Colorimetric assay for cellular transglutaminase.
A colorimetric assay for cellular transglutaminase using 5-(biotinamido)pentylamine and polyvinylidine difluoride membranes for crude cellular preparations and purified enzyme has been developed. The biotinpentylamine substrate was incorporated into N,N-dimethylcasein by transglutaminase, the biotinylated products were adsorbed onto the membrane disks and conjugated with streptavidin-beta-galactosidase, and the absorbance resulting from the formation of p-nitrophenol from hydrolysis of p-nitrophenyl-beta-D-galactopyranoside was measured at 405 nm. The validity of the assay was established by showing a good correlation, gamma = 0.922, between the colorimetric procedure and the commonly used radiometric filter paper method for the enzyme. The procedure offers a rapid, sensitive, and nonisotopic method for the estimation of cellular transglutaminase activity in as low as 20 ng of purified guinea pig liver transglutaminase and 10 micrograms of crude fibroblast cytosol protein.
Jeon
W M
WM
Biomedical Division, Samuel Roberts Noble Foundation, Inc., Ardmore, Oklahoma 73402.
Lee
K N
KN
Birckbichler
P J
PJ
Conway
E
E
Patterson
M K
MK
D000818:Animals; D002457:Cell Extracts; D002478:Cells, Cultured / Q000201:enzymology; D003124:Colorimetry / Q000379:methods*; D006168:Guinea Pigs; D006801:Humans; D008297:Male; D008567:Membranes, Artificial; D051381:Rats; D011919:Rats, Inbred Strains; D011503:Transglutaminases / Q000032:analysis*
D002457:Cell Extracts; D008567:Membranes, Artificial; D011503:Transglutaminases