N. Sakihama et al., MATURE FERREDOXIN NADP REDUCTASE WITH A GLUTAMINYL RESIDUE AT N-TERMINUS FROM SPINACH-CHLOROPLASTS, Photosynthesis research, 46(1-2), 1995, pp. 323-328
When 35%-acetone extract of spinach chloroplasts was separated by SDS-
PAGE, ferredoxin-NADP reductase (FNR) appeared as a single band at a m
olecular mass of 35 kDa. After the polypeptides on the SDS-PAGE plate
were electroblotted onto PVDF membrane, the FNR band was cut out and a
nalyzed for N-terminal structure in a gas-phase protein sequencer. Two
different FNR peptides were identified: one with glutamine at its N-t
erminus (Gln-FNR) and the other with gamma-pyroglutamic acid (<Glu-FNR
). Next, tightly bound FNR (tFNR) fraction was extracted from chloropl
asts with their loosely bound FNR (IFNR) fraction removed in advance.
The tFNR fraction contained Gln-FNR only. The Gln-FNR could be highly
purified by affinity chromatography using a ferredoxin column. The pur
ified Gln-FNR was digested with arginyl endopeptidase for peptide mapp
ing and partial sequence analysis. Primary structure of Gln-FNR differ
ed from that of <Glu-FNR previously reported by Karplus et al. (1984)
only in that it has an unblocked N-terminus. Highly purified Gln-FNR g
ave a molecular mass of 35 kDa in SDS-PAGE analysis, but its apparent
molecular mass was estimated to be 38.5 kDa by gel-filtration HPLC ana
lysis. This larger apparent molecular mass of Gln-FNR could be ascribe
d to its N-terminal moiety with around 15 amino acid residues protrudi
ng outside of a globular FNR molecule.