Abstract
A NAD(P) reductase-like protein with a molecular mass of 34.146 ± 34 Da was purified to homogeneity from the appendix of the inflorescence of the Sauromatum guttatum. On-line liquid chromatography/electrospray ionization-mass spectrometry was used to isolate and quantify the protein. For the identification of the protein, liquid chromatography/electrospray ionization-tandem mass spectrometry analysis of tryptic digests of the protein was carried out. The acquired mass spectra were used for database searching, which led to the identification of a single tryptic peptide. The 12 amino acid tryptic peptide (FLPSEFGNDVDR) was found to be identical to amino acid residues at the positions 108–120 of isoflavone reductase in the Arabidopsis genome. A BLAST search identified this sequence region as unique and specific to a class of NAD(P)-dependent reductases involved in phenylpropanoid biosynthesis. Edman degradation revealed that the protein was N-terminally blocked. The amount of the protein (termed RL, NAD(P) reductase-like protein) increased 60-fold from D-4 (4 days before inflorescence-opening, designated as D-day) to D-Day, and declined the following day, when heat-production ceased. When salicylic acid, the endogenous trigger of heat-production in the Sauromatum appendix, was applied to premature appendices, a fivefold decrease in the amount of RL was detected in the treated section relative to the non-treated section. About 40 % of RL was found in the cytoplasm. Another 30 % was detected in Percoll-purified mitochondria and the rest, about 30 % was associated with a low speed centrifugation pellet due to nuclei and amyloplast localization. RL was also found in other thermogenic plants and detected in Arabidopsis leaves. The function of RL in thermogenic and non-thermogenic plants requires further investigation.
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Abbreviations
- ACN:
-
Acetonitrile
- D-Day:
-
The day of inflorescence opening and heat-production
- IP:
-
Isopropanol
- ESI:
-
Electrospray ionization
- LC:
-
Liquid chromatography
- MaxEnt:
-
Maximum entropy software for deconvolution of multiply charged electrospray envelopes
- MS:
-
Mass spectrometry
- NIPIA:
-
N-isopropyl iodoacetamide
- OBs:
-
Oil bodies
- ODs:
-
Osmiophilic deposits
- PVP:
-
Polyvinylpyrrolidone
- RL:
-
NAD(P) reductase-like protein
- RP-HPLC:
-
Reversed-phase high-performance liquid chromatography
- RuBisCO:
-
Ribulose-1,5-bisphosphate carboxylase oxygenase
- SA:
-
Salicylic acid
- SDS-PAGE:
-
SDS-polyacrylamide gel electrophoresis
- TIC:
-
Total ion current
- TFA:
-
Trifluoroacetic acid
- TPCK:
-
L-1-tosylamido-2-phenylethyl chloromethyl ketone
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Acknowledgments
We thank the University of Washington, Department of Medicinal Chemistry, Mass Spectrometry Center for their assistance in the isolation, purification and quantification of RL; and the Harvard Medical School, Department of Genetics, Biopolymers Facility for their assistance in the identification of the tryptic peptide; and the Biomolecular Research Facility at the University of Virginia for their Edman degradation analysis.
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Skubatz, H., Howald, W.N. Purification of a NAD(P) Reductase-Like Protein from the Thermogenic Appendix of the Sauromatum guttatum Inflorescence. Protein J 32, 197–207 (2013). https://doi.org/10.1007/s10930-013-9472-7
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DOI: https://doi.org/10.1007/s10930-013-9472-7