Journal of Cereal Science vol:32 issue:1 pages:31-42
A second, distinct, aspartic proteinase (G1AP 2) associated with wheat gluten was partially purified by ammonium sulphate precipitation, anion exchange and hydrophobic interaction chromatography, a pH precipitation step and gel permeation chromatography. The enzyme has a molecular mass of c. 67 kDa and shows maximal hydrolytic activity towards haemoglobin at pH 3.0-3.5 and c. 50 degrees C. The enzyme is completely inhibited by pepstatin A and is less active towards haemoglobin than a previously purified gluten aspartic proteinase (G1AP). G1AP 2 is highly specific in its action towards oxidized insulin B-chain. This synthetic substrate is cleaved only at the Leu(15)-Tyr(16) position. The selectivity of G1AP 2 is therefore much higher than that of other plant APs. Furthermore, in contrast to the previously described G1AP. G1AP 2 shows almost no activity towards wheat gluten although it hydrolysed high molecular weight glutenin subunit 7. One Met-Ile peptide bond in the N-terminal domain and probably two Val-Leu peptide bonds in the repetitive domain of this subunit were cleaved by G1AP 2. (C) 2000 Academic Press.