Diamine Oxidase and Transglutaminase Activities in White Lupine Seedlings with Respect to Crosslinking of Proteins

Enzymes extracted from 10-d-old Lupinus albus seedlings were able to (1) polymerize casein and (2) incorporate [ 14 C]putrescine into dimethylcasein (modified casein). High molecular weight polymers formed were visualized by SDS-polyacrylamide gel electrophoresis. [ 14 C]Putrescine incorporation was not only due to transglutaminase activity but also due to diamine oxidase. The use of diamine oxidase inhibitor allowed us to localize a transglutaminase activity in the pellet after centrifugation at 41400g of the filtered homogenate of seedlings and a diamine oxidase activity mainly in the supernatant. Covalent conjugation of monodansylcadaverine and [ 14 C]putrescine to dimethylcasein by enzymes contained in the 41400g pellet was vizualized by fluorescent detection or by autoradiography in SDS-polyacrylamide gel electrophoresis. The enzyme contained in the pellet fraction was able to polymerize not only casein but also spinach ribulose-1,5-bisphosphate carboxylase/oxygenase and 7S soybean globulins. As a control we used purified diamine oxidase from porcine kidney to check the inability of this enzyme to catalyze casein polymerization. Moreover, 54% of transglutaminase activity contained in the pellet has been solubilized by 5% (v/v) detergent (Triton X-100) contrary to high concentrated salts. Transglutaminase was recovered in the 108000g supernatant. This suggests that this enzyme was an integral membrane protein.