HEAT-TREATMENT COULD AFFECT THE BIOCHEMICAL-PROPERTIES OF CALDESMON

Smooth muscle caldesmon (CaD) exhibits apparent heat stability, A wide ly used purification procedure of CaD involves extensive heat treatmen t (Bretscher, A. (1984) J. Biol. Chem. 259, 12873-12880). CaD thus pur ified co-sediments with actin, inhibits actomyosin ATPase activity, an d interacts with Ca2+/calmodulin, similarly to the unheated protein, O n the other hand, heat-treated CaD binds to actin filaments in a tethe r-like fashion, whereas lengthwise binding dominates in vivo (Mabuchi, K., Lin, J. J.-C., and Wang, C.-L. A. (1993) J. Muscle Res. Cell Moti l. 14, 54-64), suggesting that differences do exist between heat purif ied CaD and the native protein, We have isolated, without heat treatme nt, full-length recombinant chicken gizzard CaD overexpressed in insec t cells (High-Five(TM)) using a baculovirus expression system, We foun d that such unheated CaD interacts with calmodulin 10 times stronger t han does the heated CaD; its inhibitory action on actomyosin ATPase is reversed by a much lesser amount of calmodulin. Moreover, electron mi croscopic examination indicated that actin binding at the N-terminal r egion is more frequent in the unheated CaD, resulting in more lengthwi se binding. These findings point to the fact that CaD is not entirely heat-stable; the C-terminal CaM-binding regions and the N-terminal act in binding region are possibly affected by heat treatment.