Ubiquitin-peptide extensions as enzyme substrates

Details Ubiquitin-peptide extensions as enzyme substrates Ubiquitin-peptide extensions as enzyme substrates

1991-11-13

1994-11-22

Wityshyn, Michael G.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving hydrolase

435 15, 435101, 435105, 435193, 530335, C12Q 137, C12Q 148, C12P 1904

Patent

active

053668715

ABSTRACT:
A method for assaying for enzymes that modify peptide chains, such as protein kinases and enzymes which modify the C-terminus of the Ha-RAS protein, is defined. This is done by incubating an extract in which the enzyme being assayed for may be present contained in a reaction mixture. The reaction mixture is made up of a buffer solution, a ubiquitin peptide extension, wherein the peptide contains a sequence known to be modified by an agent in the presence of the enzyme being assayed for, and the agent known to modify the peptide extension when the enzyme is present. The incubation is stopped and the ubiquitin peptide extension is separated from the solution and analyzed for the presence of the agent modified peptide. The extent of peptide modification can be both qualitative and quantative of the enzyme being assayed for. Protein kinases can be assayed for using a ubiquitin pepide extension containing the sequence (SEQ ID NO:1), Ser-Glu-Glu-Glu-Glu-Glu in the presence of a phosphorylating agent. Farnesyl-protein transferase can be assayed for using a ubiquitin-peptide extension with the sequence: Pro-Gly-Cys-Met-Ser-Cys-Lys-Cys-Val-Leu-Ser, (SEQ ID NO:11) which are the eleven C-terminal residues of the RAS molecule, in the presence of a farnesylating agent such as .sup.3 H-farnesyl pyrophosphate. Carboxyl methyl transferase can be assayed for using a farnesylated ubiquitin-peptide extension in the presence of a methylating agent such as tritium labeled as [.sup.3 H-methyl]AdeMet.

REFERENCES:
patent: 5043268 (1991-08-01), Stock
patent: 5141851 (1992-08-01), Brown
patent: 5185248 (1993-02-01), Barbacid
Yoo, Y., Synthesis of Peptides as Cloned Ubiquitin Extensions, J. of Biol Chemistry, vol. 264 #29 1989, pp. 17078-10783.
Marin, O., Synthetic Peptides Including Acidic Clusters . . . Int J. Protein Res 36 #4, 1990, pp. 374-380.
Yoo, Y., Ubiquitin-Ras Peptide Extensions as Substrates . . . Biochem Journal vol. 285 Part 1 1992 pp. 55-60.

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