In vitro poly(ADP-ribosyl)ation of seminal ribonuclease.
The site of in vitro ADP-ribosylation of seminal ribonuclease was determined. Seminal enzyme was found to be a good receptor of [14C]ADP-ribose residues under the reaction conditions used. The recovery of [14C]ADP-ribosylated RNase was about 65% after purification. After tryptic digestion of modified enzyme, a fraction containing [14C]ADP-ribosylated peptides was separated from the others by ion-exchange chromatography on M82 resin. Radioactive peptides were then purified by affinity chromatography on anti-poly(ADP-ribose)IgG-Sepharose. High performance liquid chromatography of a mixture obtained after pronase digestion of purified ADP-ribosylated peptides revealed only one radioactive peptide whose amino acid composition corresponded to a peptide that has equimolar quantities of aspartic acid, serine, and glycine. Carboxypeptidase Y digestion of this peptide showed that its amino acid sequence was Asp-Ser-Gly. Only position 14-16 of seminal RNase corresponded to this sequence. The chemical stability of the ADP-ribose/enzyme linkage indicated that aspartic acid 14 is the modification site in seminal RNase.
Suzuki
H
H
Quesada
P
P
Farina
B
B
Leone
E
E
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D007700:Kinetics; D008297:Male; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D012260:Ribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D012661:Semen / Q000201:enzymology*; D013045:Species Specificity; D014357:Trypsin
D009702:Nucleoside Diphosphate Sugars; D010446:Peptide Fragments; D011064:Poly Adenosine Diphosphate Ribose; D012260:Ribonucleases; D014357:Trypsin