IDENTIFICATION OF AMINE ACCEPTOR PROTEIN SUBSTRATES OF TRANSGLUTAMINASE IN LIVER EXTRACTS - USE OF 5-(BIOTINAMIDO) PENTYLAMINE AS A PROBE

Transglutaminase is a calcium-dependent enzyme which catalyzes amine i ncorporation and cross-linking of proteins. To isolate the amine accep tor protein substrates of transglutaminase in mammalian livers, a biot in-labeled primary amine substrate of transglutaminase, 5-(biotinamido ) pentylamine, was used for biotin labeling of proteins in the liver e xtracts by endogenous transglutaminase activity. The biotin-labeled pr oteins were isolated and recovered by biotin-avidin-affinity chromatog raphy. The obtained proteins were separated by SDS-PAGE. Proteins with molecular masses of 15, 24, 35, 40, 44, 93, and 134 kDa were the main components of labeled proteins in mouse liver extract. In rat and gui nea pig liver extracts, 32-, 38-, 40-, 44-, and 134-kDa proteins and 2 8-, 40-, 44-, 55-, 60-, 91-, and 134-kDa proteins were the main compon ents of labeled proteins, respectively. Using amino-terminal amino aci d sequence analyses and sequence homology searches, the 38-kDa protein from rat liver was identified as a subunit of glyceraldehyde-3-phosph ate dehydrogenase (EC 1.2.1.12), and the 28-kDa protein from guinea pi g liver was identified as a subunit of glutathione S-transferase (clas s theta) (EC 2.5.1.18). Both the glyceraldehyde-3-phosphate dehydrogen ase from rabbit muscle and glutathione S-transferase (class pi) from h uman placenta also could be amine accepters in the amine incorporation catalyzed by guinea pig liver transglutaminase. These results suggest that these enzymes can be modified post-translationally by cellular t ransglutaminase. (C) 1998 Academic Press.