Biosynthetically active glutamine synthetase in the marine diatom Phaeodactylum tricornutum: optimization of the forward-reaction assay

The activity of glutamine synthetase (GS) was measured in the marine diatom Phaeodactylum tricornutum Bohlin (Strain SME) by a biosynthetic assay, based on -glutamyl hydroxamate synthesis, and referred to as the forwardreaction assay. The effects of pH, temperature and different homogenizing buffer preparations on enzyme activity, linearity of reactions, and substrate-saturation kinetics were investigated. The resultant data provide the basis for establishing optimum experimental conditions for a standard assay. Affinities of P. tricornutum GS for glutamate, ATP and Mg²⁺ were similar to those recorded elsewhere for a variety of other phytoplankton species using true biosynthetic assays based on release of inorganic phosphate, whereas the affinity for hydroxylamine was two orders of magnitude lower than that for ammonium, with an apparent K_m value in the millimolar range. This, together with negative results obtained during earlier attempts to detect GS activity in P. tricornutum using the true biosynthetic assay, indicates that the GS of this alga has a lower affinity for ammonium than that of other phytoplankton species. Dual substrate kinetics demonstrated that apparent K_m and V_m values for glutamate were directly proportional to the concentration of ATP, thus giving indirect evidence of a correlation between GS activity and the adenylate energy charge. Comparisons between synthetase activities obtained with the optimized forward-reaction assay and transferase activities reported from other studies on various phytoplankton species revealed discrepancies which, to a great extent, probably arise from differences in the growth conditions of the organisms.