PREPARATION AND COMPARISON OF BIOLOGICAL PROPERTIES OF RECOMBINANT CARP (CYPRINUS-CARPIO) GROWTH-HORMONE AND ITS CYS-123 TO ALA MUTANT

Carp growth hormone (cGH) cDNA, in which Cys-123 was mutated to Ala, w as prepared, transferred to the expression vector, expressed in Escher ichia coli and the mutant was purified to homogeneity. The mutation on ly slightly improved yield of the monomeric fraction, indicating that Cys-123 is not involved in improper refolding. As compared to cGH, the mutant (cGH-C123A) exhibited lower binding affinity toward homologous liver receptors and lower bioactivity in a 3T3-F442A preadipocyte bio assay despite the fact that both hormones exhibited almost identical c ross-reactivity with anti-cGH antibodies. These results, along with th ose of a structural comparison to hGH, suggest that Cys-123 is located in the hydrophobic core of the hormone, and is most likely affecting the conformation of the binding site. Dimeric forms of the hormone and its mutant were less active than their respective monomers. Homologou s binding experiments using a carp liver microsomal fraction revealed a single receptor population with Kd = 0.77 nM and B(max) = 241 fmol/m g microsomal protein.