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

Sucrose-phosphate synthase (SPS) activity measured under limiting substrate and in the presence of inorganic phosphate as an allosteric inhibitor (V-l im activity) from the leaves of Prosopis juliflora was earlier observed to respond rapidly and reversibly to light/dark transitions (Sinha et al, 1997 b,c), The experiments therefore, were conducted to study the potential regu lation 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 V-lim activity when the ext ract was preincubated and an additional inactivation when incubated with AT P, The spontaneous inactivation is not inhibited by phosphatase inhibitors but the ATP-dependent inactivation was abolished when either 5'-p-fluorosul phonylbenzoadenosine (FSBA) or glucose-6-phosphate (G6P), (both reported as inhibitors for the SPS-protein kinase from spinach) was included during pr eincubation, 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 V-max condition remained relatively unaffected by A TP or FSBA, The desalted extract prepared from darkened leaves on the other hand, when preincubated at 25 degrees C showed a time-dependent increase i n the V-lim activity and the activation state of the enzyme. The spontaneou s activation observed during preincubation appears to be due to the dephosp horylation of the enzyme and is strongly inhibited by okadaic acid, a poten t protein phosphatase inhibitor. Alternately, feeding okadaic acid to excis ed leaves in the dark also blocked the subsequent light activation of V-lim activity, These results are consistent with the assumption that the light/ dark regulation of V-lim activity observed in the leaves of P. juliflora wa s mediated through a dephosphorylation/phosphorylation mechanism.