Activation of sucrose-phosphate synthase from Prosopis juliflora in light: Effects of protein kinase and protein phosphatase inhibitors

Abstract

Sucrose-phosphate synthase (SPS) activity measured under limiting substrate and in the presence of inorganic phosphate as an allosteric inhibitor (Vlim activity) from the leaves of Prosopis juliflora was earlier observed to respond rapidly and reversibly to light/dark transitions (Sinha et al. 1997b,c). The experiments therefore, were conducted to study the potential regulation of the enzyme by a mechanism of phosphorylation/dephosphorylation. The desalted extract of the enzyme prepared from irradiated leaves showed a time-dependent spontaneous inactivation of the Vlim activity when the extract was preincubated and an additional inactivation when incubated with ATP. The spontaneous inactivation is not inhibited by phosphatase inhibitors but the ATP-dependent inactivation was abolished when either 5′-p-fluorosulphonylbenzoadenosine (FSBA) or glucose-6-phosphate (G6P), (both reported as inhibitors for the SPS-protein kinase from spinach) was included during preincubation. FSBA also prevented the dark inactivation of SPS in the leaves of P. juliflora when fed through the transpiration stream. The activity of SPS measured under the Vmax condition remained relatively unaffected by ATP or FSBA. The desalted extract prepared from darkened leaves on the other hand, when preincubated at 25°C showed a time-dependent increase in the Vlim activity and the activation state of the enzyme. The spontaneous activation observed during preincubation appears to be due to the dephosphorylation of the enzyme and is strongly inhibited by okadaic acid, a potent protein phosphatase inhibitor. Alternately, feeding okadaic acid to excised leaves in the dark also blocked the subsequent light activation of Vlim activity. These results are consistent with the assumption that the light/dark regulation of Vlim activity observed in the leaves of P. juliflora was mediated through a dephosphorylation/phosphorylation mechanism.