Abstract

ADP-ribosylation reactions of proteins can be carried out by specific bacterial toxins, e.g., cholera toxin and E. coli enterotoxin or by enzymes in a variety of tissues and organs. Our interest is to study the process in skeletal muscle, because we found activities present in this tissue that carry out ADP- ribosylation and de-ADP ribosylation reactions consistent with a regulatory function for these reactions. We have purified these enzymes and propose to use these enzymes to delineate the role(s) of these enzymatic reactions in skeletal muscle. We will examine, e.g., Ca+2 regulated processes, protein phosphorylation and evaluate the molecular basis of these types of reactions. Specifically we plan to study the following topics: I. ADP-ribosylation and de ADP-ribosylation in skeletal muscle A. Studies in membranes B. Studies with exogenous protein substrates 1. Phosphorylase kinase 2. Glycogen phosphorylase II. Properties of ADP-ribosyltransferase the hydrolase A. Kinetic studies B. Chemical characterization III. Chemical aspects of mono ADP-ribosylation and factors influencing the specificity of the enzymatic reactions A. Specificity B. Affinity HPLC C. Active site reagents - ADP-ribosylation

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034262-05
Application #
3284938
Study Section
Biochemistry Study Section (BIO)
Project Start
1985-01-01
Project End
1991-03-31
Budget Start
1989-04-01
Budget End
1990-03-31
Support Year
5
Fiscal Year
1989
Total Cost
Indirect Cost

  Institution

Name
Iowa State University
Department
Type
Earth Sciences/Resources
DUNS #
City
Ames
State
IA
Country
United States
Zip Code
50011

  Publications

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
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
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
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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