The NADH-dependent Fe(3+)-chelate reductases of tomato roots.
The NADH-dependent Fe(3+)-chelate reductase (NFCHR) of tomato (Lycopersicon esculentum L.) roots, a strategy I species, was investigated. The Fe(3+)-citrate reductase (FeCitR) assay was strongly inhibited by p-hydroxymercuribenzoic acid (PHMB); moreover, the inhibitor was found to be more specific to the FeCitR assay than to the Fe(3+)-EDTA reductase assay, which was catalyzed by at least another reductase of 46 kDa. After high-speed centrifugation of tomato root membranes, high FeCitR activities were detected in pellets and lower activities in supernatants. After two-phase partitioning of microsomes, FeCitR activity (91 nmol.min-1.mg-1) was less active in the upper phase (plasma membrane) than in the lower phase (277 nmol.min-1.mg-1). However, only the activity of the plasma-membrane-associated NFCHR (FeCitR) was significantly enhanced (2.6-fold) in iron-deficient tomato plants, whereas that of NFCHR in non-plasma-membrane rich fractions was unaffected by this treatment. The NFCHR obtained from lysophosphatidylcholine-solubilized plasma membrane was present as a 200-kDa protein complex following fast protein liquid chromatography on Superdex 200, or as a 28-kDa form following Blue Sepharose CL-6B chromatography. Both preparations were more active following iron starvation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the 28-kDa protein purified from solubilized tomato microsomes or supernatant fractions by a final Mono Q step consisted of a single band of 32 kDa. Tomato root NFCHR resembled the NFCHR of maize (a strategy II plant, P Bagnaresi and P Pupillo, 1995, J Exp Bot 46: 1497-1503) in several properties: relative molecular mass, hydrophilicity, chromatographic behaviour, sensitivity to mercurials, specificity for electron donors and acceptors (e.g. cytochrome c), and a ferricyanide reductase-to-FeCitR ratio of 2.5. Preincubation with NADH partially protected NFCHR from PHMB-induced inactivation. Our data show that strategy I and II plants seem to share similar NFCHR proteins, which appear to belong to the cytochrome b5 reductase flavoprotein group.
Bagnaresi
P
P
Dipartimento di Biologia, Universita'di Bologna, Italy.
Basso
B
B
Pupillo
P
P
D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D038181:FMN Reductase; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007700:Kinetics; D018551:Solanum lycopersicum / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D018517:Plant Roots / Q000201:enzymology
D004791:Enzyme Inhibitors; D006902:Hydroxymercuribenzoates; D009243:NAD; C030601:4-hydroxymercuribenzoate; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases; C023903:ferric citrate iron reductase