Characterization of calmodulin-binding components in the pituitary gonadotrope.
We have used an 125I-calmodulin gel overlayer assay to identify calmodulin-binding component in the rat pituitary. Tissue-specific and Ca2+ -dependent patterns of 125I-calmodulin binding were observed, with five major Ca2+-dependent 125I-calmodulin-labeled components of subunit Mr greater than 205,000, 200,000, 135,000, 60,000, and 52,000. Ca2+-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 microM) or pimozide (1 microM). Some Ca2+-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 Ca2+-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 Mrs were found for four of the components (the largest component is lacking in the bovine system).
Wooge
C H
CH
Department of Pharmacology, University of Iowa College of Medicine, Iowa City 52242.
Conn
P M
PM
D000818:Animals; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000502:physiology; D002148:Calmodulin-Binding Proteins / Q000032:analysis*; D002417:Cattle; D005591:Chemical Fractionation; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007457:Iodine Radioisotopes; D008970:Molecular Weight; D009928:Organ Specificity; D010902:Pituitary Gland / Q000032:analysis* / Q000166:cytology; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D007457:Iodine Radioisotopes; D002118:Calcium