Biochimica et Biophysica Acta, 588 (1979) 1--11 © Elsevier/North-Holland Biomedical Press
BBA 29088
DENSITY LABELLING STUDIES OF THE PHOTOCONTROL OF L-PHENYLALANINE AMMONIA-LYASE IN DISCS OF POTATO (SOLANUAt TUBEROSUM) TUBER PARENCHYME
CHRISTOPHER J. LAMB a and THERESA K. MERRITT b
a University of Oxford, Department of Biochemistry, South Parks Road, Oxford OX1 3QU and b University of Oxford, School of Botany, South Parks Road, Oxford OX1 3RA (U.K.) {Received November 7th, 1978) (Revised manuscript received June l l t h , 1979)
Key words: Phenylalanine ammonia lyase; Density labeling; Photoregulation; (Solanum tu berosu m)
Summary 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 2H20. Labelling of enzyme was measured by analysis of the equilibrium distribution of enzyme activity in CsC1 density gradients as a function of buoyant density corrected with respect to the distribution of the external marker enzyme fi-galactosidase (fi-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. Introduction The regulation of the biosynthesis, from L-phenylalanine, of phenylpropanoid compounds such as chlorogenic acid, lignin and flavonoids is a model system for study of the control of enzyme levels in higher plants [1--3]. In excised discs of potato (Solanum tuberosum) tuber parenchyme, accumulation of chlorogenic acid is accompanied by a transient increase in the activity of L-phenylalanine ammonia-lyase (EC 4.3.1.5), the first enzyme of the biosynthetic sequence [4]. Continuous illumination with white light causes a further marked stimulation of phenylalanine ammonia-lyase activity accompanied by more rapid accumulation of chlorogenic acid, and phenylalanine ammonia-lyase is thought to be a key element in the photocontrol of chlorogenic acid levels [4,5].
Temporal changes in enzyme levels can be described by the equation: (dE/dt) = k s -- k d E
(1)
where ( d E / d r ) is the rate of change of enzyme level per unit of tissue (E) with respect to time (t), ks is the rate constant for the de novo production of active enzyme and kd is the rate constant for removal of active enzyme [6,7]. Density labelling studies have demonstrated that the initial rapid increase of phenylalanine ammonia-lyase activity in continuously illuminated discs reflects an increase in the rate constant for de novo production of active enzyme in the absence of either activation of pre-existing inactive enzyme molecules or removal of active enzyme [8]. The abrupt transition to a phase of decline in enzyme activity is caused by a reduction in the rate constant for enzyme production simultaneous with a dramatic increase in the rate constant for removal of active enzyme. It follows that the effects of light-to
BBA 29088
DENSITY LABELLING STUDIES OF THE PHOTOCONTROL OF L-PHENYLALANINE AMMONIA-LYASE IN DISCS OF POTATO (SOLANUAt TUBEROSUM) TUBER PARENCHYME
CHRISTOPHER J. LAMB a and THERESA K. MERRITT b
a University of Oxford, Department of Biochemistry, South Parks Road, Oxford OX1 3QU and b University of Oxford, School of Botany, South Parks Road, Oxford OX1 3RA (U.K.) {Received November 7th, 1978) (Revised manuscript received June l l t h , 1979)
Key words: Phenylalanine ammonia lyase; Density labeling; Photoregulation; (Solanum tu berosu m)
Summary 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 2H20. Labelling of enzyme was measured by analysis of the equilibrium distribution of enzyme activity in CsC1 density gradients as a function of buoyant density corrected with respect to the distribution of the external marker enzyme fi-galactosidase (fi-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. Introduction The regulation of the biosynthesis, from L-phenylalanine, of phenylpropanoid compounds such as chlorogenic acid, lignin and flavonoids is a model system for study of the control of enzyme levels in higher plants [1--3]. In excised discs of potato (Solanum tuberosum) tuber parenchyme, accumulation of chlorogenic acid is accompanied by a transient increase in the activity of L-phenylalanine ammonia-lyase (EC 4.3.1.5), the first enzyme of the biosynthetic sequence [4]. Continuous illumination with white light causes a further marked stimulation of phenylalanine ammonia-lyase activity accompanied by more rapid accumulation of chlorogenic acid, and phenylalanine ammonia-lyase is thought to be a key element in the photocontrol of chlorogenic acid levels [4,5].
Temporal changes in enzyme levels can be described by the equation: (dE/dt) = k s -- k d E
(1)
where ( d E / d r ) is the rate of change of enzyme level per unit of tissue (E) with respect to time (t), ks is the rate constant for the de novo production of active enzyme and kd is the rate constant for removal of active enzyme [6,7]. Density labelling studies have demonstrated that the initial rapid increase of phenylalanine ammonia-lyase activity in continuously illuminated discs reflects an increase in the rate constant for de novo production of active enzyme in the absence of either activation of pre-existing inactive enzyme molecules or removal of active enzyme [8]. The abrupt transition to a phase of decline in enzyme activity is caused by a reduction in the rate constant for enzyme production simultaneous with a dramatic increase in the rate constant for removal of active enzyme. It follows that the effects of light-to
