Plasmalemma Redox Activity in the Diatom Thalassiosira

Abstract

Plasmalemma redox activity in the diatom Thalassiosira is competitively inhibited by antiserum prepared against algal nitrate reductase (NR), and fluorescent labeling experiments reveal the binding of NR antiserum to the cell surface. Furthermore, the external electron acceptor Cu bathophenanthroline disulfonate causes immediate inhibition of intracellular primary amine production. A model is proposed in which plasmalemmabound nitrate reductase reduces extracellular electron acceptors and intracellular nitrate and also acts as a trans-plasmalemma proton pump.A considerable body of literature has accumulated in the past 10 to 15 years concerning the redox systems of higher plants and animals (6, 7). Plasmalemma redox enzymes can transfer electrons from an internal or, in some cases, an external reductant to artificial electron acceptors such as DCPIP,3 ferricyanide, and Cyt c. Often, transplasmalemma electron transport appears to be accompanied by proton extrusion from the cell (16,22,24). Based on the latter observation, it has been proposed that the plasmalemma redox enzyme(s) may act as an alternate proton pump and form the energetic basis for active transport of nutrients across the plasmalemma (7).Enzymically mediated redox activity at the plasmalemma of eukaryotic phytoplankton has recently been demonstrated in our laboratory (12,13). Redox activity in phytoplankton exhibits saturation kinetics (half-saturation constants in the range 2 to 20 ,UM for Cu [II].phen derivatives) is inhibited by mild heat treatment and membrane-impermeable thiol binding reagents, and the enzyme active site has a standard reduction potential within the range -0.1 to + 0.1 V. We noted (12) (Pharmacia). The purified antiserum was passed through a Sephadex G-25 column, and the collected preparation was used without further purification. In redox inhibition experiments with anti-NR, 25 ,uL crude antiserum or 75 ,uL purified antiserum was added to 1.5 mL culture and incubated for 15 min before commencing the assay measurements. Previous studies using this antiserum (8) showed that purified anti-NR gave only one precipitin band in Ouchterlony assays with Chlorella whole cell extracts and that the band showed identity with that obtained with purified NR. Further tests of cross-reactivity of the crude and purified anti-NR with the diaphorase moiety of cellular redox enzymes were made using a range of redox enzymes purchased from Sigma; these were lipoamide dehydrogenase (type V from Torula yeast), glutathione reductase (type III from baker's yeast), Cyt c reductase (type I from porcine heart), and malic dehydrogenase (from porcine heart mitochondria). Only the diaphorase activity of lipoamide dehydrogenase was inhibited by anti-NR, and this inhibition was strictly non-competitive (data not shown) and thus was not due to anti-NR binding at the metal reduction site of the enzyme.Cells were prepared for fluorescence microscopy by treatment with purified anti-NR as described above. After incubation, the cells were collected by cent...

Nitrate regulation of Fe reduction and transport by Fe‐limited Thalassiosira oceanica

“…The ability of NH 4 ϩ -grown T. oceanica to reduce Fe, although at a slower rate, may imply that they too have low levels of a NR-like redox enzyme in the plasmalemma. Consistent with this hypothesis are observations of the insensitivity to NH 4 ϩ of basal plasmalemma NR activity of NH 4 ϩ -grown cells (Tischner et al 1989;Corzo et al 1991;Jones and Morel 1988).…”

“…Ostensibly, NR reduces NO 3 Ϫ to NO 2 Ϫ , using electrons derived from NAD(P)H (Taiz and Zeiger 1991). In higher plants and algae, both soluble NR (Solomonson and Vennesland 1972;Redinbaugh and Campbell 1983;Castignetti and Smarrelli 1984) and plasmalemma bound NR (Jones et al 1987;Jones and Morel 1988;Corzo et al 1991;Stöhr et al 1993), can catalyze the reduction of Fe bound to organic complexes. In heterotrophic bacteria, mutants lacking NR are deficient in the Fe reductase pathway (Ottow 1968(Ottow , 1970.…”

“…Phytoplankton reduce Fe and Cu bound to organic compounds, including EDTA (Anderson and Morel 1980;Jones et al 1987), by extracellular redox enzymes that chemically resemble NAD(P)H cytochrome c reductases (Jones et al 1987;Jones and Morel 1988). Our results suggest that the Fe reductase of T. oceanica may also be a plasmalemma bound form of NR (or a reductase that is regulated by N metabolic pathways), similar to that proposed for Cu reductase of T. weissflogii (Jones and Morel 1988). As outlined below, support for this model comes from our observations of greater Fe reductase activity in NO 3 Ϫ dependent cells.…”

Linking chlorophyll-nutrient dynamics to the RedÞeld N:C ratio with a model of optimal phytoplankton growth

“…NR is located partly at the plasmalemma and partly in the cytoplasm in Thalassiosira spp. (Jones & Morel 1988), but NR has also been located in chloroplasts of several dinoflagellates and green algae (Fritz et al 1996). Thus, the dependence of ζ on nitrogen source seen in Fig.…”

The role of organic complexation on ambient iron chemistry in the equatorial Pacific Ocean and the response of a mesoscale iron addition experiment

“…This type of reductive activity has been reported by several investigators who suggest it is mediated by one or more plasmalemma redox enzymes (Jones et al 1987;Jones and Morel 1988). These are proteins that span the cell membrane and can transfer electrons from an internal reductant (such as NADH) to an external acceptor.…”