Calmodulin and calmodulin binding proteins in amphibian rod outer segments

Abstract

The calmodulin (CaM) content of fully intact frog rod outer segments (ROS) has been measured. The molar ratio between rhodopsin and total CaM in ROS is 800:1. This is in good agreement with the data reported for bovine ROS CaM [Kohnken, R. E., Chafouleas, J. G., Eadie, D. M., Means, A. R. and McConnell, D. G. (1981) J. Biol. Chem. 256, 12517-12522]. In the absence of Ca2+, the ROS membrane fraction contains only 4% of total ROS CaM. In contrast, in the presence of Ca2+, 15% of total ROS CaM is found in the membrane fraction. For half-maximal binding of CaM to CaM-depleted ROS membranes, 3 .times. 10-7 M Ca2+ is required. This CaM binding is inhibited by trifluoperazine. CaM binding proteins in the ROS membrane fraction are identified by using two different methods: the overlay method and the use of 3,3''-dithiobis(sulfosuccinimidyl propionate) (DTSSP), a bifunctional cross-linking reagent. Ca2+-dependent CaM binding proteins with apparent molecular weights of 240,000, 140,000, 53,000, and 47,000 are detected in the ROS membrane fraction by the overlay method. Anomalous, Ca2+-independent CaM binding to rhodopsin is also detected with this method, and this CaM binding is inhibited by the presence of Ca2+. With the bifunctional cross-linking reagent, DTSSP, three discrete proteins with molecular weights of 240,000, 53,000, and 47,000 are detected in the native ROS membrane fraction. CaM binding to rhodopsin is not detected with this method. Moreover, while the Mr 140,000 band is not detected with DTSSP, a smeared band with a molecular weight between 78,000 and 93,000 is identified (with DTSSP) in the ROS membrane fraction. These data suggest that both the Ca2+-independent binding of CaM to rhodopsin and the Ca2+-dependent binding of CaM to the Mr 140,000 protein (detected by the overlay method) represent binding of CaM to a site(s) which is (are) exposed only after denaturation. However, Ca2+-dependent CaM binding to the smeared band is detected only in the native conformation. Ca2+-dependent CaM binding in the cytoplasmic fraction is also evaluated with the overlay method. The amount of soluble CaM binding proteins detected in this manner is less than 5% of the total membrane-bound CaM binding proteins. These data suggest that CaM and its binding proteins participate in the regulation of Ca2+-sensitive processes primarily on the ROS disk membranes.