Abstract
When 35%-acetone extract of spinach chloroplasts was separated by SDS-PAGE, ferredoxin-NADP reductase (FNR) appeared as a single band at a molecular mass of 35 kDa. After the polypeptides on the SDS-PAGE plate were electroblotted onto PVDF membrane, the FNR band was cut out and analyzed for N-terminal structure in a gas-phase protein sequencer. Two different FNR peptides were identified: one with glutamine at its N-terminus (Gln-FNR) and the other with γ-pyroglutamic acid (<Glu-FNR). Next, tightly bound FNR (tFNR) fraction was extracted from chloroplasts with their loosely bound FNR (lFNR) 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 purified Gln-FNR was digested with arginyl endopeptidase for peptide mapping and partial sequence analysis. Primary structure of Gln-FNR differed 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 gave 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 analysis. This larger apparent molecular mass of Gln-FNR could be ascribed to its N-terminal moiety with around 15 amino acid residues protruding outside of a globular FNR molecule.
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Abbreviations
- CBB:
-
Coomassie Brilliant Blue
- dabsyl chloride:
-
(dimethylamino)azobenzenesulfonyl chloride
- FNR:
-
ferredoxin-NADP reductase
- Gln-FNR:
-
FNR with glutamine at N-terminus
- Ile-FNR:
-
with isoleucine at N-terminus
- lFNR:
-
loosely bound FNR
- tFNR:
-
tightly bound FNR
- <Glu-FNR:
-
FNR with γ-pyroglutamic acid at N-terminus
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Sakihama, N., Nishimura, I., Obata, S. et al. Mature ferredoxin-NADP reductase with a glutaminyl residue at N-terminus from spinach chloroplasts. Photosynth Res 46, 323–328 (1995). https://doi.org/10.1007/BF00020447
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DOI: https://doi.org/10.1007/BF00020447