Antiamylase-pullulanase enzyme monoclonals which specifically inhibit amylase or pullulanase activity.
Monoclonal antibodies against amylase-pullulanase enzyme from Bacillus circulans F-2 have been produced to locate and characterize the catalytic sites of the enzyme. The antibodies have been examined for inhibition of both enzyme activities of amylase and pullulanase and then classified into four types: Type I which inhibited amylase activity, Type II which inhibited pullulanase activity, Type III which inhibited both enzyme activities, and Type IV which had no effect on either enzyme activity. Only two monoclonal antibodies (MAP-12 and MAP-17) as Type I and two antibodies (MAP-3 and MAP-5) as Type II were isolated. The inhibitory activities of the antibodies were characterized and compared. In Type II antibodies, the maximal demonstrated inhibition on the pullulanase activity was 88% for MAP-3 with 1 microg of antibody and 90% for MAP-5 with 2 microg of antibody, but did not inhibit the amylase activity. In Type I antibodies, in contrast, the maximal demonstrated inhibition on the amylase activity was 94% for MAP-12 and 97% for MAP-17 with 1 microg of antibody, respectively, but no inhibition of the pullulanase was noted. MAP-12 recognized sequential epitope, while MAP-17 recognized conformation-dependent epitope of amylase activity-related regions. However, both MAP-3 and MAP-5 recognized the conformation-dependent epitope of the pullulanase activity-related region. Furthermore, the antibodies of MAP-3, MAP-5, MAP-12, and MAP-17 did not compete with one another for binding to the enzyme, indicating that they have different target epitopes on the enzyme. Antibody binding of MAP-12 and MAP-17 to the enzyme was not specifically affected by any of the antiamylase compounds tested: (a) nojirimycin; and (b) 1-deoxynojirimycin. Kinetic analysis of their effects provides evidence that both antibodies of MAP-12 and MAP-17 decrease the catalytic rate of enzyme activity and have little or no effect on substrate binding.
Kim
Cheorl-Ho
CH
Department of Biochemistry and Molecular Biology, Dongguk University COM, National Research Laboratory for Glycobiology, Korean Ministry of Science and Technology, Sukjang-Dong 707, Kyung-Ju City, Kyungbuk 780-714, Republic of Korea. chkimbio@dongguk.ac.kr
Chang
Young-Chae
YC
Lee
Young-Choon
YC
D000681:Amylases / Q000037:antagonists & inhibitors / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000276:immunology* / Q000378:metabolism; D005260:Female; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000276:immunology*; D007700:Kinetics; D051379:Mice
D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D000681:Amylases; D006026:Glycoside Hydrolases; C100181:pullulanase