Partial characterization of a major gut thiol proteinase from larvae of Callosobruchus maculatus F.

Much of the proteolytic activity in the digestive tract of Callosobruchus maculatus larvae can be attributed to a thiol proteinase(s) that hydrolyzes [3H]methemoglobin optimally at pH 5.0. Maximal hydrolysis of [3H]methemoglobin, [3H]alpha‐casein, and N‐benzoyl‐DL‐arginine napthylamide‐(BANA) required the presence of thiol reducing agents. Larval gut proteinase activity was strongly inhibited by p‐hydroxymercuribenzoic acid (pHMB), Nethylmaleimide (NEM), and iodoacetic acid (IAA) but was unaffected by the Bowman‐Birk and Kunitz proteinase inhibitors from soybeans or by lima bean trypsin inhibitor. L‐Trans‐epoxysuccinyl‐leucylamido‐(4‐guanidino)‐butane (E‐64), a specific inhibitor of thiol proteinases, potently inhibited proteolysis of [3H]methemoglobin by larval gut homogenates. Proteolytic activity in the larval gut was located in the lumen contents and thus appears to play a major role in extracellular digestion. The pH of the larval midgut is slightly acidic, and midgut contents exhibit a negative redox potential, conditions supporting the activity of a thiol proteinase. The significance of these findings is discussed with reference to the vulnerability of this digestive proteinase as a target for existing or genetically engineered plant chemical defenses.