Purification and characterization of hordolisin, a subtilisin-like serine endoprotease from barley

A protease was purified from green barley (Hordeum vulgare L.) malt with an apparent molecular mass or 74 kD and a pI of 6.9. Activity was assayed usi ng an internally quenched fluorogenic peptide substrate, and the inhibitor profile indicated that the catalytic site contained a serine residue. This was confirmed by labelling with [C-14]-diisopropyl fluorophosphate. The N-t erminal amino acid sequence of the purified protease was homologous to plan t subtilisin-like serine endoproteases, such as cucumisin. The barley prote ase (trivial name hordolisin) had a pH optimum of 6 and tvas stable up to 6 0 degrees C. The substrate specificity of hordolisin was determined from P- 4 to P-3' using a series of internally quenched fluorogenic peptide substra tes. Hordolisin was similar to Savinase and subtilisin BPN', except that Al a was better accepted at P-1, and Arg was preferred at P-1'. Hordolisin doe s not appear to be important in the degradation of the hordein storage prot eins during barley grain germination.