Proteolytic degradation of ferredoxin-NADP reductase during purification from spinach.
Ferredoxin-NADP reductase (FNR) was rapidly isolated from spinach leaves with special care to suppress proteolytic degradation. The molecular mass of this FNR preparation was estimated to be 35 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Limited proteolysis of 35-kDa FNR to 33-kDa FNR was effectively suppressed by high pH (at pH 9.3), concentrated salts, and low temperature. On the basis of these observations, a new isolation procedure was designed to obtain 35-kDa FNR in a preparative scale. The resulting final preparation still contained two FNR components. One appeared to correspond to the longest polypeptide so far reported for spinach FNR (Karplus et al., 1984, Biochemistry 23, 6576-6583) while the other lacked a gamma-pyroglutamyl residue from its amino terminus. Conventional preparation procedure without suppression of proteolytic action yielded an FNR preparation with a molecular mass of 33 kDa. This FNR preparation consisted of three components. They lacked 11 to 17 amino-terminal residues, while their carboxyl-terminal structure was retained intact. These results showed that proteolytic degradation of the spinach FNR molecule during purification took place exclusively at its amino-terminal moiety and further suggested that 35-kDa FNR with Karplus' structure should be the mature FNR molecule functional in the chloroplast thylakoids.
Shin
M
M
Department of Biology, Faculty of Science, Kobe University, Japan.
Tsujita
M
M
Tomizawa
H
H
Sakihama
N
N
Kamei
K
K
Oshino
R
R
D000595:Amino Acid Sequence; D002268:Carboxypeptidases / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005287:Ferredoxin-NADP Reductase / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification*; D010447:Peptide Hydrolases / Q000494:pharmacology*; D010944:Plants / Q000187:drug effects / Q000201:enzymology*; D011489:Protein Denaturation
D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases; D002268:Carboxypeptidases; D010447:Peptide Hydrolases; C022633:serine carboxypeptidase