Proteomic identification of plant proteins probed by mammalian nitric oxide synthase antibodies.
Several studies suggest that a mammalian-like nitric oxide synthase (NOS) exists in plants. Researchers have attempted to verify its presence using two approaches: (i) determination of NOS functional activity and (ii) probing with mammalian NOS antibodies. However, up to now, neither a NOS-like gene nor a protein has been found in plants. While there is still some controversy over whether the NOS functional activity seen is due to nitrate reductase, using the mammalian NOS antibodies in western blot analysis, several groups have reported the presence of immunoreactive protein bands in plant homogenates. Based on these results, immunohistochemical studies using these antibodies have also been used to localize NOS in plant tissues. However, plant NOS has never been positively identified or characterized. Thus, we used a proteomic approach to verify the identities of plant proteins that cross-reacted with the mammalian NOS antibodies. Proteins extracted from maize (Zea mays L.) embryonic axes were separated by two-dimensional gel electrophoresis and subjected to western blot analysis with the mammalian neuronal NOS and inducible NOS antibodies. Twenty immunoreactive protein spots recognized on a corresponding Coomassie blue-stained two-dimensional gel were subjected to tryptic digestion, followed by identification using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Fifteen proteins were successfully identified and they have described functions that are unrelated to NO metabolism. The remaining five proteins could not be identified. The amino acid sequences of these identified proteins and those used to raise the antibodies were aligned. However, no homologous region could be found. Our results demonstrate that the mammalian NOS antibodies recognize many NOS-unrelated plant proteins. Therefore, it is inappropriate to infer the presence of plant NOS using this immunological technique.
Butt
Yoki Kwok-Chu
YK
The Proteomic Task Force, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, China.
Lum
John Hon-Kei
JH
Lo
Samuel Chun-Lap
SC
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D015342:DNA Probes / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured; D003313:Zea mays / Q000737:chemistry / Q000201:enzymology / Q000235:genetics
D000911:Antibodies, Monoclonal; D015342:DNA Probes; D010940:Plant Proteins; D019001:Nitric Oxide Synthase