ANTIBODY RECOGNITION OF CALCIUM-BINDING PROTEINS DEPENDS ON THEIR CALCIUM-BINDING STATUS

Previous studies have revealed changes in immunohistochemical stains f or calcium-binding proteins after manipulations that influence intrace llular calcium. Cases have been revealed in which these changes in imm unoreactivity were not correlated with changes in protein amounts. The present experiments examined whether these effects might be explained by changes in antiserum recognition due to calcium-induced changes in protein conformation. Calretinin, calbindin D28k, and parvalbumin inc ubated in high calcium were recognized by antisera better than when th ey were incubated in low calcium. Using a calbindin D28k antibody, it was shown that this effect occurs within physiological calcium concent rations. Formalin fixation of the proteins in the presence of calcium resulted in greater antibody recognition than did fixation of proteins in calcium-free states. The calretinin antiserum appeared to recogniz e a portion of the molecule previously shown to undergo calcium-depend ent conformational changes, A calcium-insensitive antiserum was made t o a different fragment of calretinin. These results indicate that some antibodies to calcium-binding proteins preferentially recognize parti cular calcium-induced protein conformations. Given the potential for w ide fluctuations in neuronal calcium, the present results indicate tha t quantitative estimates of intracellular calcium-binding proteins obt ained from immunohistochemical studies of neurons must be interpreted with caution.