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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM038497-03
Application #
3294930
Study Section
Biochemistry Study Section (BIO)
Project Start
1987-09-21
Project End
1991-08-31
Budget Start
1989-09-01
Budget End
1991-08-31
Support Year
3
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715