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

The chemical, physical and biological factors, which can affect the activity of transglutaminase (R-glutaminyl-peptide:amine γ-glutamyl-transferase, EC 2.3.2.13) in chloroplasts (ChlTGase) when photosynthesis is active, were assayed in chloroplasts isolated from the leaves of Helianthus tuberosus. Chloroplasts were incubated with putrescine (PU) in the presence of light to monitor the transglutaminase-catalysed incorporation of this polyamine into endogenous proteins. The enzyme was identified using a monoclonal antibody raised against the active site sequence of TGase K and was found to contain a thiol group, which can be slightly activated by Ca2+ and severely inhibited by EGTA. Mg2+ had a slight inhibitory effect. The enzymic activity, monitored by the isolation of glutamyl-putrescine, while already detectable above pH 7 was found to increase sharply from pH 8.0 to 9.5, with an optimal temperature of 45 °C. A hyperbolic curve was observed when the activity was measured as a function of the putrescine concentration, the apparent Km being 1 mM. A biphasic relationship was obtained between the TGase activity and the concentration of the substrate (endogenous proteins) as well as the time of assay. The reaction products of the TGase assay, carried out at three pH values, were analysed for the presence of γ-glutamyl-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 inhibitors were also tested and found to reduce the post-translational modification of the substrates.