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Purification and partial characterization of arginine-specific ADP-ribosyltransferase from skeletal muscle microsomal
J E Peterson1, J S Larew, D J Graves
1Biochemistry and Biophysics Department, Iowa State University, Ames 50011.
Abstract:
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.
Insights
Rabbit skeletal muscle microsomes yielded an integral membrane-associated arginine-specific mono-ADP-ribosyltransferase. This enzyme was purified and characterized, revealing two distinct molecular weight forms.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Integral membrane proteins play crucial roles in cellular processes.
- Mono-ADP-ribosyltransferases (ARTs) are enzymes involved in post-translational modification.
- Rabbit skeletal muscle microsomes are a rich source of cellular enzymes.
Purpose of the Study:
- To purify and characterize the integral membrane-associated arginine-specific mono-ADP-ribosyltransferase from rabbit skeletal muscle microsomes.
- To identify and analyze different molecular weight forms of the enzyme.
Main Methods:
- Enzyme solubilization using sodium deoxycholate.
- Multi-step purification including ion-exchange chromatography (DE52), affinity chromatography (concanavalin A-agarose, 3-aminobenzamide-agarose), and size-exclusion HPLC.
- Enzyme identification via zymographic assay and HPLC assay.
- Molecular weight determination using SDS-PAGE.
Main Results:
- The enzyme was purified to high homogeneity (>95% for alpha-form, >80% for beta-form).
- Two molecular weight forms, alpha (approx. 39,000 Da) and beta (approx. 38,500 Da), were isolated.
- Significant purification fold achieved (200-fold for alpha, 645-fold for beta).
- Enzyme activity confirmed through specific assays and characterized by substrate and inhibitor studies.
Conclusions:
- A rapid and effective purification protocol for rabbit skeletal muscle mono-ADP-ribosyltransferase was established.
- The existence of distinct molecular weight forms of this enzyme was demonstrated.
- The enzyme's specificity and sensitivity to reducing agents were highlighted.