Chemical and immunological characterization of the 21-kDa ADP-ribosylation factor of adenylate cyclase.
The ADP-ribosylation factor (ARF) is a 21-kDa GTP-binding protein cofactor in the cholera toxin-catalyzed ADP-ribosylation of the stimulatory regulatory subunit of adenylate cyclase. Purified bovine brain ARF was digested with cyanogen bromide, and peptides were purified and sequenced. Approximately 25-30% of the protein was sequenced in this manner. Peptides contained consensus sequences for GTP-binding proteins but were distinct from any of the previously published GTP-binding proteins. Antibodies were raised in rabbits against both protein and synthetic peptide fragments of ARF. Specific ARF immunoreactivity was detected in every eukaryotic tissue or cell examined, including yeast, slime mold, and man. No ARF immunoreactivity was observed when Escherichia coli proteins were tested. Immunoblotting revealed the majority of ARF to be present in the 100,000 x g supernatant. Immunological cross-reactivity with the cytosolic factor indicate that it and ARF are likely to be the same protein. ARF is shown to be myristylated at the amino terminus. The potential role of myristylation in cellular localization is discussed.
D020727:ADP-Ribosylation Factors; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003429:Cross Reactions; D003488:Cyanogen Bromide; D019204:GTP-Binding Proteins / Q000032:analysis; D007163:Immunosorbent Techniques; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D011817:Rabbits
D008565:Membrane Proteins; D009227:Myristic Acids; D019814:Myristic Acid; D000246:Adenosine Diphosphate Ribose; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D000262:Adenylyl Cyclases; D003488:Cyanogen Bromide