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An in vitro nitrate reductase assay for marine macroalgae: optimization and characterization of the enzyme for Fucus gardneri (Phaeophyta)

Citation

Hurd, CL and Berges, JA and Osborne, J and Harrison, PJ, An in vitro nitrate reductase assay for marine macroalgae: optimization and characterization of the enzyme for Fucus gardneri (Phaeophyta), Journal of Phycology, 31, (5) pp. 835-843. ISSN 0022-3646 (1995) [Refereed Article]

Copyright Statement

Copyright 1995 Journal of Phycology

DOI: doi:10.1111/j.0022-3646.1995.00835.x

Abstract

Measurement of the activity of the enzyme nitrate reductase (NR) may provide a useful index of nitrogen metabolism in marine macroalgae. In several species, including Fucus gardneri P. C. Silva, in vitro assays previously failed to detect NR activity, necessitating the use of in situ (or so-called"in vivo") assays, which are more loosely controlled and lead to difficulties in assessing enzyme characteristics such as the half-saturation constant (Km). In this paper, we describe an in vitro NR assay developed for F. gardneri, in which tissue was homogenized using liquid nitrogen prior to the assay. In contrast to previous studies, enzyme activity was always detectable in F. gardneri collected directly from the field at levels up to 30 nmol nitrate converted to nitrite·min−1·g−1 wet weight. The effect of a variety of compounds, commonly added to NR extraction buffers, were tested. Additions of protease inhibitors, bovine serum albumin, and ethylenediamine tetraacetic acid had no consistent effects on NR activity, while polyvinyl pyrrolidone, potassium ferricyanide, and flavin adenine dinucleotide significantly decreased activity. The half-saturation constant (Km) for NADH was 0.18 (± 0.05) mM and for nitrate, Km = 0.99 (±0.41) mM. Significant NR activity was detected without the addition of nitrate, suggesting that internal pools of nitrate averaging approximately 20 μmol NO3·g−1 wet weight were present in F. gardneri in February. The distribution of NR activity within the plant was highly variable between individuals, but activities were approximately 5-fold lower in the stipe than in midregions. In plants freshly sampled from the field, NR activity increased 7-fold from February to March, then fell to near-February levels by April. These changes in activity may correspond to seasonal changes in growth rate. The assay, optimized for F. gardneri, was used in several different macroalgal species from different taxa: Porphyra sp., Coralina vancouveriensis Yendo, Ulva sp., Enteromorpha intestinalis (Linnaeus) Nees, Macrocystis integrifolia Bory; and Costaria costatum (C. Agardh) Saunders. For all species tested, NR activity was detectable and, except for one species (Porphya sp.), was equal to or greater than activities measured by other workers using in vivo or in vitro assays for plants under similar conditions.

Item Details

Item Type:Refereed Article
Keywords:seaweed, enzyme assay, nitrate reductase, nitrogen physiology
Research Division:Biological Sciences
Research Group:Plant Biology
Research Field:Phycology (incl. Marine Grasses)
Objective Division:Expanding Knowledge
Objective Group:Expanding Knowledge
Objective Field:Expanding Knowledge in the Biological Sciences
Author:Hurd, CL (Associate Professor Catriona Hurd)
ID Code:93159
Year Published:1995
Web of Science® Times Cited:31
Deposited By:IMAS Research and Education Centre
Deposited On:2014-07-14
Last Modified:2014-08-01
Downloads:0

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