Frank Leonard Siegel, PhD - US grants

This application describes a comprehensive biochemical study of the regulation of calmodulin activity. Calmodulin, a calcium-binding protein, has been shown to mediate the calcium activation of many enzymes and physiological processes. Preliminary experiments have indicated that calmodulin undergoes two types of post-translational modification - N-methylation and carboxymethylation, which accompanies tardive dyskinesia, is related to an increase in membrane-bound calmodulin. We will investigate the N-methylation and carboxymethylation of calmodulin in an attempt to determine the functional sugnificance of these alterations in calmodulin structure. In other studies we will examine the regulation and enzymology of calmodulin regulation, the factors responsible for binding of calmodulin to neural membranes, and the factors which govern calmodulin biosynthesis and degradation. The effects of acute and chronic administration of neuroactive drugs affecting receptor sensitivity on these aspects of calmodulin modification, function and synthesis will be determined.

The research described in this application constitutes a study of calmodulin-stimulated protein methylation. Calmodulin is a calcium-binding protein which activates a number of calcium- dependent enzymes which underwrite cellular phosphorylation and dephosphorylation reactions as well as the methylation of selected proteins in liver, kidney and lung. These calmodulin-stimulated protein methylations are inhibited by a dialyzable inhibitor. Calmodulin-stimulated protein methylation does not occur in fetal or regenerating liver or in transplantable hepatoma. We will purify and characterize the major substrate of calmodulin- stimulated protein methylation, a major liver cytosolic protein of Mr 29,000, using sequential ion-exchange chromatography, gel filtration and fast protein liquid chromatography (FPLC). The number and identity of the methylated amino acid residues, the amino composition and amino acid sequence will be determined. The amino acid sequencing will be done using a gas-phase sequenator in the laboratory of Dr. John Collins. Polyclonal antibodies to MAP29 will be raised to develop a radioimmunoassay for MAP29 and this assay will be used to determine the tissue and subcellular distribution of this protein. We will characterize MAP29 methyltransferase in terms of its kinetic parameters, substrate specificity and amino acid composition. We will also purify and characterize the inhibitor of calmodulin-stimulated protein methylation and attempt to determine the mechanism of this reaction - the manner in which MAP29, MAP29 methyltransferase, calmodulin and the inhibitor interact. Finally, we describe studies of the regulation of MAP29 methylation in developing liver and hepatoma 7777 in which we hope to determine the relation between methylation of this protein and cellular proliferation. These studies are relevant to an understanding of the actions of calmodulin and also t those factors which operate in cancer and normal cellular proliferation of developing tissues.

This application describes research designed to determine the biochemical significance of the N-methylation of calmodulin. Calmodulin is a calcium-binding protein and also an important regulator of many calcium-dependent enzymes, particularly those enzymes which underwrite phosphorylation and dephosphorylation reactions. Calmodulin is structurally characterized by the presence of a single residue of trimethyllysine; the biological significance of this post-translational modification is poorly understood, although we have begun to make inroads in this area. Five specific aims define this proposal. The first of these is the purification and characterization of calmodulin N- methyltransferase, which will be accomplished by a number of sequential chromatographic procedures, including fast protein liquid chromatography (FPLC) and affinity chromatography. The second aim is to clarify the biochemical significance of the N- methylation of calmodulin. To achieve this aim we will use purified calmodulin N-methyltransferase to methylated des (methyl)calmodulin and then compare the activities of methylated and non-methylated calmodulins with respect to the activation of several calmodulin-dependent enzymes. The third aim is to determine the relative abundance of non-N-methylated calmodulin in rat tissues (we have prior evidence for the presence of des(methyl)calmodulin). The two calmodulins will be separated and quantitated by sequential melittin-Sepharose chromatography and ion-exchange FPLC. The fourth aim is a comparison of the binding of calmodulin and des(methyl)calmodulin to target proteins in rat tissues. This will be done by iodinating both calmodulins and using them as probes on gel overlay assays. The final aim is to determine those factors which regulate the activity of calmodulin N-methyltransferase. The activity of this enzyme is markedly elevated in liver tumors and in fetal tissues; the difference between enzyme activity in normal and tumor tissue is much greater for this enzyme than for calmodulin itself. The available evidence is that calmodulin is the only substrate for calmodulin N-methyltransferase and we have placed a high priority to determining the significance of the elevation of enzyme activity in rapidly proliferating cells. This research is relevant to basic biochemical processes in cancer and normal developmental processes.

electron microscopy; histology; cell biology; biomedical facility; light microscopy; tissue /cell culture; tissue /cell preparation; immunocytochemistry; neurosciences; immunoelectron microscopy; bioimaging /biomedical imaging;