INTERACTION OF CALCYCLIN AND ITS CYANOGEN-BROMIDE FRAGMENTS WITH ANNEXIN-II AND GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

The structural properties of calcyclin protein are quite well characte rized but its function remains obscure. To help elucidate the biologic al role of calcyclin we have performed the in vitro studies of the Ca2 +-dependent interaction of Ehrlich ascites tumor cells calcyclin and i ts cyanogen bromide fragments with two potential calcyclin targets: an nexin II and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), The bin ding of annexin II, evidenced by the reaction with I-125-calcyclin, wa s found to be very weak and occurred only for intact calcyclin. On the other hand the interaction between calcyclin and GAPDH was of high af finity and could be assigned to the N-terminal region of calcyclin. In tact calcyclin and its N-terminal fragment bound to GAPDH in the gel o verlay and affinity chromatography assay. When examined in the presenc e of a crosslinking agent the interaction resulted in the formation of 46K covalent adduct between calcyclin monomer and GAPDH subunit. Fluo rescence of 5-iodoacetamido-fluorescein-labelled calcyclin was efficie ntly quenched by GAPDH in the presence of Ca2+, Titration experiments revealed the stoichiometry of one calcyclin monomer binding to each of GAPDH subunits with a binding constant of 10(8) M(-1). The results of this work suggest that the binding between calcyclin and GAPDH may ha ve bearing on calcyclin function.