Allosteric modulation of Callinectes sapidus hemocyanin by binding of L-lactate.

摘要: Hemocyanin of the blue crab Callinectes sapidus has typical structure crustacean hemocyanins in that its smallest vivo is a hexamer subunits each having molecular mass approximately 75 000. As found blood, hemocyanin consists mixture hexamers and dodecamers (typically 1:4). other hemocyanins, affinity with which oxygen binds to binuclear copper site been reported be very sensitive pH variety inorganic allosteric effectors. We report here interaction L-lactate, natural metabolite,with native chromatographically purified dodecamers. Under ionic conditions approximate those physiologically, addition 10 mM L-lactate substantially increases (Δ log P(50) = -0.28). The data from lactate titrations were fit theoretical equation,and best was obtained dissociation constant 1.8 for oxy state 2.2 binding sites every 6 sites. Independent measurements by ultrafiltration techniques indicated 3.2 2.8 per two sets clearly indicate there less than one site. titration not improved assumption more class are equilibrium stable after separation gel-filtration chromatography. Polyacrylamide gel electrophoresis dissociated shows five major bands.Two these bands, constitute one-sixth total dodecameric hemocyanin, do appear upon hexamers. affinities hexameric forms similar another but show differences their sensitivity L-lactate.The increased appreciably glycolate,D-lactate, pyruvate (listed decreasing order effectiveness).Propionate, acetate, succinate, D-alanine, L-alanine without effect, thus illustrating selectivity effect on hemocyanin. magnitude Bohr unchanged over range 7.5-8.0. Moreover, no significant aggregation or 340-nm copper-oxygen absorption band.The foregoing results consistent role as specific effector acts preferential stereospecific