Evidence for complex formation between rabbit lung flavin-containing monooxygenase and calreticulin.
Rabbit lung flavin-containing monooxygenase (FMO, EC 1.14.13.8) was denatured, reduced, carboxymethylated, digested with endoproteinase Glu-C or trypsin, and subjected to mass spectrometric analysis. The amino acid sequences of selected peptides were determined by tandem mass spectrometry. Over 90% of rabbit lung FMO was mapped by liquid secondary ion mass spectrometry (LSIMS). The FMO N-terminal amino acid was found to be N-acetylated, and the N-terminal 23 amino acid peptide contained an FAD binding domain consisting of Gly-X-Gly-X-X-Gly. Another peptide was found to contain a NADP+ binding domain consisting of Gly-X-Gly-X-X-Ala. The mapped and/or sequenced peptides were found to be completely consistent with the peptide sequence deduced from the cDNA data and the previously published gas-phase sequencing data. Further mass spectrometry and protein analytical work unambiguously showed that rabbit lung FMO existed in tight association with a calcium-binding protein, calreticulin. Over 68% of rabbit lung calreticulin was mapped by LSIMS. Tandem mass spectrometric and gas-phase sequencing studies provided direct evidence for the identification of the N-terminal and other rabbit lung calreticulin-derived peptide sequences that were identical to other previously reported calreticulins. The complexation of calreticulin to rabbit lung FMO could account for some of the unusual physical properties of this FMO enzyme form.
Guan
S H
SH
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
Falick
A M
AM
Williams
D E
DE
Cashman
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D037282:Calreticulin; D003432:Cross-Linking Reagents; D005260:Female; D008168:Lung / Q000201:enzymology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009124:Muscle Proteins / Q000737:chemistry / Q000302:isolation & purification; D010105:Oxygenases / Q000737:chemistry* / Q000302:isolation & purification; D010449:Peptide Mapping; D011247:Pregnancy; D011817:Rabbits
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D009124:Muscle Proteins; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)