Calmodulin-binding proteins in subcellular fractions of zones of the adrenal cortex

The guinea pig adrenal cortex consists of a steroidogenic ACTH-responsive outer zone and an ACTH-unresponsive inner zone. It has been suggested that calmodulin plays an important role in ACTH-stimulated steroidogenesis. Thus, in an effort to examine the calmodulin 'system' in the guinea pig adrenal cortex model, Ca2+-dependent binding of calmodulin to proteins in subcellular fractions of the outer and inner zones was examined by the [125I]iodocalmodulin overlay technique and compared to similar studies utilizing pancreas, brain and liver tissue. Although the general pattern of calmodulin-binding proteins was similar for the two adrenocortical zones, quantitatively there was a striking difference with greater binding in the outer zone; this was particularly noteworthy for the mitochondrial fraction. The two most prominent calmodulin-binding proteins isolated from cytosol by calmodulin-Sepharose column chromatography had Mr of 60,000 and 47,000. The size of these two proteins suggested the presence of Ca2+/calmodulin-dependent protein kinase II. Western blot analysis, however, failed to demonstrate calmodulin kinase II in either zone, although it was clearly detectable in brain cytosol. The 60 K calmodulin-binding protein in the adrenal cortex also suggested the presence of the calmodulin-binding A subunit of the Ca2+/calmodulin-stimulated protein phosphatase, calcineurin. Western blot analysis did reveal the presence of calcineurin in the outer adrenocortical zone; it was not detectable, however, in the inner adrenocortical zone. The relation between the striking zonal differential for calmodulin-binding proteins and the zonal differential in ACTH-stimulated steroidogenesis in the guinea pig adrenal cortex will require further investigation.