Partial purification and some properties of gamma-glutamyl peptide-hydrolysing enzyme from Actinobacillus actinomycetemcomitans.
A gamma-glutamyl peptide-hydrolysing enzyme was partially purified from Actinobacillus actinomycetemcomitans. The enzyme required metal ions, and 1 to 2 mM Mn2+ ions, especially, were essential for hydrolytic reaction. Its distribution by treatment of cells with lysozyme-EDTA suggested that the enzyme was a membrane-bound protein. The pI of the enzyme was in range of pH 5.1 to 5.6, and the apparent Km value for gamma-glutamyl-p-nitroanilide was 3.0 x 10(-4) M. The enzyme hydrolysed specifically gamma-glutamyl residues from the N-terminal of gamma-glutamyl compounds such as gamma-Glu-Met, gamma-Glu-Ala, gamma-Glu-Leu and gamma-Glu-Tyr, but did not catalyse the transpeptidation reaction. Neither free amino acids such as Ala-, Pro-, Gly- and Leu-p-nitroanilide nor alpha-glutamyl derivatives were hydrolysed. Its activity was strongly inactivated by metal chelators (EDTA or o-phenanthroline) and amino acids (Glu, Gln). In addition, the activity was specifically inactivated by gamma-glutamyl affinity-labelling reagents such as AT-125, 6-diazo-5-oxo-L-norleucine and azaserine, which are inhibitors of the gamma-glutamyl donor sites of mammalian gamma-glutamyl transpeptidase. Antibodies against bovine kidney gamma-glutamyl transpeptidase decreased the activity of the bacterial enzyme by 65%. These results suggested that the active sites in the bacterial enzyme were similar to those in mammalian gamma-glutamyl transpeptidase.
Mineyama
R
R
Department of Oral Microbiology, School of Dentistry, Nippon Dental University, Niigata, Japan.
Mikami
K
K
Saito
K
K
D016976:Aggregatibacter actinomycetemcomitans / Q000201:enzymology*; D000818:Animals; D002417:Cattle; D006160:Guanosine Triphosphate / Q000276:immunology / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D013379:Substrate Specificity; D005723:gamma-Glutamyltransferase / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
D006160:Guanosine Triphosphate; D005723:gamma-Glutamyltransferase