Purification, characterization, and localization of an ADP-ribosylactin hydrolase that uses ADP-ribosylated actin from rat brains as a substrate.
Mammalian ADP-ribosylation is poorly understood. An ADP-ribosylprotein hydrolase that acted on ADP-ribosylated 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 ADP-ribose, but not by AMP. Mg2+ increased Vmax. Purified ADP-ribosylactin hydrolase catalyzed the hydrolysis of ADP-ribosylated subunits Gsalpha, Gialpha, and Goalpha and elongation factor-2. After de-ADP-ribosylation by the purified ADP-ribosylactin hydrolase, the proteins were re-ADP-ribosylated by brain mono-ADP-ribosyltransferases and bacterial toxins. The actin that was de-modified by ADP-ribosylactin 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.
Okamoto
H
H
Laboratory of Molecular Biology, Department of Veterinary Science, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 593, Japan.
Fujita
H
H
Matsuyama
S
S
Tsuyama
S
S
D000199:Actins / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001921:Brain / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D007150:Immunohistochemistry; D008297:Male; D009699:N-Glycosyl Hydrolases; D051381:Rats; D017208:Rats, Wistar; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity
D000199:Actins; D000911:Antibodies, Monoclonal; D000246:Adenosine Diphosphate Ribose; D006026:Glycoside Hydrolases; D009699:N-Glycosyl Hydrolases; C046422:ADP-ribosylarginine hydrolase