Eur. J. Biochem. 63, 137-145 (1976)

Regulation of Phenylalanine Ammonia-Lyase Activity in Cell-Suspension Cultures of Petroselinum hortense Apparent Rates of Enzyme Synthesis and Degradation Klaus HAHLBROCK Biologisches Institut I1 der Universitiit Freiburg (Received July 4/November 19, 1975)

The time courses for induced changes in the phenylalanine ammonia-lyase activity at five different stages during the growth cycle of cell-suspension cultures from parsley (Petroselinum hortense Hoffm.) were investigated. Large increases in the enzyme activity, induced either by irradiation or by dilution of a cell culture into fresh medium, were followed by an exponential decline to the initial low level. The maximum inducible level of specific enzyme activity varied within a range of about six-fold, depending on the mode of induction and the growth stage of the cell culture. The general shapes of the curves for the changes in enzyme activity were similar under the various experimental conditions. However, the precise positions of the peaks in the activity varied from about 12-27 h after the onset of induction. Proportionally large variations were found for the peak widths at half-maximum and, in the case of induction by continuous irradiation, for the periods of time required for half-maximal induction of the system with light. The apparent half-life of the enzyme, as calculated from the rate of decline of the activity subsequent to the peak, remained approximately constant under all conditions investigated. A general model is proposed which would explain the regulation of phenylalanine ammonialyase activity in Petroselinum hortense cell cultures by an interplay of potentially large variations in the rate of synthesis and an approximately constant rate of degradation of the enzyme. This conclusion is supported both by the various experimental results and by theoretical derivations of curves for the apparent changes in the enzyme-synthesizing activity and in the rates of provision of this activity at the site of enzyme synthesis.

Changes of enzyme levels in eukaryotic cells are regulated by variations in the rates of either synthetic or degradative processes, or both (for recent reviews see [I-91). Although many examples exist for the modulation of the rates of protein degradation under certain conditions [3-71, changes in the rates of enzyme synthesis seem to be the more common mechanisms by which cells respond to rapid, differential variations in metabolic requirements. ~

~~

Definilions. Induction is used in this context as an operational term to describe the increase in enzyme activity in response to a specific treatment of the cells, e.g. with light. This terminology does not upriori imply a specific molecular mechanism, even though a defined sequence of reactions is proposed in the Discussion. The HM,, value indicates the length of the light period required for the induction of half-maximal cnzyme activity under the experimental conditions employed. The calculation of this value by a linear transformation of the data obtained is merely a conscquencc of the naturc of thc experimental results without any implications regarding their molecular basis. Enzyme. Phenylalanine ammonia-lyase (EC 4.3.1.5).

One example for the induction of a specific independent metabolic pathway is the rapid rate of flavonoid accumulation upon irradiation of various higher plants including Petroselinum hortense [lo- 121. Phenylalanine ammonia-lyase, which catalyzes the formation of trans-cinnamic acid from phenylalanine, is the first in the sequence of enzymes involved in the flavonoid production [12,13]. Large changes in the activity of this enzyme occur upon continuous [13,14] or short-term [15- 171 irradiation of cultured Petroselinum hortense cells with ultraviolet light, or upon dilution of a cell culture in the absence of light either into fresh medium [18] or into water [19J Under standardized conditions of irradiation [13 - 15J or dilution [18,19] a peak in the enzyme activity was always observed after about 12- 15 h, irrespective of the mode of induction. Such time-course experiments were routinely conducted with cell cultures whose age corresponded approximately to a late stage of the linear growth phase (stage 111 in Fig. 1). For

