Kinetics for glutamine-synthetase inhibition by phosphinothricin and measurement of other enzyme activities in situ in isolated asparagus cells using a freeze-thaw technique

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Abstract
Kinetics for the inhibition of glutamine synthetase (EC 6.3.1.2) in situ by the herbicidal glutamate analogue, phosphinothricin, have been generated, and produce an inhibitor dissociation constant (Ki) of 6.5 μM. This has been achieved through the development of a rapid technique for the isolation of mesophyll cells from the cladophylls of young asparagus (Asparagus sprengeri) plants to provide starting material for the direct measurement of enzyme activities in situ. A modification of the technique developed by Rhodes and Stewart (Planta 118, 133–144 (1974) for the direct determination of enzyme activities in higher-plant tissues has been applied to these asparagus cells. Treatment of the cells by a single freezing in liquid nitrogen for a very short period (10 s), followed by thawing, alters the permeability of cell and organelle membranes allowing enzymes to become accessible to many small molecules, and yet remain concentrated and active within the cell. The activities of enzymes known to be located specifically in the organelles as well as the cytoplasm can be measured in asparagus cells treated in this way. Comparisons have been made between the activity and inhibition of glutamine synthetase in situ, and the enzyme isolated and partially purified from asparagus cells by fast protein liquid chromatography. Similarities in Km and Ki values obtained between these two emphasize the efficacy of the freeze-thaw technique. There is only a single glutamine-synthetase isoenzyme in asparagus mesophyll cells, which copurifies with the one normally associated with the chloroplast (GS2).