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

-(In vitro assay of nitrate reductase enzyme and effect of nitrate and phosphate availability in colour strains of Hypnea musciformis (Wulfen) J. V. Lamour. (Gigartinales, Rhodophyta)). 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-defi cient 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 -2 s -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 (K M ) for NADH of 0.2068 and 0.0837 μM, and K M for nitrate of 0.0492 and 0.0294 mM. The results indicate that the NR of MA strain has higher affi nity 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.Key words -colour strain, Hypnea, nitrate, nitrate reductase, phosphate RESUMO -(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)). A enzima nitrato redutase (NR) catalisa a redução do nitrato a nitrito e controla a taxa de assimilação do nitrato. O ensaio in vitro da nitrato redutase foi otimizado para a linhagem selvagem (marrom, MA) e para a linhagem defi ciente em fi coeritrina (verde-clara, VC) de Hypnea musciformis. As duas linhagens foram cultivadas em temperatura de 23 ± 2 °C, fotoperíodo de 14 horas, irradiância de 60-90 μmol fótons m -2 s -1 , e meio composto por água do mar esterilizada (30 ups) enriquecida com a solução de von Stosch na concentração de 50% (VSES/2). As condições ótimas de ensaio para ambas as linhagens foram: 40 μM de NADH; 10 min de incubação do extrato bruto (EB) e 100 μL de EB. A atividade ótima da NR ocorreu em 4 e 2 mM de nitrato para a linhagem VC e MA, respectivamente. As linhagens VC e MA apresentaram, respectivamente, constante aparente de Michaelis-Menten (K M ) para NADH de 0,2068 e 0,0837 μM, e K M para nitrato de 0,0492 e 0,0294 mM. Os resultados indicam que a NR da linhagem MA tem maior afi nidade pelo substrato do que a NR da linha...

Nitrate Assimilation: The Role of In Vitro Nitrate Reductase Assay as Nutritional Predictor

“…Lartigue & Sherman (2005) observed the same trend in Enteromorpha sp. A like inducing response, under the same conditions, has also been observed in other macroalgae (Gao, Smith & Alberte 1995;Lartigue & Sherman, 2005;Young et al, 2007;Martins et al, 2009;Cabello-Pasini et al, 2011). Nitrate reductase activity in Arctic species appears to be directly enhanced by nitrate addition, with relatively little feedback from the N-status of the cell (Gordillo et al, 2006).…”

“…This high activity has been reported in Laminaria saccharina (Davison & Davison 1987), Fucus vesiculosus (Collén & Davison 2001), and L. digitata, Fucus serratus, Fucus vesiculosus and Fucus spiralis (Young et al (2007). Macroalgae in tropical and sub-tropical environments presented high temperature tolerance during assaying (Lopes et al, 1997;Chow et al, 2007;Granbom et al, 2004;Martins et al, 2009), the optimum usually being higher than normal. Nitrate reductase activity can also vary considerably among and within species, depending on natural or laboratory growing conditions.…”

Characterization of nitrate reductase activity (NR) in foliar and radicular tissues of Physalis angulata L.: diurnal variations and protocol optimization

“…The highest enzymatic activity, with a prolonged incubation period may be related to a time requirement for complete infiltration of the reaction medium (Oliveira et al, 2005), being that period variable according to the species and tissues studied. For many plants such as coffee, peachpalm, pineapple, Brachiaria and macroalgae, similar performances of increased enzyme activity with longer incubation periods have been observed (Meguro and Magalhães, 1982;Nievola and Mercier, 2001;Oliveira et al, 2005;Martins et al, 2009;Cazetta and Villela, 2004). According to the obtained results, an incubation time of 75 minutes was adopted for all subsequent analyses.…”

Ritmos circadianos em Gracilaria birdiae (Rhodophyta): oscilação do desempenho fotossintético e caracterização enzimática da nitrato redutase

“…Outras algas apresentaram uma resiliência menor a esta variação de temperatura (CHOW; OLIVEIRA; PEDERSÉN, 2004;GRANBOM et al, 2004;HURD et al, 1995a;MARTINS;YOKOYA, 2009;NAKAMURA;IKAWA, 1993). Geralmente a atividade máxima se dá na média de temperatura do ambiente onde a alga foi coletada.…”