Characterization of a monoclonal antibody that specifically inhibits pullulanase activity of Bacillus circulans amylase-pullulanase enzyme.
A monoclonal antibody (MAb) against amylase pullulanase enzyme from Bacillus circulans, which hydrolyzes not only the alpha-1,6-glycosidic linkage but also the alpha-1,4-glycosidic linkage to the same extent, has been produced by the fusion of BALB/c mouse spleen cells immunized with the native enzyme and P3x63Ag8U1 myeloma cells, and examined for inhibition of pullulanase activity in order to characterize the catalytic site of the pullulanase. The MAb recognizes active enzyme, but not the SDS-denatured or heat-inactivated protein, indicating that the antibody is highly conformational-dependent, specific for active enzyme. The antibody inhibited the pullulanase activity, but not amylase activity. The monoclonal antibody immunoblotted the enzyme and immunoprecipitated the enzyme. The immunoprecipitation was inhibited in the presence of substrate, pullulan, and the MAb competitively inhibited the binding of pullulan to the enzyme. The MAb, therefore, recognizes the pullulan-binding site of the enzyme. Kinetic analysis showed that the MAb inhibited pullulanase activity with inhibition constant (Ki) of 0.77 microgram/mL, providing evidence that the antibody decreases the catalytic rate of enzyme activity and has an effect on substrate binding. These results strongly confirm the previous observations that APE may have two different active sites responsible for the expression of amylase and pullulanase activities (Kim, C.H. and Kim, Y.S. Eur. J. Biochem. 1995, 227, 687-693).
Kim
C H
CH
Department of Biochemistry and Molecular Biology, DongGuk University, Kyungpook, Korea. Chkimbio@email.dongguk.ac.kr
Lee
T K
TK
D000681:Amylases / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D005260:Female; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D011233:Precipitin Tests
D000911:Antibodies, Monoclonal; D000681:Amylases; D006026:Glycoside Hydrolases; C100181:pullulanase