Cloning and characterization of human colon glyoxalase-I.
Glyoxalase-I cDNA clones were isolated from a human colon cDNA library using polyclonal antibodies raised against the protein purified from human colon tissue. Positive clones were purified, subcloned, and their nucleotide sequence determined. The glyoxalase-I cDNA encodes a 184-amino acid protein with a predicted molecular weight of 20,774, corresponding to the monomeric subunit weight of the purified protein from human colon glyoxalase-I. The human enzyme showed 51% homology at the nucleotide level and 42% at the amino acid level with bacterial glyoxalase-I. Transfection of COS-1 cells with the 622-base pair cDNA containing the entire coding region cloned into a pMT2 vector produced an immunoreactive protein and an approximate 180-fold increase in glyoxalase-I enzyme activity as determined with methylglyoxal as a substrate. Transfection of a truncated cDNA lacking 94 base pairs of the 5'-coding sequence also produced an approximately 15-kDa immunoreactive protein, but with no detectable increase in enzyme activity. Northern analysis of the RNA showed an approximately 12-fold increase of the 2.2-kilobase glyoxalase-I transcript in carcinoma when compared to normal colon tissue from the same patient. Examination of colon carcinomas for the amplification of the glyoxalase-I gene by Southern blot analysis revealed no change in gene copy number. These results suggest induction of the glyoxalase-I gene expression in colon carcinomas.
Ranganathan
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S
Department of Pharmacology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Walsh
E S
ES
Godwin
A K
AK
Tew
K D
KD
D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003106:Colon / Q000201:enzymology*; D004247:DNA; D006801:Humans; D007791:Lactoylglutathione Lyase / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D004247:DNA; D007791:Lactoylglutathione Lyase