–60%), adsorption by hydroxyapatite, DEAE Sephadex ionic exchange chromatography, and Sephacryl S 300 gel filtration. Only one glutamine synthetase form was found, with Km-values of 5.5 mM, 0.8 mM and 0.4 mM for glutamate, ATP and ammonium, respectively. The in vitro activity depended on Mg2+ or Mn2+ concentrations with positive cooperativity. Enzyme assay pH optima were 7.2 for synthetase, and 6.0 for transferase. The activation energy of the enzymatic reaction was 33.2 kJ/mol. ADP appeared to be a competitive inhibitor with a Ki-value of 0.4 mM. Enzyme inhibition was also produced by aspartate, alanine and phosphoserine.
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