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Phosphoramidon-sensitve endothelin-converting enzyme of bovine endothelial cells showed substrate selectivity for big endothelin-1 (ET-1) when compared to big ET-1(1–380, big ET-2(1–37), big ET-2(1–38) and big ET-3(1–41). To investigate the big ET-1 structure important for specific conversion by the endothelin-converting enzyme, we synthesized a series of truncated analogues of big ET-1, measured the hydrolysis of their Trp21-Val22 bonds, and found that a 16-residue peptide, big ET-1(19–34), is the minimal peptide sequence. This suggests that an unusually long carboxy-terminal sequence is required for big ET-1 conversion. Alanine substitution for individual amino acids in the carboxy-terminal region of big ET-1(19–34) demonstrated that Hiss27, Val29, Pro30, Tyr31, Gly32, Leu33 and Gly34 are more important than Asn23, Thr24, Pro25, Glu26 and Val28 for eliciting efficient hydrolysis of the Trp21-Val22 bond, even though the former residues are located at more distant positions from the cleavage sites than are the latter. These results, together with the fact that big ET-2 and big ET-3 show heterogeneity in the big ET-1 residues His27, Val28, Val29 and Gly34, suggest that the His27-Val-Val-Pro-Tyr-Gly-Leu-Gly34 sequence in the carboxy-terminal region of big ET-1 plays the most important role in selective conversion by endothelin converting enzyme. [ABSTRACT FROM AUTHOR]

A plasminogen activator inhibitor (PAI) was purified from bovine endothelial cell conditioned medium by a simple procedure in the absence of protein denaturant. The yield was 2.2 mg from 1.61 conditioned medium in a typical experiment. The purified inhibitor showed a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and reverse fibrin autography with an apparent molecular mass of 45 Wa. The amino-terminal 40-amino-acid sequence was determined and found to be 70% similar to the reported corresponding sequence of human PAI-1. The amino acid composition also revealed a close relationship between bovine PAI and human PAI-1. The purified PAI was substantially inactive (570 U/mg) but it could be activated by treatment with protein denaturants such as 1% SDS (1.8 × 105 U/mg) and 4 M guanidine-HCl (1.5 × 105 U/mg). A more effective activation of this latent PAI was achieved by heat treatment at 100°C for 2.5 min, generating the specific activity of 1.0 × 106 U/mg. The heat-activated PAI lost its activity during incubation at 56°C for 30 min, but repeated heat at 100°C for 2.5 min could regenerate about 70% of the initial activity. Treatment at 37°C, 56°C and 80°C. however, failed to activate the latent PAI at all. These findings suggest that the buried reactive site of the latent PAI is exposed as a result of a heat-induced, specific conformational change, but tends to be masked again during renaturation under mild conditions, i.e. the PAI protein takes on preferentially a latent form. [ABSTRACT FROM AUTHOR]