PLANT PHYSIOLOGY , Vol 105, Issue 1 415-424, Copyright © 1994 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
A Metabolic Control Analysis of the Glutamine Synthetase/Glutamate Synthase Cycle in Isolated Barley (Hordeum vulgare L.) Chloroplasts
A. C. Baron, T. H. Tobin, R. M. Wallsgrove and A. K. Tobin
School of Biological Sciences, S3.614 Stopford Building, University of Manchester, Manchester M13 9PT, United Kingdom (A.C.B., T.H.T., A.K.T.)
Ammonia assimilation in chloroplasts occurs via the glutamine
synthetase/glutamate synthase (GS/GOGAT) cycle. To determine the extent to
which these enzymes contribute to the control of ammonia assimilation, a
metabolic control analysis was performed on isolated barley (Hordeum
vulgare L.) leaf chloroplasts. Pathway flux was measured polarographically
as ammonium-plus-2-oxoglutarate-plus-glutamine-dependent O2 evolution in
illuminated chloroplasts. Enzyme activity was modulated by titration with
specific, irreversible inhibitors of GS (phosphinothricin) and GOGAT
(azaserine). Flux control coefficients (CJ0E0) were determined (a) by
differentiation of best-fit hyperbolic curves of the data sets (flux versus
enzyme activity), and (b) from estimates of the deviation indices
(D/[prime]E0). Both analyses gave similar values for the coefficients. The
control coefficient for GS was relatively high and the value did not change
significantly with changes in 2-oxoglutarate concentration (C/0E0 = 0.58 at
5 mM 2-oxoglutarate and 0.40 at 20 mM 2-oxoglutarate). The control
coefficient for GOGAT decreased with decreasing glutamine concentrations,
from 0.76 at 20 mM glutamine to 0.19 at 10 mM glutamine. Thus, at high
concentrations of glutamine, GOGAT exerts a major control over flux with a
significant contribution also from GS. At lower concentrations of
glutamine, however, GOGAT exerts far less control over pathway flux.
