Density labelling studies of the photocontrol of L-phenylalanine ammonia-lyase in discs of potato (Solanum tuberosum) tuber parenchyme.

The photocontrol of L-phenylalanine ammonia-lyase (EC 4.3.1.5) activity in discs of potato (Solanum tuberosum) tuber parenchyme has been investigated by density labelling with 2H from 2H2O. Labelling of enzyme was measured by analysis of the equilibrium distribution of enzyme activity in CsCl density gradients as a function of buoyant density corrected with respect to the distribution of the external marker enzyme beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23). It is demonstrated that the white-light mediated stimulation of phenylalanine ammonia-lyase activity involves stimulation of the rate of de novo production of active enzyme.

Synthesis and removal of phenylalanine ammonia-lyase activity in illuminated discs of potato tuber parenchyme.

(1) The synthesis and removal of phenylalanine ammonia-lyase (EC 4.3.1.5) in illuminated discs of potato (Solanum tuberosum cv King Edward) tuber tissue has been investigated by density labelling with deuterium (2H) from deuterium oxide (2H2O) followed by centrifugation to equilibrium in a CsC1 density gradient. (2) Temporal changes in enzyme level have been described in terms of the equation (dE/dt) = ks-kdE where (dE/dt) is the rate of change of enzyme level per unit of tissue (E) with respect to time (t), ks is the rate constant for synthesis of the enzyme and kd is the rate constant for the removal of active enzyme. (3) The optimal concentration of 2H2O was determined by analysis of the relationship between 2H2O concentration, development of enzyme activity and the magnitude of the increase in buoyant density of phenylalanine ammonia-lyase. A concentration of 2H2O of about 40% (v/v) was found to be optimal, allowing achievement of maximal or near maximal increases in the buoyant density of the enzyme without inhibition of the development of enzyme activity, thereby circumventing the major drawback of 2H2O as a source of density label. (4) The overlapping distribution profiles of enzyme activity after density gradient centrifugation were resolved by an iterative method of best fit which allows estimation of the proportions of pre-existing, unlabelled enzyme and newly synthesised, labelled enzyme at the end of the labelling period. This technique has been developed to obtain the rate constants for enzyme synthesis and for removal of active enzyme throughout the period of rapid change in enzyme level. (5) It is demonstrated that the initial rapid increase in phenylalanine ammonia-lyase activity in illuminated discs reflects an increase in the rate constant for enzyme synthesis in the absence of activation of pre-existing enzyme and in the absence of removal of active enzyme. The abrupt transition to a phase of decline in enzyme activity is caused by (a) a reduction in the rate constant for enzyme synthesis and (b) a dramatic increase in the rate constant for removal of active enzyme. The subsequent stabilisation of the enzyme is caused by decay of both rate constants to relatively low levels. (6) The results are consistent with hypothesis that rapid modulation of enzyme levels during tissue differentiation is achieved by simultaneous changesin the rate constants for both enzyme synthesis and for removal of active enzyme.