摘要
AIM To study the relationship between insulinA chain regions and insulin biological activities,we designed a series of insulin analogues withchanges at A21,A12-18 of C-terminal helicalregion and A8-10 Iocated in the region of A6-A11intra-chain disulphide bond.METHODS Insulin A-chain analogues wereprepared by stepwise Fmoc solid-phase manualsynthesis and then combined with natural B-chain of porcine insulin to yield correspondinginsulin analogues.Their biological activitieswere tested by receptor binding,mouseconvulsion and immunological assay.RESULTS[A21Ala]Ins retains 70.3% receptorbinding capacity and 60% in vivo biologicalactivity.[DesA13-14,A21Ala]Ins and[DesA12-13-14-15,A21Ala]Ins still have definite biologicalactivity,7.9% and 4.0% receptor binding,and6.2% and 3.3% in vivo biological activityrespectively.[A15Asn,A17Pro,A21Ala]Insmaintains 10.4% receptor binding and 10% invivo biological activity.[A8His,A9Arg,A10Pro,A21Ala]Ins,[A8His,A9Lys,A10Pro,A21Ala]Insand [A8His,A9Lys,A10Arg,A21Ala]Ins have51.9%,44.3% and 32.1% receptor bindingrespectively,50%,40% and 30% in vivobiological activity respectively,and 28.8%,29.6% and 15.4% immunological activityrespectively. CONCLUSION A21Asn can be replaced bysimple amino acid residues.The A chains withgradually damaged structural integrity in A12-18helical region and the demolition of the A12-18helical region by the substitution of Pro and Asnfor A17Glu and A15Gln respectively can combinewith the B chain and the combination productsshow definite biological activity,the helicalstructure of A12-18 is essential for biologicalactivities of insulin.A8-10 is not muchconcerned with biological activities,but is muchmore important antigenically in binding to itsantibodies,these results may help us design anew type of insulin analogue molecule.
AIM:To study the relationship between insulin A chain regions and insulin biological activities, we designed a series of insulin analogues with changes at A21, A12-18 of C terminal helical region and A8-10 located in the region of A6-A11 intra-chain disulphide bond.METHODS:Insulin A-chain analogues were prepared by stepwise Fmoc solid phase manual synthesis and then combined with natural B-chain of porcine insulin to yield corresponding insulin analogues. Their biological activities were tested by receptor binding, mouse convulsion and immunological assay.RESULTS: A21Ala Ins retains 70.3% receptor binding capacity and 60% in vivo biological activity.DesA13-14, A21Ala Ins and DesA12-13-14-15, A21Ala Ins still have definite biological activity,7.9% and 4.0% receptor binding,and 6.2% and 3.3% in vivo biological activity respectively. A15Asn, A17Pro, A21Ala Ins maintains 10.4% receptor binding and 10% in vivo biological activity. A8His, A9Arg, A10Pro, A21Ala Ins, A8His, A9Lys, A10Pro, A21Ala Ins and A8His, A9Lys, A10Arg, A21Ala Ins have 51.9%, 44.3% and 32.1% receptor binding respectively,50%, 40% and 30% in vivo biological activity respectively, and 28.8%, 29.6% and 15.4% immunological activity respectively.CONCLUSION:A21Asn can be replaced by simple amino acid residues.The A chains with gradually damaged structural integrity in A12-18 helical region and the demolition of the A12-18 helical region by the substitution of Pro and Asn for A17Glu and A15Gln respectively can combine with the B chain and the combination products show definite biological activity, the helical structure of A12-18 is essential for biological activities of insulin. A8-10 is not much concerned with biological activities, but is much more important antigenically in binding to its antibodies, these results may help us design a new type of insulin analogue molecule.
基金
the"Eighth Five Year Plan"Key Research Project,No.KS 852017
National Natural Science Foundation of China.No.3880193,NO.39270157,No.39700028
Chinese Academy of Sciences,KJ951-B1-606.
