D M Watterson

Biosynthesis of calmodulin in normal and virus-transformed chicken embryo fibroblasts.

We report here that the higher levels of calmodulin in transformed chicken embryo fibroblasts are due to an increase in the rate of synthesis of calmodulin that results from an increased amount of calmodulin-specific mRNA in transformed cells. Transformation of several types of eucaryotic cells by oncogenic viruses results in a two- to threefold increase in the intracellular levels of calmodulin. We used the normal chicken embryo fibroblast and its Rous sarcoma virus-transformed counterpart to examine the biosynthesis of calmodulin. We show that the higher levels of calmodulin found in transformed fibroblasts appear to be the consequence of a selective increase in the rate of synthesis of calmodulin above that of total soluble or total cellular protein. A significant difference in the rate of degradation of calmodulin or total protein between transformed and normal cells was not detected. We also examined the mechanism of the increased synthesis rate of calmodulin and show that the levels of calmodulin mRNA are increased in transformed fibroblasts as measured by both translational activity and hybridization to a calmodulin cDNA probe. It is suggested by these data that the higher levels of calmodulin in transformed cells may result from a specific increase in the rate of either calmodulin gene transcription or mRNA processing.

Calmodulin Structure and Function

Calcium is a mediator of many cellular responses and a regulator of major importance in cellular homeostasis. In order to elucidate the mechanisms of calcium control and the role of calcium in stimulus-response coupling, it is necessary to examine the cellular receptors for calcium. Thermodynamic, kinetic, and structural data strongly suggest that the cytoplasmic receptors for calcium acting as a biological messenger are a class of calcium-binding proteins referred to as calcium-modulated proteins. Calcium-modulated proteins reversibly bind calcium with dissociation constants in the micromolar range under relatively physiological conditions. Because most calcium-modulated proteins are intracellular and have dissociation constants that span the range of intracellular free calcium concentrations, they are postulated to be the major signal transducers of biological calcium signals. Most calcium-modulated proteins characterized to date are not enzymes but are effector proteins capable of transducing a calcium signal into a cellular response by their ability to regulate or modulate the activity of other macromolecules, including enzymes, in a calcium-dependent manner. Examples of calcium-modulated proteins are troponin C, parvalbumin, S100, vitamin D-dependent calcium-binding protein, and calmodulin.

Perturbation of the calmodulin system in transformed cells.

In eukaryotic cells, calcium acts as an intracellular signal transducer primarily through its interaction with a class of calcium binding proteins, of which calmodulin (CaM) is the most highly conserved, phylogenetically ubiquitous member (for reviews, see Van Eldik et al., 1982; Van Eldik and Roberts, 1988; Cohen and Klee, 1988). CaM transduces a calcium signal into a biological response by its ability to regulate the activity of other proteins in a calcium dependent manner. Because CaM is probably the most widely distributed mediator of intracellular calcium signals, fundamental insights can be derived from an enhanced knowlege about the genetic encoding, biosynthetic assembly and regulation of CaM-modulated calcium response pathways. Regardless, in order to understand fully the roles of CaM in the eukaryotic cell and obtain insight into how CaM-modulated pathways can respond differentially to calcium signals, it is necessary to be able to describe in some detail all of the CaM pathways for at least one biological system. This has not been done yet for any biological system. Chicken embryo fibroblasts (CEF) represent one biological system for which a relatively extensive body of information has been described. Also, CEF transformed by Rous sarcoma virus exhibit a number of phenotypic alterations that are potentially mediated by CaM and CaM binding proteins, and perturbations in CaM regulation have been described for normal and transformed CEF. In a more general sense, because perturbations of CaM pathways occur in many kinds of virus-transformed cells, knowledge of how alterations in CaM expression are coupled to oncogene expression may yield insight into how CaM-regulated calcium response pathways are involved in mechanisms of oncogenic transformation.

Immunoreactive levels of myosin light-chain kinase in normal and virus-transformed chicken embryo fibroblasts.

