C - Chemistry – Metallurgy – 12 – N
Patent
C - Chemistry, Metallurgy
12
N
150/13, 167/40,
C12N 9/10 (2006.01) C07K 9/00 (2006.01) C12Q 1/48 (2006.01) G01N 33/573 (2006.01) G01N 33/574 (2006.01)
Patent
CA 1339109
An onco-developmentally regulated .alpha.-N-acetyl-galactosaminyltransferase isolated as a component of a particulate membrane fraction separated from cell and tissue homogenates and having the following characteristics: (a) Activity in Various Cells and Tissues -- present in human fetal lung fibroblasts, hepatoma tissues and placenta, but absent from normal adult liver, lung, kidney and spleen tissues; (b) Substrate Specificity -- acts on polypeptides comprising a sequence val-thr-his-pro-gly-tyr by catalyzing .alpha.-N-acetylgalactosaminylation at the thr residue of the sequence; (c) Requirements for Metal Ion -- requires metal ion for activity in 25 mM tris buffer, pH 7.6; (d) Optimal pH -- optimal pH is about 7. 6 assayed in hepatoma cell homogenate in tris buffer and at a pH of about 6 to about 7 assayed in hepatoma cell homogenate; enzyme activity is higher in 2(N-morpholino)ethanesulfonic acid than in cacodylate buffer; and (e) Rm -- apparent Km for UDP - GalNAc is about 48 µM. A method of detecting tumor cells comprising the steps of: (1) homogenizing tissue suspected of having tumor cells; (2) incubating the homogenate with labeled .alpha.-N-acetylgalactosamine (.alpha.-GalNAc) and a polypeptide substrate comprising a sequence of val-thr-his-pro-gly-tyr under conditions sufficient to catalyze .alpha.-N-acetylgalactosaminylation of the thr residue to thereby form labeled polypeptide substrate comprising val-thr(.alpha.-GalNAc)-his-pro-gly-tyr; and (3) determining the presence of said labeled polypeptide substrate. A diagnostic test kit for detecting tumor cells in a sample suspected of containing tumor cells comprising as separate components: (a) labeled .alpha.-N-acetylgalactosamine; and (b) polypeptide substrate comprising a sequence of val-thr-his-pro-gly-tyr. - 40 -
602670
Hakomori Sen-Itiroh
Matsuura Hidemitsu
Fred Hutchinson Cancer Research Center
G. Ronald Bell & Associates
The Biomembrane Institute
University Of Washington
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