Structural organization of aldehyde dehydrogenases probed by limited proteolysis.
Incubation of cytosolic and mitochondrial aldehyde dehydrogenases with trypsin or Glu-C protease under native conditions causes a time-dependent loss of dehydrogenase activity and the production of protein fragments. For evaluation of the results, termination of the reactions with a specific protease inhibitor is especially important in the case of the Glu-C protease. Cleavage site determination by SDS/polyacrylamide gel electrophoresis and sequence analysis identified protease-sensitive amino acid residues at two internal regions spanning positions 248-268 (region 1) and 397-399 (region 2) and at positions in the N-terminal segment (region 3). Region 1 encompasses several cleavages and is sensitive to both proteases in both aldehyde dehydrogenases. Further, it is in a conserved segment and correlates with reactive residues and regions ascribed functional roles. It also correlates with exon borders in the corresponding genes. Combined, the results define region 1 as an important and highly accessible segment of the protein. Region 2 is also adjacent to a conserved segment but lacks further correlation with special properties and appears just to represent an accessible region. The internally cleaved subunits retain a tetrameric configuration as calculated from exclusion chromatography and polyacrylamide gel electrophoresis under native conditions, suggesting that the quaternary structure is not dependent on covalently linked domains within the subunits. Furthermore, the fragments can bind to AMP-Sepharose, suggesting that some functional properties are retained within the cleaved tetramers. However, cleavage at position 35 appears to cause a large fragment (36-263) to be released from the tetramer, suggesting a role of an N-terminal segment or arm (at or before region 3) in subunit interactions.
Loomes
K
K
Department of Chemistry I, Karolinska Institutet, Stockholm, Sweden.
Jörnvall
H
H
D000444:Aldehyde Dehydrogenase / Q000737:chemistry*; D000595:Amino Acid Sequence; D002918:Chymotrypsin; D010450:Endopeptidases; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D008930:Mitochondria, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D014357:Trypsin
D010446:Peptide Fragments; D000444:Aldehyde Dehydrogenase; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin