Proteolysis and the diurnal decrease of asparaginase activity

The mechanism responsible for the decrease in asparaginase (EC 3.5.1.1) activity in darkened leaves of Pisum sativum L. cv. Little Marvel was investigated. Asparaginase activity, obtained from half-expanded leaves harvested at the end of the dark period, or during the light periods, was inactivated by bromelain (EC 3.4.22.4), ficin (EC 3.4.22.3), both thiol proteases, and trypsin (EC 3.4.21.4), a serine protease. Thrombin (EC 3.4.21.5), pepsin (EC 3.4.23.1), or carboxypeptidase A (EC 3.4.17.1) had no effect on dark- or light-harvested asparaginase preparations. Inactivation of asparaginase activity in crude or purified preparations by ficin was not observed in the presence of leupeptin (an inhibitor of thiol proteases). Supplying leupeptin to detached half-expanded leaves had no effect on the increase of asparaginase observed at the start of the light period, while it maintained asparaginase activity at high levels in leaves excised during or at the end of the light period. These results suggest that decreased asparaginase activity in vivo is brought about by thiol-dependent proteases.