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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 soluble 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 proteinase inhibitor (...
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Published in: | The Journal of biological chemistry 1993-10, Vol.268 (29), p.21560-21568 |
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Main Authors: | , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | A recent report described a thrombin inhibitory activity in the soluble 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 proteinase 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 125I-thrombin was incubated with
the cytosolic fraction of a monkey kidney epithelial cell line, BSC-1. This complex was not observed in either the particulate
cell fraction extracted with 0.2% Triton X-100 or medium conditioned by cells, suggesting that the thrombin-complexing factor
is confined to the cytoplasm. The cytoplasmic antithrombin activity was purified to apparent homogeneity from the cytosol
of BSC-1 cells previously pulsed with [35S]methionine by a combination of heparin-agarose chromatography, Mono Q fast protein
liquid chromatography, and anhydrotrypsin-Affi-Gel 10 affinity chromatography. Analysis of the affinity-purified preparation
by SDS-polyacrylamide gel electrophoresis and fluorography revealed a single protein with an apparent molecular mass of 38
kDa. The purified 38-kDa protein inhibited the amidolytic activities of thrombin, trypsin, urokinase, and factor Xa but not
that of elastase. Incubation of the 38-kDa protein with excess thrombin identified approximately 60% of the labeled 38-kDa
protein in an SDS-stable 67-kDa complex. The purified 38-kDa inhibitor was cleaved with cyanogen bromide and the isolated
peptides subjected to microsequencing. Amino acid sequence obtained for a region within this protein exhibited significant
homology with human antithrombin III and plasminogen activator inhibitors 1 and 2. This homologous peptide contained the full
complement of residues designated as highly conserved in helix F of the greater serine proteinase inhibitor superfamily. In
addition, an internal sequence of GGGGDIHQGF was found in the monkey cytoplasmic inhibitor, which is identical to that reported
for an internal sequence of the human placental inhibitor. These findings confirm the existence of a novel cytoplasmic serine
proteinase inhibitor in mammalian cells and provide additional details of its molecular properties. The physiological function
of this novel serine proteinase inhibitor in cytoplasm is unknown. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(20)80578-X |