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

Abstract

Relat~ve in wtro actlv~ty levels for the enzyme glutamate synthase (COGAT) with the cofactors NADH NADPH and reduced ferredoxln were determined wlth a I4C method in 6 specles of phytoplankton from 3 taxonomic classes The NADH form of the enzyme was the dominant isozyme in all of the marlne and estuanne specles, whde the ferredoxln form was dominant in the 1 fresh-water species tested Significant amounts of an NADPH specific GOCAT were not detected In any of the orgalusms A sensihve fluorometnc assay for m wtro measurements was subsequently u a z e d to determme the substrate lunetics, pH ophmum and temperature-dependent activation energy for NADH-GOGAT m the diatom Skeletonema costatum Good agreement was obtained with the optlmal m wtro assay condit~ons for achvlty measurements of NADH-GOGAT on a conlmon phytoplankton extract with the I4C and fluorometnc assay methods The sensitlvltles of the 2 assays (l e fluorometric and "C) make them suitable for apphcatlon in studies of nltrogen asslmilat~on and phytoplankton growth rate dynamcs in aquatic ecosystems

Dissolved inorganic nitrogen uptake by intertidal microphytobenthos: nutrient concentrations, light availability and migration

“…The influences of temperature on relative and maximal assimilation rates of ammonium have been demonstrated previously in natural diatom-dominated communities (Lomas & Glibert 1999b, Harrison et al 1996. This regulation is related to activity of the enzyme involved in ammonium assimilation (glutamate synthase, GOGAT), which may have optimal activity at around 25°C (Clayton & Ahmed 1986). Hence, summer temperatures would have been close to the optimal temperature, while winter low temperatures may have led to a reduction in uptake rate.…”

The Response of Diatom Central Carbon Metabolism to Nitrogen Starvation Is Different from That of Green Algae and Higher Plants

“…Transcript levels of glnII (encoding GSII) are higher in diatom cells assimilating nitrate than in those assimilating ammonium directly (Takabayashi et al, 2005). Diatoms appear to have an NAD(P)Hdependent GOGAT [NAD(P)H-GOGAT] and also a ferredoxin-dependent form for this enzyme (Fd-GOGAT), which is thought to be plastid localized (Clayton and Ahmed, 1986;Zadykowicz and Robertson, 2005); however, our knowledge of this enzyme is limited. The activity of the GS/GOGAT cycle requires the input of carbon skeletons in the form of 2-oxoglutarate, while oxaloacetate is also important in the production of amino acids.…”

“…Nitrite is then transported into the chloroplast and further reduced to ammonium by a cyanobacterium-like ferredoxindependent nitrite reductase (Fd-NiR; Milligan and Harrison, 2000;Bowler et al, 2010). The joint action of glutamine synthetase (GS) and glutamate synthase (GOGAT) is thought to be the main route of ammonium assimilation into amino acids and other nitrogenous compounds (Dortch et al, 1979;Clayton and Ahmed, 1986;Zehr and Falkowski, 1988). Diatoms possess a plastid-localized GSII that is of red algal origin (Robertson et al, 1999;Robertson and Tartar, 2006;Siaut et al, 2007) and thought to be responsible for the assimilation of ammonium produced by nitrate reduction.…”

Changes in intracellular amino acids and glutamine : glutamate during N‐deprivation and feeding in Candida nitratophila

“…Furthermore, the pattern of change in levels of major amino acids in response to N-deprivation and refeeding is identical in both C. nitratophila and the various microalgae so far studied (Flynn & Al-Amoudi, 1988;Al-Amoudi & Flynn, 1989;Flynn, unpublished). The existence of a GS-GOGAT pathway has been suggested for several species of microalgae (Clayton & Ahmed, 1986 and Ahmed & Hellebust (1988) suggest that the GS-GOGAT pathway is the major route of NH,,* assimilation in such organisms. Magasanik (1988) suggests that in E. coli it is the relative proportions of 2-ketoglutarate to Gin that regulate the response of this organism to Ndeprivation.…”