Biological and immunological activity of fructose 1,6-bisphosphatase. Effect of structural changes on the quantitative displacement radioimmunoassay of fructose 1,6-bisphosphatase.

Abstract

The relation between the catalytic activity in the bioassay and the immunologic activity in the immuno‐displacement assay of fructose 1,6‐bisphosphatase was investigated. After biological inactivation by long‐time exposure to room temperature or by treatment with 1% acetic acid, the enzyme could still be quantitatively determined by the immunoassay. Biological inactivation and structural changes were demonstrated after treatment with 4 M and 8 M urea as well as 5 M guanidine hydrochloride solution. The sedimentation coefficient dropped from 7.5 S to about 2 S, and high‐speed sedimentation equilibrium runs yielded a molecular weight of 35000 and 33000 for the enzyme denatured in 8 M urea and 5 M guanidine hydrochloride, respectively, which shows that the enzyme is dissociated into subunits prior to the incubation with the antiserum. Measurements of optical rotatory dispersion revealed a pronounced change of the absorption trough at 233 nm compared to the native enzyme. In the radioimmunoassay fructose 1,6‐bisphosphatase denatured by urea and guanidine hydrochloride followed the standard curve of the native enzyme very closely. In contrast, that inactivated and denatured by 5% sodium dodecylsulfate did not displace 135I‐labelled fructose 1,6‐bisphosphatase from the antibody.A sensitive standard curve for the bisphosphatase was also obtained in the presence of 4 M urea in the assay medium, while higher concentrations interfered.The data indicate that there is no correlation between the catalytic activity and the capability to displace 126I‐labelled fructose 1,6‐bisphosphatase from the antibody. It can be concluded that catalytically inactive enzymes can be quantitatively determined by a specific radioimmuno assay; however, this technique is insensitive to detect conformational changes in the structure of the enzyme.