Hepatocyte growth factor (HGF) activator is a serine protease that is produced and secreted by the liver and circulates in the blood as an inactive zymogen. In response to tissue injury, the HGF activator zymogen is converted to the active form by limited proteolysis. The activated HGF activator converts an inactive single chain precursor of HGF to a biologically active heterodimer in injured tissue. The activated HGF may be involved in the regeneration of the injured tissue. In this study, we purified an inhibitor of HGF activator from the conditioned medium of a human MKN45 stomach carcinoma cell line and molecularly cloned its cDNA. The sequence of the cDNA revealed that the inhibitor has two well defined Kunitz domains, suggesting that the inhibitor is a member of the Kunitz family of serine protease inhibitors. The sequence also showed that the primary translation product of the inhibitor has a hydrophobic sequence at the COOH-terminal region. Inhibitory activity toward HGF activator was detected in the membrane fraction as well as in the conditioned medium of MKN45 cells. These results suggest that the inhibitor may be produced as a membrane-associated form and secreted by the producing cells as a proteolytically truncated form.

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We have previously shown that the Saccharomyces cerevisiae USO1 gene required in the protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus encodes a 200-kDa protein (1,790 amino acids) which is present in a nonglobular high molecular mass complex. Antibodies against an N-terminal portion of Uso1 protein recognized a 100-kDa protein in Western blot of the temperature-sensitive uso1-1 mutant cell lysate. The nucleotide sequence of uso1-1 indicated the 951st codon was UAG (amber) in place of CAG (glutamine) in USO1. Deletion study of USO1 gene indicated that such truncated Uso1 polypeptides are sufficiently functional at 25 degrees C but not at 37 degrees C. Mutant Uso1-1 protein displayed an apparent molecular mass of 400-500 kDa in gel filtration while it cosedimented with a globular 6S marker protein, horseradish peroxidase (44 kDa), in sucrose density gradient centrifugation. These results indicated that truncated Uso1-1 protein is still present in a nonglobular high molecular mass complex, similar to the wild-type Uso1 protein.

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