Using periodate-oxidized nucleotide as affinity label for the nucleotide site of proteins.
The active site of pigeon liver malic enzyme was labeled with a fluorescent affinity label, the periodate-oxidized aminopyridine adenine dinucleotide phosphate. The modified enzyme was subjected to proteolytic digestion with trypsin. The resulted peptides were then separated with reversed-phase high-performance liquid chromatography on Waters muBondapak C18 column. Two pure fluorescent peptides were obtained after three runs of the chromatography. The peptides were then subjected to automatic Edman degradation on a Beckman peptide sequencer and subsequently separated and identified with phenylthiohydantoin C18 column. No sequence was obtained. The possible reasons for the failure in sequencing the periodate-oxidized nucleotides labeled active site peptide and some possible pitfalls in using these reagents were discussed.
Lin
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Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Chang
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D000227:Adenine Nucleotides / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D010856:Columbidae; D005456:Fluorescent Dyes; D008099:Liver / Q000201:enzymology; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D010504:Periodic Acid
D000227:Adenine Nucleotides; D000345:Affinity Labels; D005456:Fluorescent Dyes; D009711:Nucleotides; D010504:Periodic Acid; D009249:NADP; C039995:3-aminopyridine-1,N(6)-ethenoadenine dinucleotide phosphate; C009288:metaperiodate; D008291:Malate Dehydrogenase; C030574:malate dehydrogenase (decarboxylating)