caa a22 4500 47574392X CHVBK 20180406123507.0 cr unu---uuuuu 170329e20000801xx s 000 0 eng 10.1023/A:1026501515450 doi (NATIONALLICENCE)springer-10.1023/A:1026501515450 Steady-State Kinetic Analysis of Human Ubiquitin-Activating Enzyme (E1) Using a Fluorescently Labeled Ubiquitin Substrate [Elektronische Daten] [Kevin Wee, Zhihong Lai, Kurt Auger, Jianhong Ma, Kurumi Horiuchi, Randine Dowling, Cristy Dougherty, Jeanne Corman, Richard Wynn, Robert Copeland] We report the synthesis of fluorescently labeled ubiquitin (Ub) and its use for following ubiquitin transfer to various proteins. Using Oregon green (Og) succinimidyl ester, we prepared a population of Ub mainly labeled by a single Og molecule; greater than 95% of the Og label is associated with Lys 6 of Ub. We demonstrate that Og-Ub is efficiently accepted by Ub-utilizing enzymes, such as the human ubiquitin-activating enzyme (E1). We used this fluorescent substrate to follow the steady-state kinetics of human E1-catalyzed Ub-transfer to the ubiquitin-carrier enzyme Ubc4. In this reaction, E1 uses three substrates: ATP, Ubc4, and Ub. The steady-state kinetics of Og-Ub utilization by E1 is presented. We have also used analytical ultracentrifugation methods to establish that E1 is monomeric under our assay condition (low salt) as well as under physiological condition (150 mM NaCl). Plenum Publishing Corporation, 2000 Ubiquitin nationallicence human E1 nationallicence fluorescence nationallicence steady-state kinetics nationallicence ubiquitin-activating enzyme nationallicence activity assay nationallicence Wee Kevin Chemical Enzymology, DuPont Pharmaceuticals Company, P.O. Box 80400, 19880-0400, Wilmington, Delaware aut Lai Zhihong Chemical Enzymology, DuPont Pharmaceuticals Company, P.O. Box 80400, 19880-0400, Wilmington, Delaware aut Auger Kurt Cancer Research, DuPont Pharmaceuticals Company, P.O. Box 80400, 19880-0400, Wilmington, Delaware aut Ma Jianhong Chemical Enzymology, DuPont Pharmaceuticals Company, P.O. Box 80400, 19880-0400, Wilmington, Delaware aut Horiuchi Kurumi Chemical Enzymology, DuPont Pharmaceuticals Company, P.O. Box 80400, 19880-0400, Wilmington, Delaware aut Dowling Randine Chemical Enzymology, DuPont Pharmaceuticals Company, P.O. Box 80400, 19880-0400, Wilmington, Delaware aut Dougherty Cristy Chemical Enzymology, DuPont Pharmaceuticals Company, P.O. Box 80400, 19880-0400, Wilmington, Delaware aut Corman Jeanne Applied Biotechnology, DuPont Pharmaceuticals Company, P.O. Box 80400, 19880-0400, Wilmington, Delaware aut Wynn Richard Applied Biotechnology, DuPont Pharmaceuticals Company, P.O. Box 80400, 19880-0400, Wilmington, Delaware aut Copeland Robert Chemical Enzymology, DuPont Pharmaceuticals Company, P.O. Box 80400, 19880-0400, Wilmington, Delaware aut Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/6(2000-08-01), 489-498 0277-8033 19:6<489 2000 19 10930 https://doi.org/10.1023/A:1026501515450 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1023/A:1026501515450 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Wee Kevin Chemical Enzymology, DuPont Pharmaceuticals Company, P.O. Box 80400, 19880-0400, Wilmington, Delaware aut NATIONALLICENCE 700 1- Lai Zhihong Chemical Enzymology, DuPont Pharmaceuticals Company, P.O. Box 80400, 19880-0400, Wilmington, Delaware aut NATIONALLICENCE 700 1- Auger Kurt Cancer Research, DuPont Pharmaceuticals Company, P.O. Box 80400, 19880-0400, Wilmington, Delaware aut NATIONALLICENCE 700 1- Ma Jianhong Chemical Enzymology, DuPont Pharmaceuticals Company, P.O. Box 80400, 19880-0400, Wilmington, Delaware aut NATIONALLICENCE 700 1- Horiuchi Kurumi Chemical Enzymology, DuPont Pharmaceuticals Company, P.O. Box 80400, 19880-0400, Wilmington, Delaware aut NATIONALLICENCE 700 1- Dowling Randine Chemical Enzymology, DuPont Pharmaceuticals Company, P.O. Box 80400, 19880-0400, Wilmington, Delaware aut NATIONALLICENCE 700 1- Dougherty Cristy Chemical Enzymology, DuPont Pharmaceuticals Company, P.O. Box 80400, 19880-0400, Wilmington, Delaware aut NATIONALLICENCE 700 1- Corman Jeanne Applied Biotechnology, DuPont Pharmaceuticals Company, P.O. Box 80400, 19880-0400, Wilmington, Delaware aut NATIONALLICENCE 700 1- Wynn Richard Applied Biotechnology, DuPont Pharmaceuticals Company, P.O. Box 80400, 19880-0400, Wilmington, Delaware aut NATIONALLICENCE 700 1- Copeland Robert Chemical Enzymology, DuPont Pharmaceuticals Company, P.O. Box 80400, 19880-0400, Wilmington, Delaware aut NATIONALLICENCE 773 0- Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/6(2000-08-01), 489-498 0277-8033 19:6<489 2000 19 10930 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer