Isoforms of mammalian ubiquitin-activating enzyme.
Ubiquitin-activating enzyme, "E1," is the first enzyme in the pathway leading to formation of ubiquitin-protein conjugates and represents a potential target for regulation in the metabolic control of the conjugation reaction. Antiserum raised against human E1 recognizes two immunoreactive proteins in extracts from several human cell lines and animal tissues. We have characterized these two immunoreactive proteins in HeLa cells and present evidence that they are isoforms of E1. We have designated these isoforms as "E1(110 kDa)" and "E1(117 kDa)" to reflect their apparent molecular masses determined from SDS-polyacrylamide gel electrophoresis. These two immunoreactive proteins are immunologically similar, have nearly identical peptide maps, and comigrate with enzymatic activity characteristic of E1 in native polyacrylamide gel electrophoretic separations. Pulse-labeling experiments reveal that both isoforms are long-lived in vivo with degradation rates which are inconsistent with a proenzyme/enzyme model. Furthermore, their rates of degradation, which vary depending on the cell line studied, are kinetically distinguishable in contact-inhibited human lung fibroblasts. This work represents the first demonstration of E1 isoforms in a non-plant species and carries important implications for studies of the regulatory mechanisms controlling ubiquitin conjugation.
Cook
J C
JC
Section on Metabolic Regulation, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Chock
P B
PB
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D006367:HeLa Cells; D006801:Humans; D007074:Immunoglobulin G; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008025:Ligases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D009928:Organ Specificity; D010449:Peptide Mapping; D051381:Rats; D017207:Rats, Sprague-Dawley; D013737:Testis / Q000201:enzymology; D013950:Thymus Gland / Q000201:enzymology; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases
D007074:Immunoglobulin G; D007527:Isoenzymes; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes