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 incorporation and cross-linking of proteins. To isolate the amine acceptor protein substrates of transglutaminase in mammalian livers, a biotin-labeled primary amine substrate of transglutaminase, 5-(biotinamido) pentylamine, was used for biotin labeling of proteins in the liver extracts by endogenous transglutaminase activity. The biotin-labeled proteins were isolated and recovered by biotin-avidin-affinity chromatography. 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 guinea pig liver extracts, 32-, 38-, 40-, 44-, and 134-kDa proteins and28-, 40-, 44-, 55-, 60-, 91-, and 134-kDa proteins were the main components of labeled proteins, respectively.Using amino-terminal amino acid sequence analyses and sequence homology searches, the 38-kDa protein from rat liver was identified as a subunit of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and the 28-kDa protein from guinea pig liver was identified as a subunit of glutathione S-transferase (class theta) (EC 2.5.1.18). Both the glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle and glutathione S-transferase (class pi) from human placenta also could be amine acceptors in the amine incorporation catalyzed by guinea pig liver transglutaminase. These results suggest that these enzymes can be modified posttranslationally by cellular transglutaminase.
Ikura
K
K
Faculty of Engineering and Design, Kyoto Institute of Technology, Matsugasaki, Kyoto, 606-8585, Japan. ikura@ipc.kit.ac.jp
Kita
K
K
Fujita
I
I
Hashimoto
H
H
Kawabata
N
N
D000588:Amines / Q000378:metabolism*; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives* / Q000378:metabolism; D015153:Blotting, Western; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000302:isolation & purification / Q000378:metabolism; D006168:Guinea Pigs; D006632:Histamine / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008110:Liver Extracts / Q000378:metabolism*; D051379:Mice; D015335:Molecular Probes / Q000378:metabolism*; D009928:Organ Specificity; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*
D000588:Amines; D007527:Isoenzymes; D008110:Liver Extracts; D015335:Molecular Probes; D010446:Peptide Fragments; D011506:Proteins; C076022:5-(biotinamido)pentylamine; D001710:Biotin; D006632:Histamine; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase; C496557:Gstp1 protein, mouse; C496558:Gstp1 protein, rat; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins