Extracellular conversion of epidermal growth factor (EGF) to des-Arg53-EGF by carboxypeptidase M.
Epidermal growth factor (EGF) is a 53-amino-acid mitogenic polypeptide present in a variety of tissues and fluids including kidney, urine, and amniotic fluid. An EGF isoform, des-Arg53-EGF, has been identified in urine and is the earliest metabolite generated in target cells upon EGF binding. In this study, purified carboxypeptidase M efficiently released the COOH-terminal arginine residue from EGF with a Km = 56 microM, kcat = 388 min-1, and kcat/Km = 6.9 microM-1 min-1. When EGF was incubated with urine or amniotic fluid, des-Arg53-EGF was the only metabolite detected. This conversion was blocked by immunoprecipitation with specific antiserum to carboxypeptidase M or by 10 microM DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (a carboxypeptidase M inhibitor), indicating that the major EGF metabolizing enzyme in these fluids is carboxypeptidase M. When incubated on a confluent monolayer of Madin-Darby canine kidney (MDCK) cells, EGF was readily converted to a single metabolite, des-Arg53-EGF, by carboxypeptidase M. To investigate one possible functional consequence of this conversion, mitogenic activities of EGF and des-Arg53-EGF were tested. Both peptides were equipotent in stimulating [3H]thymidine incorporation in MDCK cells at all doses tested. In addition, inhibition of the conversion of EGF to des-Arg53-EGF by the carboxypeptidase M inhibitor did not affect the mitogenic potency of EGF. These data indicate that carboxypeptidase M, present in a variety of cells and biological fluids, can convert EGF to des-Arg53-EGF. However, in contrast to many other peptide hormones whose activity depends on a final carboxypeptidase processing step, removal of Arg53 of EGF is not required for its mitogenic activity.
McGwire
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Department of Pharmacology, University of Illinois, College of Medicine, Chicago 60612, USA.
Skidgel
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D000653:Amniotic Fluid / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D004285:Dogs; D004815:Epidermal Growth Factor / Q000378:metabolism*; D058851:GPI-Linked Proteins; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008666:Metalloendopeptidases / Q000494:pharmacology*; D011817:Rabbits; D013936:Thymidine / Q000378:metabolism
D058851:GPI-Linked Proteins; D004815:Epidermal Growth Factor; C058479:carboxypeptidase M; D008666:Metalloendopeptidases; D013936:Thymidine