Ammonium induction of a novel isoform of glucose‐6P dehydrogenase in barley roots

Abstract

The effects of ammonium and glutamine supply on amino acid levels and the activity of glucose‐6P dehydrogenase (G6PDH EC 1.1.1.49), the main regulated enzyme of the oxidative pentose phosphate pathway, were investigated in barley roots (Hordeum vulgare cv. Alfeo). Feeding ammonium to barley plants increased the contents of glutamine, asparagine and G6PDH in roots. These effects were abolished by using inhibitors of glutamine synthetase. Glutamine‐fed barley roots showed a similar increase in G6PDH activities to ammonium‐fed plants. Two G6PDH enzymes (G6PDH 1 and 2) were partially purified and characterized from ammonium‐fed and glutamine‐fed roots. The isozymes had different pH optima and apparent Km values for glucose‐6P. G6PDH 2 showed similar kinetic parameters to the G6PDH present in root extracts of barley grown without any nitrogen source, while G6PDH 1 exhibited different kinetic parameters, suggesting the appearance of a second G6PDH isoform in response to ammonium. Western blot analysis demonstrated the existence of two G6PDH subunits of different molecular mass in barley roots grown in the presence of ammonium or glutamine, while only one isoform could be detected in roots grown without any nitrogen source. The results suggest a primary role of ammonium and/or glutamine in the appearance of a novel G6PDH isoform; this enzyme (G6PDH 1) shows kinetic parameters similar to those measured previously for chloroplastic and plastidic isoforms and seems to be induced by changes in glutamine content or a related compound(s) in the roots.