Jochem F J, Smith G J, Gao Y, Zimmerman R C, Cabello-Pasini A, Kohrs D G, Alberte R S
The University of Texas at Austin, Marine Science Institute, Port Aransas, Texas 78373, USA.
Cytometry. 2000 Mar 1;39(3):173-8.
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.
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.
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.
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.
浮游植物对硝酸盐的摄取是生物海洋学中的一个核心问题，因为它对初级生产和生物源碳的垂直通量至关重要。硝酸还原酶催化硝酸盐同化的第一步，即将NO(3)还原为NO(2)。本文介绍了一种用于检测和量化海洋中心硅藻中肋骨条藻硝酸还原酶（NR）蛋白含量相对变化的细胞计数方法。
用针对中肋骨条藻NR产生的多克隆抗体对NR蛋白进行免疫标记。比较了对NR蛋白亚基和NR多肽序列具有特异性的抗血清，并将NR蛋白丰度的细胞计数结果与蛋白质免疫印迹分析结果相关联。在一个上升流模拟实验中，跟踪中肋骨条藻细胞NR丰度和活性的变化，该实验中中肋骨条藻从以氨为主要氮源转变为以硝酸盐为主要氮源。
在以NO(3)培养的细胞中可以检测到NR蛋白，而在以NH(4)培养的细胞中，通过蛋白质免疫印迹密度测定法只能检测到极低水平、几乎难以分辨的NR蛋白。该方法能够观察到上升流模拟过程中NR诱导的早期阶段。营养物质转变后，NR丰度增加，96小时后达到新的生理“稳态”。NR活性呈现出日变化，在中午达到最大值。通过流式细胞术和蛋白质免疫印迹分析估计的NR丰度与NR活性呈双曲线关系。这种模式表明NR蛋白存在翻译后激活。
本文介绍的方法能够区分以NH(4)和NO(3)培养的藻类，以及监测硝酸盐同化能力诱导的早期阶段。
