In vitro enzyme activation with calbindin-D 28k, the vitamin D-dependent 28 kDa calcium binding protein

Abstract

Purified porcine erythrocyte membrane Ca 2+-ATPase and 3′:5′-cyclic nucleotide phosphodiesterase were stimulated in a dose-dependent, saturable manner with the vitamin D-dependent calcium binding protein from rat kidney, calbindin-D 28k (CaBP-D 28k). The concentration of CaBP-D 28k required for half-maximal activation ( K 0.5 act.) of the Ca 2+-ATPase was 28 nM compared to 2.2 nM for calmodulin (CaM), with maximal activation equivalent upon addition or either excess CaM or CaBP-D 28k, 3′:5′-Cyclic nucleotide phosphodiesterase (PDE) also showed equivalent maximum saturable activation by calbindin ( K 0.5 act. = 90 nM) or calmodulin ( K 0.5 act. = 1.2 nM). CaBP-D 28k was shown to effectively compete with CaM-Sepharose for PDE binding. Immunoprecipitation with CaBP-D 28k antiserum completely inhibited calbindin-mediated activation of PDE but had no effect on calmodulin's ability to activate PDE. While the physiological significance of these results remains to be established, they do suggest that CaBP-D 28k can activate enzymes and may be a regulator of yet to be identified target enzymes in certain tissues.