A simple assay and histochemical localization of transglutaminase activity using a derivative of green fluorescent protein as substrate.
Histidine-tagged green fluorescent protein (His(6)-Xpress-GFP), a widely used fluorescent probe, was found to be a good substrate for transglutaminase, an enzyme that catalyzes covalent crosslinking of proteins. GFP alone did not serve as a substrate but its derivative His(6)-Xpress-GFP was readily crosslinked through the Gln and Lys residues present in the short N-terminal extension (His(6)-Xpress). His(6)-Xpress-GFP was sensitive enough to detect the transglutaminase activity in guinea pig liver homogenates. The fluorescent substrate could also be used for activity staining of transglutaminase on histological tissue sections, and such applications revealed a surprisingly wide distribution of transglutaminase in the body, especially in the extracellular matrices of various tissues, suggesting an important role for transglutaminase in maintaining the integrity of the extracellular matrix and connective tissues by crosslinking its constituent proteins.(J Histochem Cytochem 49:247-258, 2001)
Furutani
Y
Y
Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan.
Kato
A
A
Notoya
M
M
Ghoneim
M A
MA
Hirose
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005109:Extracellular Matrix / Q000201:enzymology; D005629:Frozen Sections; D049452:Green Fluorescent Proteins; D006168:Guinea Pigs; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008297:Male; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014020:Tissue Extracts / Q000378:metabolism; D011503:Transglutaminases / Q000378:metabolism*
D003432:Cross-Linking Reagents; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D014020:Tissue Extracts; D049452:Green Fluorescent Proteins; D011503:Transglutaminases