Biosynthesis of Δ-aminolevulinate in greening barley leaves IV. Isolation of three soluble enzymes required for the conversion of glutamate to Δ-aminolevulinate

  • Wei-Yeh Wang
  • Simon P. Gough
  • C. Gamini Kannangara

Abstract

The soluble enzymes converting glutamate into δ-aminolevulinate and subsequently into uroporphyrinogen were partially purified from the stroma of greening barley plastids using Sephacryl S-300 gel filtration. By affinity chromatography employing sequentially Blue Sepharose, Matrex Gel Red A and heme-Sepharose the partially purified enzymes were separated into three fractions which together are required to catalyze the synthesis of δ-aminolevulinate from glutamate: proteins binding to Blue Sepharose, proteins binding to heme-Sepharose and run-off proteins not retained by the three columns. By analysing the characteristics of these fractions the following conclusions are reached:
  1. 1.

    Conversion of glutamate into glutamate-1-semialdehyde in the presence of ATP, Mg2+ and NADPH requires at least two proteins, one binding to heme-Sepharose and one binding to Blue Sepharose.

     
  2. 2.

    Glutamate-1-semialdehyde is converted into δ-aminolevulinate by glutamate-1-semialdehyde aminotransferase, which is not retained on the affinity columns.

     
  3. 3.

    The run-off protein fraction also contains δ-aminolevulinate dehydratase and porphobilinogen deaminase.

     
  4. 4.

    The heme-Sepharose bound protein(s) probably converts glutamate to glutamate-1-phosphate in the presence of ATP and Mg2+ and the Blue-Sepharose bound protein(s), glutamate-1-phosphate to glutamate-1-semialdehyde, in the presence of NADPH.

     

Keywords

Heme-Sepharose Blue Sepharose affinity chromatography high pressure liquid chromatography glutamate-1-semialdehyde aminotransferase glutamine synthetase glutamate-1-phosphate kinase glutamate-1-phosphate dehydrogenase δ-aminolevulinate dehydratase porphobilinogen deaminase uroporphyrinogen 

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Copyright information

© Carlsberg Laboratory 1981

Authors and Affiliations

  • Wei-Yeh Wang
    • 1
    • 2
  • Simon P. Gough
    • 1
    • 2
  • C. Gamini Kannangara
    • 1
    • 2
  1. 1.Department of PhysiologyCarlsberg LaboratoryCopenhagen-Valby
  2. 2.Department of BotanyUniversity of IowaIowa CityUSA

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