Potential for ambiguity in the interpretation of immunochemical studies on multienzymic proteins. The case of uridylate synthase.
Rabbit antibodies directed against homogeneous uridylate synthase multienzyme from mouse Ehrlich ascites carcinoma precipitate both the orotidine-5'-monophosphate decarboxylase (EC 4.1.1.23) and orotate phosphoribosyltransferase (EC 2.4.2.10) activities of mouse and human erythrocyte uridylate synthase. When the partially purified human enzyme is used as antigen the two activities coprecipitate with the same apparent titer; however, when the mouse carcinoma protein was studied under the same conditions the decarboxylase activity immunoprecipitated with significantly higher avidity than did the transferase activity. Since the mouse multienzyme has been shown to be a single polypeptide that contains both activities (McClard, R.W., Black, M.J., Livingstone, L.R. and Jones, M.E. (1980) Biochemistry 19, 4699-4706), these results were, at face value, surprising. However, when the mouse orotate phosphoribosyltransferase activity (which is largely lost upon dilution into the immunoassay medium) was stabilized with 5-phosphoribosyl 1-pyrophosphate, both enzyme activities displayed the same apparent antibody titer. The immunochemical studies indicate that the antibodies, as a population, preferentially bind to a form or forms of the enzyme which contain(s) denatured transferase domains. A calculation based on a simple model yields a value of approximately 100 for the relative selectivity of the antibody for the denatured form of uridylate synthase. These results illustrate an ambiguity that is inherent in the interpretation of immunochemical studies on such multienzymic proteins; that is, it is possible to conclude incorrectly that two enzyme activities are not functionally associated if one of the catalytic domains is particularly unstable and thereby displays greater immunoreactivity for the specific antiserum.
D000818:Animals; D000936:Antigen-Antibody Complex; D002262:Carboxy-Lyases / Q000276:immunology*; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology; D000939:Epitopes / Q000032:analysis*; D007106:Immune Sera; D007700:Kinetics; D051379:Mice; D009097:Multienzyme Complexes / Q000276:immunology* / Q000378:metabolism; D009962:Orotate Phosphoribosyltransferase / Q000276:immunology* / Q000378:metabolism; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000276:immunology* / Q000378:metabolism; D010430:Pentosyltransferases / Q000276:immunology*
D000936:Antigen-Antibody Complex; D000939:Epitopes; D007106:Immune Sera; D009097:Multienzyme Complexes; C027157:uridine 5'-monophosphate synthase; D010430:Pentosyltransferases; D009962:Orotate Phosphoribosyltransferase; D002262:Carboxy-Lyases; D009964:Orotidine-5'-Phosphate Decarboxylase