Human erythrocyte transglutaminase: purification and preliminary characterisation.
Erythrocyte transglutaminase was purified by anion-exchange chromatography, size exclusion and affinity chromatography. Homogeneity was achieved by an additional step of HPLC size-exclusion chromatography. The molecular mass of the purified enzyme was calculated to be 65,000 Da by size-exclusion chromatography and sucrose-gradient centrifugation, and 92,000 Da by SDS-PAGE, thus suggesting a high degree of asymmetry. The amino-acid composition of erythrocyte transglutaminase differed substantially from that of the guinea-pig liver enzyme, notably with respect to the number of histidine, cysteine and acidic amino-acid residues. The enzyme has an absolute requirement for divalent cations for activity: calcium, manganese, and the lanthanides terbium and gadolinium activate the enzyme in decreasing order of efficacy, while no activity is displayed in the presence of magnesium. In the presence but not in the absence of calcium ions, the enzyme is rapidly inactivated by N-ethylmaleimide and by diethylpyrocarbonate suggesting that the cation influences the reactivity of amino acids essential for catalysis. When erythrocyte proteins are employed as amine acceptors in the presence of calcium, the erythrocyte transglutaminase appears to preferentially modify membrane-associated proteins, although, in the absence of calcium ions and exogenous amines, it displays a pH-dependent interaction with soluble proteins.
D000596:Amino Acids / Q000032:analysis; D002413:Cations, Divalent; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D011503:Transglutaminases / Q000097:blood* / Q000302:isolation & purification
D000596:Amino Acids; D002413:Cations, Divalent; D011503:Transglutaminases