Biosynthesis of Δ-aminolevulinate in greening barley leaves IV. Isolation of three soluble enzymes required for the conversion of glutamate to Δ-aminolevulinate
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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:
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.
Glutamate-1-semialdehyde is converted into δ-aminolevulinate by glutamate-1-semialdehyde aminotransferase, which is not retained on the affinity columns.
The run-off protein fraction also contains δ-aminolevulinate dehydratase and porphobilinogen deaminase.
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.
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- Biosynthesis of Δ-aminolevulinate in greening barley leaves IV. Isolation of three soluble enzymes required for the conversion of glutamate to Δ-aminolevulinate
Carlsberg Research Communications
Volume 46, Issue 4 , pp 243-257
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- 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