Calmodulin, a calcium-modulated effector protein, is an important mediator of the intracellular actions of calcium through its interaction with calmodulin-binding proteins. We report here that the immunoreactive levels of a calmodulin-binding protein, myosin light-chain kinase, are decreased in transformed chicken embryo fibroblasts.

Purification of calmodulin and preparation of immobilized calmodulin.

Publisher Summary This chapter focuses on the purification of calmodulin and preparation of immobilized calmodulin. Calmodulin is an acidic, low molecular weight, calcium-modulated protein, which has been isolated from a diverse number of sources, and characterized in terms of its amino acid sequence and biochemical activities. The protein is highly conserved in both structure and function, although, there are quantitative differences in some activator activities among calmodulins. As a consequence, it may be important that functional studies of calmodulin-binding proteins be carried out with calmodulins from the appropriate biological sources. Because calmodulin is a member of a family of closely related calcium-binding proteins that can coexist in the same cell, purification procedures designed to give proteins of high purity are also necessary for many studies. Purification procedures for calmodulins from different sources have appeared in preceding volumes of this series and in the literature. Recent advances in high-performance liquid chromatography (HPLC) and protein sequence analysis, however, have allowed a more detailed characterization of calmodulins.

Mutant analysis approaches to understanding calcium signal transduction through calmodulin and calmodulin regulated enzymes

An example set of site-specific mutagenesis studies of calmodulin has been discussed in terms of strategy and how the results can provide insight into the functioning of calmodulin. A set of common examples for the study of calcium binding and enzyme activation were discussed. Essentially, site-specific mutagenesis in these initial studies is a perturbation approach. From these perturbation studies, structural features can be correlated in future studies with function and mechanisms of action proposed. More importantly, the approach allows efficient testing of proposed mechanisms and further probing of the molecular aspects of the signal transduction pathways. Clearly, the key functional feature that must be addressed in future studies is how the calcium binding steps in the mechanism are coupled to the enzyme activation step, which is the final step of the calmodulin-enzyme binding mechanism.

A knowledge-based experimental design system for nucleic acid engineering

Presented in this paper is a knowledge-based experimental design system that incorporates the domain expertise used in nucleic acid engineering, thus automating the processing of error-prone, laborious low-level work, and many decision-making steps, and guiding the biologist toward a workable plan. This allows the biologist to work at a higher abstraction level, concentrating on more fundamental, difficult and challenging problems directly related to protein structure - function relationships. Cassette-based site-directed mutagenesis and synthetic gene designs are used as examples to illustrate the utility of the knowledge-based system approach to experimental design.

A knowledge-based system for cassette mutagenesis experimental design

A knowledge-based system for the design and planning of cassette mutagenesis experiments has been developed for scientists working in the field of structural biology and protein engineering. The system applies domain-specific knowledge to manage the menial details and automate most of the decision-making steps involved in the design process. This allows scientists to work at a high abstraction level, and results in significant time savings and increased productivity. The system also includes an automated documentation facility to improve the efficiency and accuracy of record keeping.

Analysis of Phosphodiesterase Reaction Mixtures by High-Performance Liquid Chromatography

Publisher Summary This chapter discusses a few technical updates and one specific example using simple HPLC procedures for the analysis of phosphodiesterase reaction mixtures. Ion-exchange HPLC columns, in contrast to reversed-phase columns, have been the columns of choice for enzymes such as adenylate cylase and phosphodiesterase because of their ability to resolve easily the various nucleotides in a simple and reproducible manner. Columns of different sizes are available from a variety of vendors. Reproducible and simple systems that use 4.6-mm-diameter columns are currently available with detailed protocols. The details of the system depend on the HPLC hardware that is available in the laboratory. The commercial availability of prepacked columns and protocols makes the set-up adaptable and easy to use. The ability to resolve metabolites and inhibitors and the rapid, unattended fractionation and quantification of components in enzyme-catalyzed reaction mixtures make it applicable to a variety of uses in the study of cyclic nucleotide metabolism.