Factors affecting transglutaminase activity catalysing polyamine conjugation to endogenous substrates in the entire chloroplast

The chemical, physical and biological factors, which can affect the activit y of transglutaminase (R-glutaminyl-peptide:amine gamma-glutamyl-transferas e, EC 2.3.2.13) in chloroplasts (ChITGase) when photosynthesis is active, w ere assayed in chloroplasts isolated from the leaves of Helianthus tuberosu s. Chloroplasts were incubated with putrescine (PU) in the presence of ligh t to monitor the transglutaminase-catalysed incorporation of this polyamine into endogenous proteins. The enzyme was identified using a monoclonal ant ibody raised against the active site sequence of TGase K and was found to c ontain a thiol group, which can be slightly activated by Ca2+ and severely inhibited by EGTA. Mg2+ had a slight inhibitory effect. The enzymic activit y, monitored by the isolation of glutamyl-putrescine, while already detecta ble above pH 7 was found to increase sharply from pH 8.0 to 9.5, with an op timal temperature of 45 degrees C. A hyperbolic curve was observed when the activity was measured as a function of the putrescine concentration, the a pparent K-m being 1 mM. A biphasic relationship was obtained between the TG ase activity and the concentration of the substrate (endogenous proteins) a s well as the time of assay. The reaction products of the TGase assay, carr ied out at three pH values, were analysed for the presence of gamma-glutamy l-putrescine; mono- and bis-derivatives were detected, showing that most of the modifications of Chl proteins are catalysed by the enzyme. Due to the stimulatory effect that proteases have on some animal TGases, protease inhi bitors were also tested and found to reduce the post-translational modifica tion of the substrates. (C) 2000 Editions scientifiques et medicales Elsevi er SAS.