Purification and Characterization of Aleurain

Abstract

Barley (Hordeum vulgare L. cv Himilaya) aleurain is a vacuolar thiol protease originally isolated as a cDNA with 65% derived amino acid sequence identity with cathepsin H (JC Rogers, D Dean, GR Heck [1985] Proc NatI Acad Sci USA 82: 6512-6516). We purified aleurain from barley leaves to homogeneity (>1000-fold) and characterized its activity against a number of substrates. Aleu- rain is best described as an aminopeptidase; it hydrolyzes three different aminopeptidase substrates with similar catalytic effi- ciency but is less efficient at hydrolyzing an NH2-blocked substrate analog and azocasein. Our values for Km and kcat for three sub- strates (arginine 4-methyl-7-coumarylamide, L-arginine,6-naphthy- lamide, and N-a-benzoyl-L-arginine #-naphthylamide) and specific activity with azocasein are all within a threefold range of those previously reported for human cathepsin H for these substrates (WN Schwartz, AJ Barrett [1980] Biochem 1 191: 487-497). Aleu- rain also shows a number of other similarities to cathepsin H including heterogeneity of charge forms, position of the NH2- terminus of the mature protein, and pH-activity profile. The similar properties of aleurain and cathepsin H suggest that these enzymes have a similar function(s) that is required by both plant and animal cells. The availability of a plant system may permit functional ablation experiments in the future to clarify the role of this enzyme in higher eukaryotes. Thiol