Subcellular distribution and isoelectric heterogeneity of the substrate for ADP-ribosyl transferase from Clostridium botulinum.
When the homogenate of bovine adrenal gland was subjected to subcellular fractionation, an Mr 21,000 substrate for botulinum ADP-ribosyl transferase was found not only in the membrane fractions but also in the cytosol; the amounts in the 10,000 x g precipitates and the 100,000 x g supernatant were about 21 and 56% of the total amount, respectively. Each fraction gave a single ADP-ribosylated protein band on SDS-polyacrylamide gel electrophoresis, but yielded on isoelectric focusing at least three bands between pH 5.5 and 6.0, suggesting the presence of multiple forms of the substrate of a similar molecular weight but different isoelectric points. ADP-ribosylated protein bands from the membrane and cytosol overlapped each other on both electrophoreses. After ammonium sulfate fractionation, the substrate from the cytosol showed requirement of divalent cations or guanine nucleotides for the reaction. Among cations tested, calcium, magnesium and manganese stimulated, whereas cadmium and lanthanum inhibited the reaction. Guanine nucleotides such as GTP, GDP and GTP-gamma-S also stimulated the substrate activity in the cytosol as that in the membrane fraction. However, no additive effects were observed when the nucleotides and cations were added together.
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000311:Adrenal Glands / Q000032:analysis / Q000648:ultrastructure*; D000818:Animals; D002413:Cations, Divalent; D002417:Cattle; D002458:Cell Fractionation; D002462:Cell Membrane / Q000032:analysis; D003014:Clostridium botulinum / Q000201:enzymology*; D003600:Cytosol / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006150:Guanine Nucleotides / Q000494:pharmacology; D007526:Isoelectric Point; D008970:Molecular Weight; D010430:Pentosyltransferases / Q000378:metabolism*; D011506:Proteins / Q000032:analysis* / Q000378:metabolism
D002413:Cations, Divalent; D006150:Guanine Nucleotides; D011506:Proteins; D000246:Adenosine Diphosphate Ribose; D036002:ADP Ribose Transferases; D010430:Pentosyltransferases