.1 A Chain Chain Disulfide Wrong Then (a11ser, A12cys) Insulin Mutants, Expression And Activity Of Epimedium Compound Antitumor Activity And The Berbamine Immune Inhibition Studies

The mutant proinsulin was constructed with the condons for A11Cys and A12Ser changed to A11Ser and A12Cys to modify the intra-A chain disulfide bond by the PCR mutagenesis method. After expression,processing and purification, Met-(A11Ser, A12Cys)-human proinsulin (Mut-HPI) was gained. It showed 78.08% radio immuno activity (RIA), but only about 5.2% of receptor binding activity (RBA) as compared with Met-human proinsulin (Met-HPI) which was prepared in the same way. The Met-(A11Ser, A12Cys)-human insulin (Mut-HI) and Met-human insulin (Met-HI) were obtained after trypsin and carboxypeptidase B (CPB) cleavage and FPLC Resource^TM Q anion-exchanged column separation. The amino acid compositions were in good agreement with those of expected. Mut-HI kept 80.2% immuno activity as well, but only 0.1% receptor binding assay (RBA) with Met-HI as control. Mut-HPI was less easily digested by trypsin and CPB than Met-HPI. The mobility of Met-HI on Native-PAGE was the same as that of HI, while Mut-HI displayed lower mobility. The conformational analysis demonstrated that the conformation of Mut-HI was quite different from that of Met-HI. These result indicate that modification of intra-A chain disulfide bond cause some chang on the conformation, especially the changs on the conformation which needed by receptor binding, which greatly decrease the affinity of Mut-HI with its receptor, while the maitenence of most immuno activity of Mut-HI indicate that though the intra-A chain disulfide bond is modified, most of native conformation of human insulin are retained. These indicate that correctly paired intra-A chain disulfide bond seems to be essential for human insulin displaying its biological activity.The inhibitiory effects of compounds isolated from the Epimedium wanshaneme and E. Ikoreanum on the tumor cell lines had been studied by MTT...