Mono ADP-ribosylation reactions of proteins and guanidine derivatives can be carried out by specific bacterial toxins, e.g., cholera toxin and E. coli enterotoxin or by enzymes present in normal tissues. The exact function of these latter enzymes is not known and we will concentrate our efforts to establish the role of the enzyme we found in skeletal muscle. A comparison will be made with reactions catalyzed by cholera toxin to learn how these processes occur and what effects these reactions could have on other enzyme-catalyzed covalent modification reactions. Specifically, we plan to study the following topics: I. NAD-dependent Mono ADP-ribosyltransferase from Skeletal Muscle A. Enzyme Isolation B. Determination of Physical, Chemical, and Catalytic Properties of the Purified Enzyme C. Identification of Natural Protein Substrates II. The Process of Mono ADP-ribosylation A. The Kinetic Mechanism B. Specificity of the Reaction 1. Action on synthetic peptides 2. Action on guanyl hydrazones 3. Modification sites in proteins C. Active Site Directed Reagents D. Reversibility of the Process III. Consequences of ADP-ribosylation Reactions A. Effects on Other Enzyme Catalyzed Covalent Modification Reactions B. On Reactions Occurring in the Glycogen Particle, Sarcoplasmic Reticulum and Sarcolemma

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM034262-01
Application #
3284931
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1985-01-01
Project End
1987-12-31
Budget Start
1985-01-01
Budget End
1985-12-31
Support Year
1
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Iowa State University
Department
Type
Earth Sciences/Resources
DUNS #
City
Ames
State
IA
Country
United States
Zip Code
50011
Huang, H Y; Graves, D J; Robson, R M et al. (1993) ADP-ribosylation of the intermediate filament protein desmin and inhibition of desmin assembly in vitro by muscle ADP-ribosyltransferase. Biochem Biophys Res Commun 197:570-7
Kharadia, S V; Huiatt, T W; Huang, H Y et al. (1992) Effect of an arginine-specific ADP-ribosyltransferase inhibitor on differentiation of embryonic chick skeletal muscle cells in culture. Exp Cell Res 201:33-42
Larew, J S; Peterson, J E; Graves, D J (1991) Determination of the kinetic mechanism of arginine-specific ADP-ribosyltransferases using a high performance liquid chromatographic assay. J Biol Chem 266:52-7
Peterson, J E; Larew, J S; Graves, D J (1990) Purification and partial characterization of arginine-specific ADP-ribosyltransferase from skeletal muscle microsomal membranes. J Biol Chem 265:17062-9
Kim, E S; Graves, D J (1990) Development of a high-performance liquid chromatography assay method and characterization of adenosine diphosphate-ribosylarginine hydrolase in skeletal muscle. Anal Biochem 187:251-7
Narayanan, J; Hartman, P A; Graves, D J (1989) Assay of heat-labile enterotoxins by their ADP-ribosyltransferase activities. J Clin Microbiol 27:2414-9
Soman, G; Graves, D J (1988) Endogenous ADP-ribosylation in skeletal muscle membranes. Arch Biochem Biophys 260:56-66
Kharadia, S V; Graves, D J (1987) Relationship of phosphorylation and ADP-ribosylation using a synthetic peptide as a model substrate. J Biol Chem 262:17379-83
Chang, Y C; Soman, G; Graves, D J (1986) Identification of an enzymatic activity that hydrolyzes protein-bound ADP-ribose in skeletal muscle. Biochem Biophys Res Commun 139:932-9
Chang, Y C; Scott, R D; Graves, D J (1986) Function of pyridoxal 5'-phosphate in glycogen phosphorylase: 19F NMR and kinetic studies of phosphorylase reconstituted with 6-fluoropyridoxal and 6-fluoropyridoxal phosphate. Biochemistry 25:1932-9

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