Multiple forms of ubiquitin-protein ligase. Binding of activated ubiquitin to protein substrates.
Enzyme activities that catalyzed the covalent attachment of ubiquitin to protein substrates (ubiquitin-protein ligase, UbL) were purified from the extracts of human red blood cells. These activities required the presence of ubiquitin-activating enzyme and ATP for activity. Four fractions (UbL A, B1, B2, and C) were resolved and showed different specificities toward added substrates [carboxymethylated bovine serum albumin (CM-BSA), G-actin, lysozyme, and alpha-lactalbumin]. The enzyme fractions gave different products with a given substrate. UbL A and UbL B1 were exclusively active with CM-BSA and alpha-lactalbumin, respectively. UbL B2 was most active toward CM-BSA with substantial activities to G-actin and alpha-lactalbumin and with no activity to lysozyme. UbL C showed significant activities with all four substrates, having a highest activity toward CM-BSA. There were many endogenous proteins present in the erythrocyte extract which were efficient substrates for ubiquitin conjugation. In particular, a pair of substrates were identified from erythrocyte extracts which were far more efficient substrates than the denatured proteins exogenously added.
D002845:Chromatography; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology; D006609:High Mobility Group Proteins / Q000097:blood*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006882:Hydroxyapatites; D007700:Kinetics; D008025:Ligases / Q000097:blood* / Q000302:isolation & purification; D008970:Molecular Weight; D011485:Protein Binding; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000097:blood* / Q000302:isolation & purification
D006609:High Mobility Group Proteins; D006882:Hydroxyapatites; D014452:Ubiquitins; D017886:Durapatite; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes