Capillary electrophoresis assay for ubiquitin carboxyl-terminal hydrolases with chemically synthesized ubiquitin-valine as substrate.
Ubiquitin is expressed in eukaryotic cells as precursors, fused via its carboxyl terminus either to other ubiquitin sequences in linear polyubiquitin arrays or to specific ribosomal proteins. In some of the polyubiquitin fusions a single amino acid (e.g., valine in humans) is attached to the carboxyl terminus. These gene products are rapidly (probably cotranslationally) cleaved by ubiquitin carboxyl-terminal hydrolase (UCH) enzymes; therefore, although ubiquitin precursors are suitable substrates for assays of UCH activity, they are difficult to isolate from nucleated cells. While the recombinant approach allows the production of ubiquitin precursors in prokaryotic cells (which do not contain the ubiquitin system), proteins produced in this manner require purification and may also be susceptible to modification by bacterial enzymes, e.g., adventitious proteolysis. As an alternative we have chemically synthesized human ubiquitin-valine. In the assay described here the cleavage of ubiquitin-valine to ubiquitin (77 and 76 residue proteins, respectively) by a purified recombinant Drosophila UCH was monitored by capillary electrophoresis. Mass spectrometry verified the precise cleavage of ubiquitin-valine, confirming that this synthetic protein is a UCH substrate. Synthetic ubiquitin-valine may serve as a generic substrate for UCHs allowing the purification and identification of new members of this enzyme family.
Franklin
K
K
Department of Biochemistry, University of Nottingham Medical School, Queen's Medical Centre, United Kingdom.
Layfield
R
R
Landon
M
M
Ramage
R
R
Brown
A
A
Love
S
S
Muir
T
T
Urquhart
K
K
Bownes
M
M
Mayer
R J
RJ
D000818:Animals; D004330:Drosophila / Q000201:enzymology; D029721:Drosophila Proteins / Q000032:analysis* / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry; D015394:Molecular Structure; D011498:Protein Precursors / Q000138:chemical synthesis / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000032:analysis* / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000032:analysis* / Q000378:metabolism; D014452:Ubiquitins / Q000138:chemical synthesis / Q000737:chemistry; D014633:Valine / Q000737:chemistry
D029721:Drosophila Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C584287:UCH protein, Drosophila; D014452:Ubiquitins; D013869:Thiolester Hydrolases; D043222:Ubiquitin Thiolesterase; D014633:Valine