Purification and partial characterization of arginine-specific ADP-ribosyltransferase from skeletal muscle microsomal membranes.
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 greater than or equal to 95% homogeneity by successive DE52, concanavalin A-agarose, 3-aminobenzamide-agarose, and size-exclusion high-performance liquid chromatography (HPLC) 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 greater than or equal to 95% homogeneity was approximately 39,000 +/- 500 as estimated by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Mr of the beta-form purified to greater than or equal 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 microsomal fraction. Positive identification of the enzyme was confirmed by utilizing a zymographic in situ gel assay and by HPLC assay of polyacrylamide gel slice incubations with an 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.
Peterson
J E
JE
Biochemistry and Biophysics Department, Iowa State University, Ames 50011.
Larew
J S
JS
Graves
D J
DJ
D036002:ADP Ribose Transferases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002772:Cholera Toxin / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D007425:Intracellular Membranes / Q000201:enzymology*; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D011817:Rabbits; D014461:Ultracentrifugation
D002772:Cholera Toxin; D036002:ADP Ribose Transferases