The interaction of the 11S globulin-like protein of kiwifruit seeds with pepsin

Abstract

In a search for aspartic proteinase inhibitors (APIs) in kiwifruit seeds, we observed pepsin inhibitory activity (PIA) in an abundant globulin fraction extracted in high salt buffer with a Mr of ∼148 kDa by gel-filtration. On a SDS-polyacrylamide gel, a major protein band of 54 kDa was observed under non-reducing conditions. This band was largely replaced by two subunits of Mr 33.5 and 20 kDa under reducing conditions. N-terminal sequencing of the smaller subunit, which had associated PIA, revealed a β-subunit of the 11S globulin-like protein (11S-GLP), legumin. After trypsin, chymotrypsin or papain digestion, the α-subunit of the kiwifruit legumin (11S-GLP) was degraded to varying degrees but there was no effect on the β-subunit, or on the PIA. This 11S-GLP also appeared to inhibit bovine spleen cathepsin D, Candida albicans secreted aspartic proteinases (SAPs) 1, 2 and 4, the plant fungus Glomerella cingulata SAP as well as apple seed aspartic proteinase, but to a much lesser extent kiwifruit seed aspartic proteinase. Through kinetic analysis of pepsin inhibition, the 11S-GLP and the purified β-subunit were found to fit a Michaelis–Menten model for competitive inhibition rather than a tight-binding model characteristic for typical proteinase inhibitors. Extrapolated complete inhibition was only obtained at an 11S-GLP concentration ∼900 times that of the pepsin. Further investigation revealed that the 11S-GLP and the β-subunit acted as weak alternative substrates for pepsin. As the 11S-GLP or the β-subunit were degraded, the PIA activity declined in parallel. We discuss these results in terms of the substrate recognition site of pepsin compared with other proteinases.