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The Receptor Binding and Biological Activity of [B1-Ala, B2-Ala]-insulin
Shang-Quan Zhu1, Xin-Tang Zhang, Ming-Hua Xu
1Shanghai Institute of Biochemistry, Academia Sinica, Shanghai 200031, China.
The receptor binding properties with human placental membrane (HPM) and the in vitro biological activity of an insulin analogue, [B1-Ala, B2-Ala]-insulin were investigated in detail and compared with those of insulin. It was found that the binding of (125)I-[B1-Ala, B2-Ala]-insulin and (125)I-insulin to HPM was time dependent reaching equilibrium after 6min at 37 degrees in the presence of bacitracin with an equilibrium maximum binding of 6.44 fmol/mg protein for [B1-Ala, B2-Ala]-insulin and 3.47 fmol/mg protein for insulin The half time (T1/2) to reach equilibrium was 19 seconds for [B1-Ala, B2-Ala]-insulin and 25 seconds for insulin. [B1-Ala, B2-Ala]-insulin and insulin competed with specific (125)I-[B1-Ala, B2-Ala]-insulin in a dose-dependent manner. From IC(50), the receptor binding activity of [B1-Ala, B2-Ala]-insulin was 139.6% compared with that of insulin. Scatchard analysis revealed that the receptor association constants of [B1-Ala, B2-Ala]-insulin in HPM at 4 degrees were 5.88x10(8)L/mol and 7.63x10(5)L/mol respectively, while those of insulin were 4.83x10(8)L/mol and 3.39x10(5)L/mol respectively. The in vitro activity of [B1-Ala, B2-Ala]-insulin was 130% of that of insulin in terms of lipogenesis in rat adipocytes.
The receptor binding properties with human placental membrane (HPM) and the in vitro biological activity of an insulin analogue, [B1-Ala, B2-Ala]-insulin were investigated in detail and compared with those of insulin. It was found that the binding of (125)I-[B1-Ala, B2-Ala]-insulin and (125)I-insulin to HPM was time dependent reaching equilibrium after 6min at 37 degrees in the presence of bacitracin with an equilibrium maximum binding of 6.44 fmol/mg protein for [B1-Ala, B2-Ala]-insulin and 3.47 fmol/mg protein for insulin The half time (T1/2) to reach equilibrium was 19 seconds for [B1-Ala, B2-Ala]-insulin and 25 seconds for insulin. [B1-Ala, B2-Ala]-insulin and insulin competed with specific (125)I-[B1-Ala, B2-Ala]-insulin in a dose-dependent manner. From IC(50), the receptor binding activity of [B1-Ala, B2-Ala]-insulin was 139.6% compared with that of insulin. Scatchard analysis revealed that the receptor association constants of [B1-Ala, B2-Ala]-insulin in HPM at 4 degrees were 5.88x10(8)L/mol and 7.63x10(5)L/mol respectively, while those of insulin were 4.83x10(8)L/mol and 3.39x10(5)L/mol respectively. The in vitro activity of [B1-Ala, B2-Ala]-insulin was 130% of that of insulin in terms of lipogenesis in rat adipocytes.