Glutamate synthesis in barley roots: the role of the plastidic glucose-6-phosphate dehydrogenase.
Evidence is provided for a close link between glutamate (Glu) synthesis and the production of reducing power by the oxidative pentose phosphate pathway (OPPP) in barley ( Hordeum vulgare L. var. Alfeo) root plastids. A rapid procedure for isolating organelles gave yields of plastids of over 30%, 60% of which were intact. The formation of Glu by intact plastids fed with glutamine and 2-oxoglutarate, both substrates of glutamate synthase (GOGAT), depends on glucose-6-phosphate (Glc-6-P) supply. The whole process exhibited an apparent K(m Glc-6-P) of 0.45 mM and is abolished by azaserine, a specific inhibitor of GOGAT; ATP caused a decrease in the rate of Glu formation. Glucose and other sugar phosphates were not as effective in supporting Glu synthesis with respect to Glc-6-P; only ribose-5-phosphate, an intermediate of OPPP, supported rates equivalent to Glc-6-P. Glucose-6-phosphate dehydrogenase (Glc6PDH) rapidly purified from root plastids showed an apparent K(m Glc-6-P) of 0.96 mM and an apparent K(m NADP)(+) of 9 micro M. The enzyme demonstrated high tolerance to NADPH, exhibiting a K(i) (NADPH) of 58.6 micro M and selectively reacted with antibodies against potato plastidic, but not chloroplastic, Glc6PDH isoform. The data support the hypothesis that plastidic OPPP is the main site of reducing power supply for GOGAT within the plastids, and suggest that the plastidic OPPP would be able to sustain Glu synthesis under high NADPH:NADP(+) ratios even if the plastidic Glc6PDH may not be functioning at its highest rates.
Esposito
Sergio
S
Dipartimento di Biologia Vegetale, Università di Napoli "Federico II", Via Foria 223, 80139 Naples, Italy. esposito@cds.unina.it
Massaro
Graziella
G
Vona
Vincenza
V
Di Martino Rigano
Vittoria
V
Carfagna
Simona
S
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001377:Azaserine / Q000494:pharmacology; D015153:Blotting, Western; D002374:Catalase / Q000378:metabolism; D046508:Culture Techniques; D003600:Cytosol / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D003576:Electron Transport Complex IV / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019298:Glucose-6-Phosphate / Q000494:pharmacology; D005954:Glucosephosphate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D005970:Glutamate Synthase / Q000378:metabolism; D018698:Glutamic Acid / Q000096:biosynthesis*; D001467:Hordeum / Q000201:enzymology* / Q000378:metabolism; D007700:Kinetics; D009584:Nitrogen / Q000378:metabolism; D010730:Phosphoenolpyruvate Carboxylase / Q000378:metabolism; D018517:Plant Roots / Q000201:enzymology* / Q000378:metabolism; D018087:Plastids / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D011755:Pyrophosphatases / Q000378:metabolism; D012267:Ribosemonophosphates / Q000378:metabolism
D004791:Enzyme Inhibitors; D012267:Ribosemonophosphates; D018698:Glutamic Acid; C031626:ribose-5-phosphate; D019298:Glucose-6-Phosphate; D001377:Azaserine; D000255:Adenosine Triphosphate; D005954:Glucosephosphate Dehydrogenase; D002374:Catalase; D005970:Glutamate Synthase; D003576:Electron Transport Complex IV; D011755:Pyrophosphatases; D010730:Phosphoenolpyruvate Carboxylase; D009584:Nitrogen