Methodological Aspects of Protein-Binding Assays for Cyclic AMP in Human Adipose Tissue

Abstract

The specificity of the protein-binding assay for cyclic AMP according to Gilman's method (11) was studied in human adipose tissue. A kit assay obtained from Boehringer-Mannheim was used for the study. Impurities in the solvents used for homogenization and extraction influenced the assay and led to serious overestimation of the actual amount of cAMP. This was corrected for by treating the standard and tissue samples in the same manner. Extracts from human adipose tissue contained material that increased the binding of cAMP to the binding protein and led to underestimation of intracellular cAMP by 60–70 per cent. This was overcome by using a protein kinase inhibitor in the assay. Furthermore, when human adipose tissue was incubated in an albumin-containing medium, intracellular cAMP was underestimated by 30 per cent, since albumin increased the binding of cAMP to the binding protein.

Key Words:

• Cyclic AMP
• human adipose tissue
• protein-binding assay