The interaction of the 11S globulin-like protein of kiwifruit seeds with pepsin Maysoon Rassam * , William A. Laing The Horticultural and Food Research Institute of New Zealand, PB 92169 Auckland, New Zealand Received 8 February 2006; received in revised form 15 June 2006; accepted 16 June 2006 Available online 12 July 2006 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 b-subunit of the 11S globulin-like protein (11S-GLP), legumin. After trypsin, chymotrypsin or papain digestion, the a-subunit of the kiwifruit legumin (11S-GLP) was degraded to varying degrees but there was no effect on the b-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 b-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 b-subunit acted as weak alternative substrates for pepsin. As the 11S-GLP or the b-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. # 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Actinidia deliciosa; Actinidia chinensis; 11S globulins; Legumin; Aspartic proteinase inhibitor; Seeds 1. Introduction Large (11S–15S) globulin-like proteins (11S-GLPs) also named legumin-like proteins, have been reported as storage proteins from a wide range of seeds including wheat [1,2], lupin [3], Ginkgo biloba [4], faba bean [5], alfalfa [6], buckwheat [7], rye and corn [1]. They are trimers or hexamers with Mr between 300 and 400 kDa. 11S globulin subunits consist of two polypeptide chains linked by at least one S–S bridge between cysteine residues at highly conserved positions in the acidic a- chain and basic b-chain. Both chains are post-translationally generated from a common precursor protein, which is the product of one member of a multigene family [8]. Aspartic proteinases are widely distributed in living organisms including viruses, fungi, plants and animals [9]. They are a class of endopeptidases, usually with an acidic pH optimum and inhibited by pepstatin, a pentapeptide from streptomyces [10]. Aspartic proteinase activity has been found in seeds from a broad variety of plant species [11–15]. Tight-binding proteinaceous inhibitors of proteolytic enzymes are ubiquitous in nature. They generally bind at a 1:1 molar ratio and at low proteinase and inhibitor concentrations often show slow binding kinetics taking minutes to completely inhibit the target enzyme. The proteinase:inhibitor complex is stable and the inhibitor is rarely degraded by the target proteinase, except in some serine PIs where the bait sequence is cleaved [16]. Some inhibitors modulate the activity of endogenous proteinases operating in various physiological processes, while others are defence proteins with protective action against harmful endogenous or pathogenic proteinases [17–19]. www.elsevier.com/locate/plantsci Plant Science 171 (2006) 663–669 Abbreviations: AP, aspartic proteinase; API, aspartic proteinase inhibitor; GLP, globulin-like protein; LSE, low salt extract; Mr, molecular mass; PIs, protease inhibitors; PIA, pepsin inhibitory activity; SAP, secreted aspartic proteinase; TCA, trichloroacetic acid; WAPI, wheat aspartic proteinase inhi- bitor * Corresponding author. Tel.: +64 9 815 4200; fax: +64 9 815 4202. E-mail address: mrassam@hortresearch.co.nz (M. Rassam). 0168-9452/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2006.06.014