Identification of new amine acceptor protein substrate candidates of transglutaminase in rat liver extract: use of 5-(biotinamido) pentylamine as a probe.
Transglutaminases (TGs) are a family of enzymes that catalyze Ca(2+)-dependent post-translational modification of proteins by introducing protein-protein crosslinks (between specific glutamine and lysine residues), amine incorporation, and site-specific deamidation. In this study, new amine acceptor protein substrates of TG were isolated from rat liver extract and identified using 5-(biotinamido) pentylamine, a biotinylated primary amine substrate, as a probe. TG protein substrate candidates labeled with biotin by endogenous TG activity were isolated and recovered by avidin column chromatography. Proteins with molecular masses of 40, 42, and 45 kDa were the main components of the labeled proteins. Determination of their partial amino acid sequences and immunoblotting analyses were done to identify them. The 45-kDa protein was identical with betaine-homocysteine S-methyltransferase (EC 2.2.2.5), which was identified in our previous study. The 40- and 42-kDa proteins were identified as arginase-I (EC 3.5.3.1) and fructose-1,6-bisphosphatase (EC 3.1.3.11) respectively. TG catalyzed incorporation of 5-(biotinamido) pentylamine into both arginase-I and fructose-1,6-bisphosphatase purified from rat liver was confirmed in vitro. These results suggest that these two enzymes are the new protein substrate candidates of TG and that they can be modified post-translationally by cellular TG.
Ichikawa
Akira
A
Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Kyoto 606-8585, Japan.
Ishizaki
Jin
J
Morita
Manabu
M
Tanaka
Kentaro
K
Ikura
Koji
K
D000588:Amines / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001119:Arginase / Q000276:immunology / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D001710:Biotin / Q000031:analogs & derivatives* / Q000378:metabolism; D019921:Biotinylation; D004591:Electrophoresis, Polyacrylamide Gel; D006597:Fructose-Bisphosphatase / Q000276:immunology / Q000378:metabolism; D006168:Guinea Pigs; D015151:Immunoblotting; D008110:Liver Extracts / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011503:Transglutaminases / Q000378:metabolism*
D000588:Amines; D000906:Antibodies; D008110:Liver Extracts; D011506:Proteins; C076022:5-(biotinamido)pentylamine; D001710:Biotin; D011503:Transglutaminases; D006597:Fructose-Bisphosphatase; D001119:Arginase