Biochemical characterization and regulation of nitrate reductase in the marine macro-stage Gracilaria tenuistipitata (Rhodophyta)

Author(s)

Patricia de Fátima Lopes

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2001-06-13

Examining board members

Pio Colepicolo; Iolanda Midea Cuccovia; Estela Maria Plastino; Marie Anne van Sluys; Clelia Ferreira Terra

• Advisor: Pio Colepicolo

Abstract

The marine red alga Gracilaria tenuistipitata var. liui Zhang et Xia) is economically important, being the main source for the production of phycocolloids. This strain is extensively cultivated in ponds in southern China and Taiwan and exhibits a wide tolerance to environmental factors. The major source of nitrogen in the ocean is in the form of nitrate being the main limiting macroalgal production. The nitrogen assimilation process occurs in a two-step reaction catalyzed by 2 enzymes working sequentially, nitrate reductase (NR) and nitrite reductase (NiR). NR catalyzes the reduction of nitrate to nitrite using NAD(P)H as electron donor. Nitrite is reduced to ammonium (by NiR) and it is immediately assimilated in organic nitrogen compounds, such as amino acids and bases. NR is considered the first and regulatory step in this assimilating process. We report the circadian oscillation of NR activity as well as NR protein content in G. tenuistipitata. Both NR activity and NR protein levels peaks at midday in algae maintained under light:dark cycle (12:12h). Toe NR activity is 30 fold higher in light period for alga growing under light-dark conditions, and the NR protein level is about 40 fold higher in extracts from the light period. The oscillation observed is under biological clock control since the fluctuation of NR activity persists after 10 days under constant light conditions. No oscillation in the NR activity is found when the algae were maintained under constant dark condition. The experiments on nitrate uptake carried out in algae growing under light:dark conditions had shown that the absorption peaks at the middle of the dark periods. So we can conclude that the process of nitrate assimilation and nitrate uptake is occurring in such an independent ways. NR from G. tenuistipitata is a NADH specific enzyme with Michaelis-Menten apparent constant of 95 µM. Toe apparent KM for nitrate was estimated to be 197 µM, which is reasonable with the values described to another algae. The isoeletric focusing revealed NR is a basic protein with pi of 8.66. The NR from was purified in four steps: ion exchange chromatography, ammonium sulfate precipitation, gel filtration chromatography, and Affigel-blue affinity column. Non-denaturated protein shows a molecular mass of about 420 kDa, and 4 identical subunits of 110 kDa, based on SDS-PAGE. Toe intracellular localization of NR in G. tenuistipitata was examined by applying both immunofluorescence and immunogold methods revealing NR associated with chloroplasts. (AU)