Purification and characterization of rat brain transglutaminase.
Transglutaminase (TGase) catalyzes an acyl-transfer reaction between peptidyl glutamine residues and primary amines including the epsilon-amino group of lysine residues in protein. TGase in the neuronal system has been suggested to be involved in neurotransmitter release, long-term potentiation, and so forth. In order to study the mammalian brain TGase at the molecular level, TGase was purified to apparent homogeneity from Sprague-Dawley rat brain, using DEAE ion exchange, and heparin and alpha-casein affinity column chromatographies. The brain TGase was concentrated 11,400-fold and had a specific activity of 11,000 nmol/h/mg protein. The purified protein migrated on SDS-PAGE to a position corresponding to a molecular size of approximately 75 kDa. The brain TGase activity was Ca(2+)-dependent (EC50 approximately 0.28 mM), and its Km values for putrescine and N,N-dimethylcasein were 0.26 and 0.065 mM, respectively. GTP inhibited the brain enzyme activity 100-fold more potently than ATP did, and the enzyme was photolabeled with [alpha-32P]8-azido-GTP, suggesting that the brain TGase is a member of the GTP-binding protein family. Monoiodo-acetate and cystamine potently inhibited the enzyme activity, suggesting that cysteine residue(s) are essential for brain TGase. Zn2+ inhibited the enzyme, while Mg2+ was not inhibitory. The rat brain TGase reacted only weakly with monoclonal and polyclonal antibodies against tissue-type TGase such as guinea pig liver TGase and human red blood cell TGase, while it did not react with antibodies against non-tissue types of TGase, namely human epidermal TGase and human coagulation factor XIIIa. The results suggest that rat brain TGase shares major characteristics with tissue-type TGase, but is nevertheless distinct from tissue-type TGase.
Ohashi
H
H
Banyu Tsukuba Research Institute, Ibaraki.
Itoh
Y
Y
Birckbichler
P J
PJ
Takeuchi
Y
Y
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002412:Cations / Q000494:pharmacology; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000201:enzymology; D005973:Glutamine; D006160:Guanosine Triphosphate / Q000378:metabolism; D006168:Guinea Pigs; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008239:Lysine; D008297:Male; D008970:Molecular Weight; D051381:Rats; D017207:Rats, Sprague-Dawley; D011503:Transglutaminases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D000345:Affinity Labels; D002412:Cations; D004791:Enzyme Inhibitors; D005973:Glutamine; D006160:Guanosine Triphosphate; D011503:Transglutaminases; D008239:Lysine