Effect of substrate size on immunoinhibition of amylase activity.
Immunoinhibition assays are hypothesized to work by antibodies blocking substrate access to enzyme active sites. To test this hypothesis, the inhibition of amylase isoenzymes by monoclonal and polyclonal antisera was assessed using substrates of varying sizes: chromogenic sustrates 3, 5, or 7 glucose units in length, novel synthetic macromolecular substrates, and starch. The synthetic macromolecular substrates consisted of small oligosaccharide substrates linked to an inert polymer that conferred a large size to substrate molecules as determined by gel filtration chromatography. When substrate size increased, amylase activity could be inhibited equivalently by antibody concentrations that are 10-fold lower. Progressively less polyclonal serum was required to inhibit amylase activity as substrate length increased from 3 to 5 to 7 glucose units and as size was increased by linkage to a polymer. Different effects of substrate size were observed with two monoclonal antibodies. One monoclonal antibody blocked amylase activity independent of substrate size, while another monoclonal antibody had little inhibitory effect except using starch as substrate. We conclude that use of larger substrates can expand the repertoire of inhibitory epitopes on enzymes and convert a noninhibitory antibody into an inhibitory one.
Warshawsky
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Clinical Pathology Department of the Warren Grant Magnusson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892-1508, USA.
Hortin
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GL
D000681:Amylases / Q000037:antagonists & inhibitors* / Q000276:immunology*; D019138:Antibodies, Blocking / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001666:Binding Sites, Antibody; D001667:Binding, Competitive / Q000276:immunology; D004789:Enzyme Activation / Q000276:immunology; D004791:Enzyme Inhibitors / Q000276:immunology; D006801:Humans; D007106:Immune Sera / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000276:immunology; D046911:Macromolecular Substances; D010179:Pancreas / Q000201:enzymology; D010316:Particle Size; D012469:Salivary Glands / Q000201:enzymology; D013213:Starch / Q000276:immunology / Q000378:metabolism; D013379:Substrate Specificity / Q000276:immunology
D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D007106:Immune Sera; D007527:Isoenzymes; D046911:Macromolecular Substances; D013213:Starch; D000681:Amylases