Identification of transglutaminase substrates in HT29 colon cancer cells: use of 5-(biotinamido)pentylamine as a transglutaminase-specific probe.
A biotinamine probe, 5-(biotinamido)pentylamine, was used for biotin-labeling of proteins in HT29 colon cancer cell extracts by endogenous transglutaminase activity. The biotin-labeled protein substrates were isolated and recovered by avidin-affinity chromatography. The proteins were separated using SDS-polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene difluoride membrane, visualized using Coomassie blue, cut out, and sequenced. Amino acid sequence data identified human fructose-1,6-bisphosphate aldolase A, an intracellular protein, as a substrate for cellular transglutaminase.
Lee
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Samuel Roberts Noble Foundation, Biomedical Division, Ardmore, OK 73402.
Maxwell
M D
MD
Patterson
M K
MK
Birckbichler
P J
PJ
Conway
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E
D000588:Amines; D000595:Amino Acid Sequence; D001710:Biotin / Q000031:analogs & derivatives; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003110:Colonic Neoplasms; D005634:Fructose-Bisphosphate Aldolase / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D015335:Molecular Probes; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000588:Amines; D015335:Molecular Probes; C076022:5-(biotinamido)pentylamine; D001710:Biotin; D011503:Transglutaminases; D005634:Fructose-Bisphosphate Aldolase