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Metabolism and Enzymology

High Performance Liquid Chromatography Resolution of Ubiquitin Pathway Enzymes from Wheat Germ ¹

Department of Horticulture, University of Wisconsin-Madison, Madison, Wisconsin 53706

The highly conserved protein ubiquitin is involved in several cellular processes in eukaryotes as a result of its covalent ligation to a variety of target proteins. Here, we describe the purification of several enzymatic activities involved in ubiquitin-protein conjugate formation and disassembly from wheat germ (Triticum vulgare) by a combination of ubiquitin affinity chromatography and anion-exchange high performance liquid chromatography. Using this procedure, ubiquitin activating enzyme (E1), several distinct ubiquitin carrier proteins (E2s) with molecular masses of 16, 20, 23, 23.5, and 25 kilodaltons, and a ubiquitin-protein hydrolase (isopeptidase) were isolated. Purified E1 formed a thiol ester linkage with ¹²⁵I-ubiquitin in an ATP-dependent manner and transferred bound ubiquitin to the various purified E2s. The ubiquitin protein hydrolase fraction was sensitive to hemin, and in an ATP-independent reaction, was capable of removing the ubiquitin moiety from both ubiquitin ¹²⁵I-lysozyme conjugates (-amino or isopeptide linkage) and the ubiquitin 52-amino acid extension protein fusion (-amino or peptide linkage). Using this procedure, wheat germ represents an inexpensive source from which enzymes involved in the ubiquitin pathway may be isolated.

¹ This research was supported by a U.S. Department of Agriculture-Competitive Research Grants Office grant (88-37262-3368) to R.D.V., a National Institutes of Health Cellular and Molecular Biology Predoctoral Training Fellowship to M.L.S., and a National Science Foundation Postdoctoral Fellowship in Plant Biology to J.C.

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