The purification and properties of ferredoxin-NADP(+)-oxidoreductase from roots of Pisum sativum L.
A ferredoxin-NADP(+)-oxidoreductase (FNR) was purified to homogeneity from pea root plastids to a specific activity of 200 nkat.mg protein-1, following acetone precipitation and ferredoxin affinity chromatography. The molecular weight of the enzyme was estimated to be 36,000 and 33,800 by SDS-polyacrylamide gel electrophoresis and molecular exclusion chromatography, respectively. The absorption spectrum of the enzyme suggests it contains flavin as a prosthetic group. The enzyme requires NADPH and did not use NADH as an electron donor. The Km values for NADPH and ferredoxin were calculated to be 28 and 5 microM, respectively. The enzyme exhibited optimal activity at pH 8.0. Although resembling the leaf enzyme in most properties, amino terminal sequencing demonstrates clear differences between the leaf and root proteins and suggests closer homology of the pea root enzyme with the enzyme from spinach roots. A polyclonal antibody against the pea root plastid enzyme was raised by the immunization of rabbits. Judging by immunodiffusion only partial identity was observed between the root plastid and chloroplast FNR. The root plastid FNR enzyme activity was precipitated with increasing concentrations of the antibody, in contrast to the chloroplast enzyme which was not inhibited. The potential usefulness of these antibodies is discussed.
Bowsher
C G
CG
Department of Cell and Structural Biology, University of Manchester, United Kingdom.
Dunbar
B
B
Emes
M J
MJ
D000595:Amino Acid Sequence; D003429:Cross Reactions; D003574:Cytochrome c Group / Q000378:metabolism; D007887:Fabaceae / Q000201:enzymology*; D005287:Ferredoxin-NADP Reductase / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D005288:Ferredoxins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D010946:Plants, Medicinal; D018087:Plastids / Q000201:enzymology*; D011233:Precipitin Tests; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D003574:Cytochrome c Group; D005288:Ferredoxins; D009249:NADP; D005287:Ferredoxin-NADP Reductase