title: Expression of guinea-pig liver transglutaminase cDNA in Escherichia coli. Amino-terminal N alpha-acetyl group is not essential for catalytic function of transglutaminase.
abstract: Transglutaminases (EC 2.3.2.13) catalyze the formation of epsilon-(gamma-glutamyl)lysine cross-links and the substitution of a variety of primary amines for the gamma-carboxamide groups of protein-bound glutamine residues. These enzymes are involved in many biological phenomena. Transglutaminase reactions also have been shown to be suitable for applied enzymology. In this study, as a first step of studies to elucidate the structure/function relationship of transglutaminase, we constructed an expression plasmid, pKTG1, containing a cDNA of guinea-pig liver transglutaminase between the NcoI and PstI sites of an expression vector, pKK233-2, and produced the liver transglutaminase as an unfused protein in Escherichia coli. The purified recombinant enzyme was indistinguishable from natural liver transglutaminase in some structural properties such as molecular mass, amino acid composition, and amino- and carboxyl-terminal sequences. However, the alpha-amino group of the amino-terminal alanine residue of the recombinant transglutaminase was not acetylated as was that of the natural enzyme. Comparison of the recombinant enzyme with the natural one did not indicate significant differences in specific activity and apparent Km values for substrates in the histamine incorporation into acetyl alpha s1-casein. The sensitivity to activation by Ca2+ and the rate of catalyzed protein cross-linking were also similar between recombinant and natural transglutaminases. These results indicated that the N alpha-acetyl group in natural liver transglutaminase has not a particular role in the catalytic function of this enzyme.
authors.affiliation: Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Ikura
authors.forename: Y
authors.initials: Y
authors.lastname: Tsuchiya
authors.forename: R
authors.initials: R
authors.lastname: Sasaki
authors.forename: H
authors.initials: H
authors.lastname: Chiba
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002384:Catalysis; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis / Q000096:biosynthesis*; D004586:Electrophoresis / Q000379:methods; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D006168:Guinea Pigs; D015151:Immunoblotting; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D011503:Transglutaminases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000502:physiology
chemical_list: D000596:Amino Acids; D011994:Recombinant Proteins; D004247:DNA; D011503:Transglutaminases