Ensaio in vitro da enzima nitrato redutase e efeito da disponibilidade de nitrato e fosfato em variantes pigmentares de hypnea musciformis (wulfen) j. v. lamour. (gigartinales, rhodophyta)

Abstract

The enzyme nitrate reductase (NR) catalyzes the reduction of nitrate to nitrite and controls the rate of nitrate assimilation. The in vitro assay of NR was optimized for the wild strain (brown, MA), and the phycoerythrin-deficient strain (light-green, VC) of Hypnea musciformis. Both strains were cultured at temperature of 23 ± 2 °C, photoperiod of 14 h, irradiance of 60-90 μmol photons m-2s-1, with medium composed by sterilized seawater (salinity 30 psu) with 50% von Stosch's enrichment solution (VSES/2). The optimal conditions for in vitro assay of NR were: 40 μM of NADH; 10 min of incubation of crude extracts (EB), and 100 μL of EB to both strains. Optimal activity of NR occurred at 4 and 2 mM of nitrate to the VC and MA strains, respectively. The VC and MA strains showed, respectively, Michaelis-Menten constants (KM) for NADH of 0.2068 and 0.0837 μM, and KM for nitrate of 0.0492 and 0.0294 mM. The results indicate that the NR of MA strain has higher affinity by the substrate than the NR of VC strain of H. musciformis. Experiments on the effects of availabilities of nitrate (5 to 105 μM) and nitrate and phosphate (0.5 to 25.5 μM, with a N:P relation of 4:1) showed that NR activity of VC and MA strain did not increase with the addition of nitrate to the medium, what can be related with their nutritional state. The NR activity was higher in treatments with phosphate addition than those with only nitrate addition, indicating that this nutrient is important to metabolic processes related to the NR activity.