Detection of glutamine synthetase activity in marine phytoplankton: optimization of the biosynthetic assay

Abstract

A comprehensive study of the key ammonium-assimilating enzyme, glutarnine synthetase (GS), was made in a variety of marine phytoplankton. GS activity was detected in 13 out of the 15 species examined comprising 6 algal classes. Both the biosynthetic GS reaction as well as the transferase reaction were measured with phytoplankton extracts for comparative purposes. In evaluating the differences between the 2 assays it was found that the biosynthetic assay was more sensitive than the transferase assay in terms of final product formed/assay. The transferase assay yielded a more stable final product but the reaction is not specific for GS (Meister, 1974). A series of kinetic studies on the GS enzyme were carried out on a number of marine species. These studies included the determination of the pH optimum, linearity of the reaction with time, apparent energies of activation (E,) and apparent Y, values for the substrates: NH,', glutamate and ATP. In addition, the optimum requirement for the divalent cation, Mgf + was ascertained. The most significant finding of these studies was the broad distribution of GS activity in marine phytoplankton. The apparent K, values for ammonium were in the micromolar range. This is in marked contrast to earlier observations of the requirement of glutamate dehydrogenase (GDH), an alternate ammonium-assimilating enzyme, which has been shown to have a K, for ammonium ions in the millimolar range. These data support the view that GS is the primary ammonium-assimilating enzyme in phytoplankton found in areas of intermittent nitrogen supply.

Detection of glutamate synthase (GOGAT) activity in phytoplankton: evaluation of cofactors and assay optimization

“…In subsequent studies with the diatom Skeletonerna costaturn Dortch et al (1979) concluded that GDH could account for less than 5 % of the NH: uptake and assimilation rates in cells. An ensuing examination of 13 marine phytoplankton species comprising 6 algal classes revealed the presence of the GS enzyme with much greater affinities for NH: (ca 2 to 8 FM) (Bressler & Ahmed 1984). Therefore, the occurrence of a highly efficient NH: assimilating enzyme system (i.e.…”

“…The NH: derived from either NO; andlor NO; reduction or direct uptake from the extracellular medium is then assimilated into amino acids. The specific pathway for this latter assimilation step has been the subject of considerable speculation, with the current consensus favoring a coupled enzymatic reaction involving glutamine synthetase (GS) and glutamate synthase (GOGAT) (Morris 1974, Ahmed et al 1977, Miflin & Lea 1980, Syrett 1981, Wheeler 1983, Falkowslu 1983, Bressler & Ahmed 1984. Both the phylogenetic occurrence of GS and sensitive assay methods for ~t s detection have been documented for a variety of phytoplankton species (Bressler & Ahmed 1984, Clayton & Ahmed unpubl.).…”

Modified assay procedure for enhanced sensitivity of in vitro glutamine synthetase (GS) activity measurements in marine phytoplankton

“…The preparation of cell-free extracts from experimental phytoplankton cultures is described in Bressler & Ahmed (1984). Briefly, a volume of phytoplankton culture was filtered onto a glass fiber filter.…”

“…Shapiro & Stadtman 1970, Falkowski & Rivkin 1976, Bressler & Ahmed 1984 or detection of the ADP end product using a fluorometric coupled enzyme assay (Falkowski & Rivkin 1976). Both approaches have limitations.…”

“…This paper summarizes efforts to achieve greater sensitivity for in vitro GS activity measurements in marine phytoplankton samples. Although similar to the parent method of Bressler & Ahmed (1984), 2 modifications have been adopted to enhance overall assay sensitivity. First, a more sensitive method for detecting the end product inorganic phosphate (P,) has been adapted to the procedure.…”