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 Natl 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. Aleurain is best described as an aminopeptidase; it hydrolyzes three different aminopeptidase substrates with similar catalytic efficiency but is less efficient at hydrolyzing an NH(2)-blocked substrate analog and azocasein. Our values for K(m) and k(cat) for three substrates (arginine 4-methyl-7-coumarylamide, l-arginine beta-naphthylamide, and N-alpha-benzoyl-l-arginine beta-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 J 191: 487-497). Aleurain also shows a number of other similarities to cathepsin H including heterogeneity of charge forms, position of the NH(2)-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.