ISOLATION AND CHARACTERIZATION OF AN INTRACELLULAR SERINE PROTEINASE-INHIBITOR FROM A MONKEY KIDNEY EPITHELIAL-CELL LINE

A recent report described a thrombin inhibitory activity in the solubl e fraction of human placenta and the cytosolic fraction of K562 cells. Isolation and characterization of the functionally inactive 35-38-kDa placental form of this protein revealed that it was a novel serine pr oteinase inhibitor (Coughlin, P. B., Tetaz, T., and Salem, H. H. (1993 ) J. Biol. Chem. 268, 9541-9547). In the present study, we observed a 67-kDa sodium dodecyl sulfate (SDS)-stable complex when I-125-thrombin was incubated with the cytosolic fraction of a monkey kidney epitheli al cell line, BSC-1. This complex was not observed in either the parti culate cell fraction extracted with 0.2% Triton X-100 or medium condit ioned by cells, suggesting that the thrombin-complexing factor is conf ined to the cytoplasm. The cytoplasmic antithrombin activity was purif ied to apparent homogeneity from the cytosol of BSC-1 cells previously pulsed with [S-35]methionine by a combination of heparin-agarose chro matography, Mono Q fast protein liquid chromatography, and anhydrotryp sin-Affi-Gel 10 affinity chromatography. Analysis of the affinity-puri fied preparation by SDS-polyacrylamide gel electrophoresis and fluorog raphy revealed a single protein with an apparent molecular mass of 38 kDa. The purified 38-kDa protein inhibited the amidolytic activities o f thrombin, trypsin, urokinase, and factor Xa but not that of elastase . Incubation of the 38-kDa protein with excess thrombin identified app roximately 60% of the labeled 38-kDa protein in an SDS-stable 67-kDa c omplex. The purified 38-kDa inhibitor was cleaved with cyanogen bromid e and the isolated peptides subjected to microsequencing. Amino acid s equence obtained for a region within this protein exhibited significan t homology with human antithrombin III and plasminogen activator inhib itors 1 and 2. This homologous peptide contained the full complement o f residues designated as highly conserved in helix F of the greater se rine proteinase inhibitor superfamily. In addition, an internal sequen ce of GGGGDIHQGF was found in the monkey cytoplasmic inhibitor, which is identical to that reported for an internal sequence of the human pl acental inhibitor. These findings confirm the existence of a novel cyt oplasmic serine proteinase inhibitor in mammalian cells and provide ad ditional details of its molecular properties. The physiological functi on of this novel serine proteinase inhibitor in cytoplasm is unknown.