Human erythrocyte transglutaminase. Purification and properties

Abstract

Transglutaminase has been isolated from human erythrocytes, and some of its molecular and catalytic properties have been determined. An enzyme preparation of about 15% purity is readily obtained in about 25% yield after DEAE-cellulose fractionation and gel filtration. In order to achieve this yield of enzyme it is essential to add to the buffers a dialyzable stabilizing factor which is present in the early enzyme fractions. This natural factor can be partly replaced by chelating compounds and totally replaced by ATP, and in practice, the purification of the enzyme is best carried out with ATP present in the buffers. The role of ATP in stabilizing the enzyme is unknown. Complete purification of the erythrocyte transglutaminase can be accomplished by preparative acrylamide gel electrophoresis. The pure enzyme has a molecular weight of 82 000 ± 5000, as established by gel filtration and SDS gel electrophoresis, and its catalytic properties are essentially identical to those of guinea pig liver transglutaminase. The guinea pig liver and the erythrocyte enzymes have also been compared as catalysts for protein modification reactions, and have been found to have quite similar specificity requirements for protein substrates. Both enzymes catalyzed significant incorporation of amines into 4 of 20 soluble proteins tested and into proteins 1, 2 and 3 of the red cell membrane. The partially purified erythrocyte enzyme has been found to be completely satisfactory for protein modification experiments, and the ready availability of outdated human blood and the simple purification procedure should make this enzyme a convenient protein-modifying or crosslinking reagent.