Characterization of calmodulin-binding components in the pituitary gonadotrope

Abstract

We have used an 125I-calmodulin gel overlayer assay to identify calmodulin-binding component in the rat pituitary. Tissue-specific and Ca 2+-dependent patterns of 125I-calmodulin binding were observed, with five major Ca 2+-dependent 125I-calmodulin-labeled components of subunit M r > 205000, 200000, 135000, 60000, and 52000. Ca 2+ dependent binding was defined as that which was abolished in the presence of 1 mM EGTA. Calmodulin binding was inhibited by calmodulin antagonists such as penfluridol (1 μM) or pimozide (1 μM). Some Ca 2+independent binding was observed and appears to be due to (nonspecific) hydrophobic interaction of calmodulin with acid-soluble proteins, principally histones. Subcellular fractionation revealed that the Ca 2+-dependent calmodulin-binding components are localized primarily in the cytosolic fraction. Separation of dispersed anterior pituitary cells by a linear metrizamide gradient yielded gonadotrope-enriched fractions; these contained all five 125I-calmodulin-binding components corresponding to the major bands in the pituitary homogenate. Studies with ovariectomized and steroid-replaced animals indicated that the tissue content of calmodulin-binding components, like those of calmodulin itself, did not appear to be differentially regulated by steroids. A comparison of rat and bovine pituitary tissue homogenates revealed that binding components migrating at the same apparent M r s were found for four of the components (the largest component is lacking in the bovine system).