Development of a sensitive peptidase assay: In search of cell associated proteases responsible for the cleavage of prepro TGF_α

Abstract

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.

Characterization and localization of mitochondrial oligopeptidase (MOP) (EC 3.4.24.16) activity in the human cervical adenocarcinoma cell line HeLa

“…4), even though the level of protein in the fractions was below measurable limits. The high sensitivity and rapid analysis of the P9 assay [Brown et al, 1992] made it a valuable tool in this study. MOP was shown to cleave between the small hydrophobic amino acids in P9 (YVA †A †A †VVSH) as well as aromatic and charged residues in neurotensin (ELYENKPR-RP †YIL) and GnRH 1-9 (EHWSY †GLRP).…”

“…Bestatin was added to the assay mixture to inhibit any contaminating aminopeptidase activity [Suda et al, 1976]. Extracts (35 µl) from the assay medium were applied to channelled TLC plates and separated by ascending chromatography [butanol:H 2 O:acetic acid, 100:30:10 (BAW), pH 2.6] [Brown et al, 1992]. Briefly, after development, the TLC plate was subjected to phosphorimaging (Storage phosphorscreen, Kodak).…”

“…As there was no detectable induction of mRNA or protein synthesis of the TGFaase following UV exposure it was suggested that there may be intracellular reservoir of the enzyme which migrates to the cell surface to explain the increased levels of activity. We therefore attempted to purify this putative TGFaase from HeLa cell extracts using a synthetic nine amino acid peptide (P9) which is cognate with the N-terminal cleavage site of preproTGFa [Brown et al, 1992]. An enzyme activity was found in HeLa cell lysates which cleaved P9, and the nature of the enzyme responsible was sought.…”

Increased ecto-metallopeptidase activity in cells undergoing apoptosis

“…The P9 octapeptide (YVAAAVVSH), which is cognate to the N-terminal cleavage site of preproTGF␣, was labeled with 125 I as previously described [Brown et al, 1992]. The rate of P9 hydrolysis by the sorted cell populations was assayed as follows: 20 µl of labeled P9 (1 µl 125 I-P9 (10 pmoles) in 20 µl HBSS) was added to a 1.5-ml Eppendorf tube along with 50 µl of FACS-sorted cell population (prewarmed to 37°C for 15 min) for 15 min (P9 final concentration 140 nM).…”

“…At the end of the experiment, 40 µl of the reaction mixture was spotted on a multichanneled thinlayer chromatography (TLC) plate. The plates were dried and the peptide fragments separated by ascending chromatography [butanol: H 2 O:acetic acid, 100:30:10, pH 2.6], then imaged and analyzed by phosphorimaging analysis as previously described [Brown et al, 1992].…”