Purification, Characterization, and Localization of an ADP-ribosylactin Hydrolase That Uses ADP-ribosylated Actin from Rat Brains as a Substrate

Abstract

Mammalian ADP-ribosylation is poorly understood. An ADP-ribosylprotein hydrolase that acted on ADPribosylated actin was purified from rat brain. The molecular weight of this enzyme was 62,000 as determined by SDS-polyacrylamide gel electrophoresis and gel filtration. Enzyme activity with ADP-ribosylated actin as a substrate was inhibited by NAD, ATP, ADP, and ADPribose, but not by AMP. Mg 2؉ increased V max . Purified ADP-ribosylactin hydrolase catalyzed the hydrolysis of ADP-ribosylated subunits G s ␣, G i ␣, and G o ␣ and elongation factor-2. After de-ADP-ribosylation by the purified ADP-ribosylactin hydrolase, the proteins were re-ADPribosylated by brain mono-ADP-ribosyltransferases and bacterial toxins. The actin that was de-modified by ADPribosylactin hydrolase could form actin filaments. Two kinds of monoclonal antibodies against ADP-ribosylactin hydrolase were prepared and characterized. In an immunohistochemical study, the plasma membranes and cytoplasmic regions of the nerve cells in the rat brain were immunoreactive. In subcellular fractionation of the brains, most of the ADP-ribosylactin hydrolase activity was found in the cytosol and synaptosome fractions. When the synaptosomes were treated with a hypotonic solution, ADP-ribosylactin hydrolase activity was found in the supernatant. Our findings suggest that brain ADP-ribosylactin hydrolase has the important function of polymerizing actin for signal transduction in the cytosol of nerve cells and synaptosomes.

Post-translational modification and regulation of actin

“…Eukaryotic ADP-ribosyltransferases affect actin in different ways – from the effects of Transferase A, which ribosylates Arg-95 and Arg-372 and delays filament formation, to arginine-specific ADP-ribosyltransferase, which modifies Arg-206 in G-actin to alter polymerization (Table S2). Enzymes that reverse actin ADP-ribosylation (ADP-ribosylactin Hydrolases) have also been described although they remain poorly understood [13]. …”

Genomic organization and promoter analysis of the mouse ADP-ribosylarginine hydrolase gene

“…More recently, an ADP-ribosylactin hydrolase which cleaves the ADPribose moiety from ADP-ribosylated actin was purified from rat brain (Okamoto et al, 1997). This enzyme differs from the ADP-ribosylarginine and ADP-ribosylcysteine hydrolases, in that it is less specific for the amino acid moiety of the substrate (Okamoto et al, 1997).…”