Biosynthesis of Δ-aminolevulinate in greening barley leaves IV. Isolation of three soluble enzymes required for the conversion of glutamate to Δ-aminolevulinate
Cite this article as: Wang, W., Gough, S.P. & Kannangara, C.G. Carlsberg Res. Commun. (1981) 46: 243. doi:10.1007/BF02906501 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:
Conversion of glutamate into glutamate-1-semialdehyde in the presence of ATP, Mg
2+ 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 Mg
2+ 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 Download to read the full article text References
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