This study describes the localization of the previously purified T cell‐specific serine proteinase, termed TSP‐1 (M. M. Simon et al., EMBO J. 1986. 5: 3267), within cytoplasmic granules of cytolytic T cell lines (CTLL). Subcellular fractionation of disintegrated CTLL (ruptured by nitrogen cavitation) was accomplished by Percoll density gradient centrifugation of cell lysates (postnuclear supernatant). Individual fractions were tested for proteinase activity on chromogenic peptide substrates and for the presence of TSP‐1 by Western blot analysis. In addition, each fraction was assayed for cytolytic activity against sheep red blood cells (SRBC), for protein and for additional marker enzymes to assess the enrichment for cellular organells. All serine enzyme‐type molecules including TSP‐1 expressed by CTLL were identified by labeling cell lysates or gradient fractions with the serine proteinase‐specific affinity ligand tritiated diisopropyl fluorophosphate ([3H]DFP) in the presence or in the absence of class‐specific or enzyme‐specific proteinase inhibitors and subsequent sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The data demonstrate that the Percoll gradient fraction, which was shown by morphological examination in the electron microscope to be highly enriched for cytoplasmic granules, also contained > 80% of proteinase activity in addition to the granule‐associated structures cytolysin and aryl‐sulfatase. The identity of the granule‐associated proteinase in two independent cell lines, CTLL HY3‐Ag3 and CTLL 1.D.9, with the serine proteinase TSP‐1 is indicated by (a) its specificity for the chromogenic substrate H‐D‐Pro‐Phe‐Arg‐p‐nitroanilide, (b) its sensitivity to class‐specific as well as TSP‐1‐specific enzyme inhibitors and (c) by its reactivity with a polyvalent TSP‐1‐specific rabbit antiserum. Both CTLL contain a [3H]DFP‐labeled protein that migrates with a molecular mass of 60 kDa under nonreducing conditions and with 30 kDa under reducing conditions and which can be inactivated by the TSP‐1‐specific inhibitor H‐D‐Pro‐Phe‐Arg‐chloromethylketone. CTLL HY3‐Ag3 (a long‐term culture CTLL with natural killer‐like activity) but not CTLL 1.D.9 (an antigen‐specific short‐term cultured CTLL) express in addition a further [3H]DFP‐binding protein which migrates with 27 kDa under nonreducing or reducing conditions. No substrate specificity was found for this molecule. The possible function of the granule‐associated serine proteinase TSP‐1 is discussed.
ASJC Scopus subject areas
- Immunology and Allergy