Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Donald J. Graves
Integral membrane-associated arginine specific mono-ADP-ribosyltransferase was purified from rabbit skeletal muscle microsomes. The ADP-ribosyltransferase was solubilized from the 100,000 x g pellet with 0.3% sodium deoxycholate and purified to ≥95% homogeneity by successive DE-52, concanavalin A agarose, 3-aminobenzamide agarose and size exclusion chromatography steps in the presence of detergents. Two molecular weight forms of the enzyme were isolated and partially characterized. The apparent Mr of the [alpha]-form of the enzyme purified to ≥95 homogeneity was about 39,000 ± 500 as estimated by silver-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis. The Mr of the [beta] form purified to ≥80% homogeneity was 38,500 ± 500. The rapid procedure resulted in a 200-fold purification for the [alpha]-form and a 645-fold purification for the [beta]-form, relative to the microsome. Positive identification of the enzyme was confirmed by utilizing a zymographic in situ gel assay and by HPLC assay of PAGE gel slice incubations with a NAD and guanylhydrazone substrate. The specificity of the mono-ADP-ribosyltransferase zymographic assay was characterized by time course incubations, hydroxylamine sensitivity, 3-aminobenzamide inhibition, and histone dependence. The ADP-ribosyltransferase is inactivated by reducing agents.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Jon Eric Peterson
Peterson, Jon Eric, "Purification and partial characterization of ARG-specific ADP-ribosyltransferase from skeletal muscle microsomal membranes " (1990). Retrospective Theses and Dissertations. 9405.