Post-translational arginylation of ornithine decarboxylase from rat hepatocytes.
Ornithine decarboxylase (ODC) was purified 6500-fold from NMRI mouse kidneys under conditions designed to inhibit degradation by proteinases. The enzyme was homogeneous by SDS/polyacrylamide-gel electrophoresis, and the specific activity was among the highest reported. The yield was 70%. A monoclonal antibody against this preparation was generated and used in studies to investigate the half-life of ODC in cultured rat hepatocytes labelled with [35S]methionine. This value was 39 +/- 4 min and was unchanged when either NH4Cl (as a lysosomotropic agent) or leupeptin (as a lysosomal proteinase inhibitor) was added to the culture medium. Thus the intracellular turnover of ODC in cultured hepatocytes occurs mainly in extra-lysosomal compartments. Arginylation of rat ODC was investigated in vitro by incubation with L-[3H]arginyl-tRNA, and the incorporation of the label was compared with that of total cytosolic proteins. Arginylated ODC had a specific radioactivity 8600 times that of the bulk of cytosolic protein. Edman degradation of this ODC showed that the post-translational arginylation occurred only at the alpha-amino end of the enzyme. The inhibitor of arginyl-tRNA:protein arginyltransferase (EC 2.3.2.8), L-glutamyl-L-valyl-L-phenylalanine, increased the half-life of ODC in cultured hepatocytes from 39 min to more than 90 min. The possible significance of the preferential post-translational arginylation of ornithine decarboxylase to its rapid turnover is discussed.
Kopitz
J
J
Physiologisch-chemisches Institut, Universität Tübingen, Federal Republic of Germany.
Rist
B
B
Bohley
P
P
D000818:Animals; D001120:Arginine / Q000378:metabolism*; D002478:Cells, Cultured; D006207:Half-Life; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D009955:Ornithine Decarboxylase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012345:RNA, Transfer, Amino Acid-Specific / Q000378:metabolism*; D012347:RNA, Transfer, Arg / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013739:Testosterone / Q000494:pharmacology
D012345:RNA, Transfer, Amino Acid-Specific; D012347:RNA, Transfer, Arg; D013739:Testosterone; D001120:Arginine; D009955:Ornithine Decarboxylase