Application of Peptides Containing the Cleavage Sequence of Pro-TNFα in Assessing TACE Activity of Whole Cells
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B. F. Becker
Abstract
Tumor necrosis factorα (TNFα) is presumably shed from cell membranes by TNFαcleaving enzyme (TACE). The peptides SPLAQAVRSSSR and DabcylLAQAVRSSSREdans, each encompassing the cleavage sequence of proTNFα recognized by TACE, were applied to intact umbilical vein endothelium (HUVEC), peripheral blood leukocytes (PBL) and the mast cell line HMC-1, which express TACE, to homogenates of rat heart tissue and to membrane and cytoplasmic extracts of PBL. Formation of SPLAQA (specific cleavage) was determined by HPLC, while cleavage (specific plus nonspecific) of DabcylTNFαEdans was followed over time by measuring fluorescence. Participation of TACE was assessed from inhibition due to the drug TAPI-2. Incubation with recombinant human TACE gave specific cleavage, fully inhibitable by TAPI-2 (IC50<0.1 M). HUVEC rapidly degraded TNFαpeptide, but in a nonspecific manner (no SPLAQA detectable) and 50 M TAPI-2 was without effect. Fluorescence was evoked when Dabcyl LAQAVRSSSREdans was incubated with HMC-1 or PBL and also with cytoplasmic and membrane fractions of lysed PBL, but in no case was there significant inhibition by TAPI-2. However, marginal (10%) inhibition of fluorescence by 50 M TAPI-2 was observed with homogenized heart tissue. This contained TACE, about 75% of which was without the inhibitory cysteine switch (Western blot). In conclusion, simple peptide analogs of proTNFα cannot be employed as substrates for measuring membrane TACE activity, largely due to extensive nonspecific proteolytic cleavage by whole cells and cell extracts.
Copyright © 2002 by Walter de Gruyter GmbH & Co. KG
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