Development of a sensitive peptidase assay: in search of cell associated proteases responsible for the cleavage of preproTGF alpha.
A radiometric assay has been developed for the detection of proteolytic activity capable of releasing transforming growth factor alpha (TGF alpha) from its membrane bound precursor. The assay is dependent upon the separation by thin layer chromatography of hydrolytic products of a nonapeptide substrate containing a radioactive iodinated tyrosine residue as a reporting group N-terminal to an octapeptide which is cognate to the N-terminal cleavage sequence of TGF alpha. We describe the selectivity of the peptidase assay with commercially purified proteases and with cell-associated peptidases, its exquisite sensitivity, and its applicability to defining peptidase activity, which may be responsible for the processing of the membrane-bound prepro TGF alpha. The activity of two different elastases had different profiles which thus may be of use in characterizing them. The characteristics of the intact and extracted HeLa cell assay with respect to time, cell density, and peptidase concentration are defined, as are conditions needed to remove endogenous, confounding, proteolytic activity from the serum used to support cell culture. Intact HeLa cell cultures exhibit both exo- and endo-peptidase activity at approximately equal levels in both sparse and dense monolayer culture without relationship to cell density, and at a level equal to 1-2% of total cell activity of these enzyme classes.
Brown
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SB
Queensland Cancer Fund Research Unit, Queensland Institute of Medical Research, Bancroft Centre, Brisbane, Australia.
Krause
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Townsend
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Ellem
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KA
D000595:Amino Acid Sequence; D002855:Chromatography, Thin Layer; D010450:Endopeptidases / Q000032:analysis*; D008722:Methods; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism*; D016211:Transforming Growth Factor alpha / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D011498:Protein Precursors; D016211:Transforming Growth Factor alpha; D010450:Endopeptidases