An introductory program has shown that the volume change, DeltaV, accompanying the sequential uptake of Ca++ to calmodulin clearly indicated an ordered binding, suggesting a cooperativity among the 4 sites. DeltaV studies, therefore, should be of unusual importance in the case of the intracellular calcium-binding proteins in general, because they all contain multidentate coordination sites in their unique and evenly-space binding loops. Moreover, the acquisition of DeltaV data has recently been advanced such that proteins like these can be studied which are not available in large supply - a problem which heretofore severely restricted the generation of this important information. By the use of densimetric techniques which lower the expenditure of protein by about 2 orders of magnitude or more, the uptake of Ca++ by intracellular calcium-binding proteins in the presence of the competing cellular cations, K+ and Mg++, is projected. Additionally, because these proteins are simple polypeptides, a correlation of DeltaV with chemical events should be possible where X-ray structures are available. The items for study include calmodulin, calmodulin fragments, parvalbumin, troponin-C (skeletal), calcineurin (phosphatase 2B) and a comparison between plant and animal calmodulins (wherein one or more amino acid substitutions elicit the functional changes). Lanthanide binding will also be imposed to test their suitability as substitutes for Ca++ by DeltaV. Finally, hydrodynamic volume changes attending the uptake of metal ions will be studied with a new magnetic viscometer which also uses very small amounts of sample per measurement; such changes have been indicated by other means upon metal-ion binding. These proteins, in particular calmodulin, modulate a variety of diverse processes in the cell upon entry of Ca++ signals; they are central to the metabolism and function of all types of cells in the body. Hence, elucidation of their uptake of Ca++ such as specific responses to Ca++ levels, whether stepwise or random, is fundamental to an understanding of their role in health and disease.
