THE EFFECT OF PLACEMENT OF TRYPTOPHAN RESIDUES IN SELECTED A-CHAIN POSITIONS ON THE BIOLOGICAL PROFILE OF INSULIN

in continuation of our efforts to study the solution structure and con formational dynamics of insulin by time-resolved fluorescence spectros copy, we have synthesized and examined the biological activity of five insulin analogues in which selected naturally occurring residues in t he A-chain have been replaced with the strongly fluorescent tryptophan residue. The potency of these analogues was evaluated in lipogenesis assays in isolated rat adipocytes, in receptor binding assays using ra t liver plasma membranes, and in two cases, in receptor binding assays using adipocytes. [A3 Trp]insulin displays a potency of 3% in recepto r binding assays in both liver membranes and in adipocytes, but only 0 .06% in lipogenesis assays as compared to porcine insulin. [A10 Trp]in sulin displays a potency of ca. 40% and ca. 25% in rat liver receptor binding and lipogenesis assays, respectively. [A13 Trp]insulin display s a potency of ca. 39% in rat liver receptor binding assays, but only ca. 9% in receptor binding in adipocytes; in lipogenesis assays, [A13 Trp] insulin displays a potency of ca. 12%, comparable to its potency in adipocyte receptor binding assays. [A15 Trp]insulin exhibits a pote ncy of 18% and 9% in rat liver receptor binding and lipogenesis assays , respectively. The doubly substituted analogue, [A14 Trp, A19 Trp] in sulin, displays a potency of ca. 0.7% in both rat liver receptor bindi ng assays and lipogenesis assays. These data suggest two major conclus ions: (1) the A3 and A15 residues lie in sensitive regions in the insu lin molecule, and structural modifications at these positions have del eterious effects on biological activity of the hormone; and (2) [A13 T rp]insulin appears to be a unique case in which an insulin analogue ex hibits a higher potency when assayed in liver tissue than when assayed in fat cells.