title: Cytometric quantification of nitrate reductase by immunolabeling in the marine diatom Skeletonema costatum.
abstract: BACKGROUND
  The uptake of nitrate by phytoplankton is a central issue in biological oceanography due to its importance to primary production and vertical flux of biogenic carbon. Nitrate reductase catalyzes the first step of nitrate assimilation, the reduction of NO(3) to NO(2). A cytometric protocol to detect and quantify relative changes in nitrate reductase (NR) protein content of the marine centric diatom Skeletonema costatum is presented.
  
  
  METHODS
  Immunolabeling of NR protein was achieved with polyclonal antibodies raised against S.costatum NR. Antisera specific to a NR protein subunit and to a NR polypeptide sequence were compared, and cytometric results of NR protein abundance were related to Western analyses. Changes in cellular NR abundance and activity were followed during an upwelling simulation experiment in which S. costatum was exposed to a shift from ammonia to nitrate as major nitrogen source.
  
  
  RESULTS
  NR protein could be detected in NO(3)-grown cells and at extremely low levels hardly discernible by Western Blot densiometry in NH(4)-grown cells. The protocol allowed observation of early stages of NR induction during an upwelling simulation. NR abundance increased after the nutrient shift to reach a new physiological "steady-state" 96 hrs later. NR activity exhibited diel variation with maxima at mid-day. NR abundance as estimated by both flow cytometry and Western analysis exhibited a hyperbolic relationship to NR activity. This pattern suggests post-translational activation of NR protein.
  
  
  CONCLUSIONS
  The presented protocol allows the differentiation of NH(4)- versus NO(3)-grown algae as well as the monitoring of early stages in the induction of nitrate assimilatory capacities.
authors.affiliation: The University of Texas at Austin, Marine Science Institute, Port Aransas, Texas 78373, USA.
authors.forename: F J
authors.initials: FJ
authors.lastname: Jochem
authors.forename: G J
authors.initials: GJ
authors.lastname: Smith
authors.forename: Y
authors.initials: Y
authors.lastname: Gao
authors.forename: R C
authors.initials: RC
authors.lastname: Zimmerman
authors.forename: A
authors.initials: A
authors.lastname: Cabello-Pasini
authors.forename: D G
authors.initials: DG
authors.lastname: Kohrs
authors.forename: R S
authors.initials: RS
authors.lastname: Alberte
mesh_terms: D017377:Diatoms / Q000166:cytology / Q000201:enzymology*; D005455:Fluorescent Antibody Technique; D007700:Kinetics; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000032:analysis / Q000378:metabolism*; D012623:Seawater
chemical_list: D009565:Nitrate Reductases; D050901:Nitrate Reductase