Characterization of plasma membrane-bound Fe3+-chelate reductase from Fe-deficient and Fe-sufficient cucumber roots

In vivo Fe3+-chelate reductase (FeR) activity in cucumber roots (Cucumis sativus L,) increased by transferring the plants from Fe-sufficient (+Fe) conditions to Fe-deficient (-Fe) conditions, This increase was inhibited by the protein synthesis inhibitor, cycloheximide, The plasma membranes were isolated from the +Fe and -Fe roots and then the enzymatic properties of plasma membrane-bound FeR were characterized, The FeR in the plasma membranes from both the +Fe and -Fe roots reduced Fe3+-citrate using NADH as an electron donor in the presence of Triton X-100, Plasma membrane-bound FeR from both types of roots showed similar K-m values for Fe3+-citrate and NADH at 70 and 100 mu M, respectively, whereas V-max of the enzyme from -Fe roots was three-fold higher than that of the enzyme from +Fe roots, The enzyme was solubilized from plasma membranes with 1.0% Triton X-100 and subsequently analyzed on an isoelectric focusing gel, The activity staining of the gel after electrophoresis showed that four FeR isozymes with different pls of 5.3, 6.8, 7.5, and 8.7 were present on the plasma membranes of both the +Fe and -Fe roots, Only the intensity of the pi 7.5 band was enhanced in the -Fe roots, These results suggested that the increase of FeR activity in the -Fe roots resulted from the increased synthesis of the FeR isoform which is constitutively present in the plasma membranes of +Fe roots.