Glucose-6-phosphate dehydrogenase in barley roots: kinetic properties and localisation of the isoforms.
Two different isoforms of glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) have been partially purified from barley (Hordeum vulgare L., cv. Alfeo) roots. The procedure included an ammonium sulfate step, Q-Sepharose and Reactive Blue agarose chromatography, and led to 60-fold and 150-fold purification for the two enzymes, respectively. The Glc6PDH 1 isoform accounts for 17% of total activity of the enzyme in roots, and is very sensitive to the effects of NADP+/NADPH ratio and dithiothreitol; the Glc6PDH 2 isoform is less affected by reducing power and represents 83% of the total activity. The isoforms showed distinct pH optima, isoelectric points, Km for glucose-6-phosphate and a different electrophoretic mobility. The kinetic properties for the two enzymes were affected by ATP and metabolites. Both enzymes are inhibited to different extents by ATP when magnesium is omitted from the assay mixture, whereas the addition of ATP-Mg2+ had no effect on Glc6PDH activities. The Glc6PDH isoforms are usually present in the plastids and cytosol of plant cells. To verify the intracellular locations of the enzymes purified from barley roots, Glc6PDH was purified from isolated barley root plastids; this isoform showed kinetic parameters coincident with those found for Glc6PDH 1, suggesting a plastid location; the enzyme purified from the soluble fraction had kinetic parameters resembling those of Glc6PDH 2, confirming that this isoform is present in the cytosol of barley roots.
Esposito
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S
Dipartimento di Biologia Vegetale, Università di Napoli, Federico II, Naples, Italy. esposito@CDS.unina.it
Carfagna
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S
Massaro
G
G
Vona
V
V
Di Martino Rigano
V
V
D015153:Blotting, Western; D050260:Carbohydrate Metabolism; D003600:Cytosol / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology*; D007526:Isoelectric Point; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010427:Pentose Phosphate Pathway; D018517:Plant Roots / Q000201:enzymology; D018087:Plastids / Q000378:metabolism
D007527:Isoenzymes; D005954:Glucosephosphate Dehydrogenase