Characterization of serine proteinase inhibitors in dry seeds of cultivated pasture grass species

Abstract

AbstractA survey of proteinaceous inhibitors of the serine proteinases, bovine trypsin and chymotrypsin, that are extractable from dry seeds of several cultivars of pasture grasses has been undertaken. Using crude extracts, most cultivars screened contained inhibitors of chymotrypsin, whereas trypsin inhibition was not detectable. Seeds from four cultivars,Lolium perenneL. cv. Grasslands Ruanui,Lolium×boucheanumcv. Grasslands Greenstone,Festuca arundinaceaSchreb. cultivars Grasslands Roa and Grasslands Garland, that contained more potent chymotrypsin inhibition were purified further. After gel filtration chromatography, both trypsin and chymotrypsin inhibition could be observed in all four cultivars, and each separated into two discrete native molecular weights; one ofca.20–22 kDa and one ofca.8–10 kDa. However, activity staining, after polyacrylamide gel electrophoresis, revealed an array of iso-inhibitors with molecular weights that ranged fromca.3 kDa to 20 kDa. One of these, a dual trypsin/chymotrypsin inhibitor ofca.12 kDa that is present in all four cultivars examined, was purified to homogeneity fromF. arundinaceacv. Grasslands Garland using anhydro-trypsin affinity chromatography and reverse-phase HPLC. The protein was found to comprise two closely related peptides and N-terminal amino acid sequencing revealed highest identity with a trypsin inhibitor identified in rye (Secale cereale) seeds.