Regulation of Phenylalanine Ammonia-Lyase

138

instance, a peak in the phenylalanine ammonia-lyase activity occurred regularly after about 12 h in each new culture, for which cells from stage I11 or IV were routinely used for inoculation [18]. Cell suspension cultures of Petroselinum hortense have been extensively used as a model system for studies of the regulation of phenylalanine ammonialyase activity [Is, 19-22]. A particular advantage of this system is the high specificity of the inducing effect of the irradiation, which seems to be confined to the enzymes of flavonoid formation. No significant effects have been observed on general protein synthesis [23], on various enzyme activities of independent metabolic pathways [20,24], nor on the growth rate of the cell culture [14]. Even considerable variations of the amount of extractable cellular protein during the growth cycle of a culture are small, when compared with the drastic, light-induced changes in the phenylalanine ammonia-lyase activity (see below). Using this system the present communication describes a number of experiments designed in order to obtain more detailed information on the regulation of phenylalanine ammonia-lyase activity by the interplay of synthesis and degradation. Especially useful in this connection was the finding that the precise pattern of changes in the enzyme activity was greatly dependent upon the growth stage of the cell culture employed for induction.

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MATERIALS AND METHODS Cell Cultures

Cell suspension cultures of Petroselinum hortense Hoffm. were propagated in the fully synthetic medium I [25] at 27 "C in the dark either in conical flasks on a rotary shaker or in a fermenter [21,26]. Seven-day-old cultures were used for inoculation with about 1 g fresh weight of cells/40 ml medium by means of a sterile sieve-spoon. The cells from culture volumes of 40 ml or more (see below) were harvested for each experiment. Part of the material was used for purposes not dealt with in the present report. The fermenter was irradiated, if desired, with 16 Osram-L 20 W/73 lamps through the glass vessel [26]. Samples of about 100 ml containing 15 - 23 g fresh weight of cells were taken aseptically from the fermenter at the appropriate times. For studying the light-independent induction in a freshly started culture, samples of 300 ml containing only about 2 g fresh weight of cells were taken. When cells from a rotary shaking culture were used, up to several volumes of 400 ml were combined aseptically in a conical flask, and portions of 40 ml were transferred to 200-ml conical flasks. These cultures were irradiated with white light from fluorescent lamps (Philips K 40 W/18 [14]) for an appropriate

Time of incubation ( h )

Fig. 1. Relationship between the growth curve (0) of a Petroselinum hortense cell culture and ( A ) changes in the light-inducible phenylalanine ammonia-lyase activity (e), the conduclivity of' the medium (A), and ( B ) changes in the amount of extractable cellular protein (0). A total number of 52 small cultures (40 ml each) were propagated as described under Methods and harvested at the appropriate times. The values for specific enzyme activity and extractable protein are means of two, those for cell fresh weight and conductivity are means of four separate measurements. The enzyme activity was determined 15 h after the onset of a continuous irradiation of duplicate cultures from the appropriate growth stage. The broken line was taken from a previous report [25] and indicates the exhaustion of nitrate from the medium. The curves for the changes in the nitrate concentration and in the conductivity of the medium coincided precisely until the time when the broken line branched off from the solid curve [25].The arrows indicate the various growth stages at which cultures were used for of the following investigations

period of time and either harvested immediately or returned to darkness, if desired. The cells were harvested on a porous-glass filter, frozen with liquid nitrogen, and stored at - 18 "C until use. The conductivity of the medium was routinely checked as an indication of the actual age-of a culture, because the time of incubation has not always been a reliable measure for a specific growth stage. The rate of decrease in the conductivity reflects the rate of depletion of the inorganic nutrients, predominantly

K. Hahlbrock

potassium nitrate, from the medium. The rate of uptake of these ionic constituents by the cells is, in turn, closely correlated with the growth rate of the culture, thus allowing growth curves to be determined by recording the conductivity changes in the medium [25,27]. The arrows in Fig. 1A indicate the various growth stages at which the experiments were conducted. Extraction and Assay of Enzyme Activity Protein was extracted from the frozen cells either by the previously used method [18] or by the following procedure. The cells (1 g or 2 g fresh weight) were homogenized with 4 ml 0.1 M sodium borate buffer, pH 8.8, for 60 min at 0 "C, using a magnetic stirrer at low speed. The debris was removed by centrifugation at 20000xg for 10 min. The supernatant solution was stirred for 20 min with 0.1 g Dowex 1 x 2 (equilibrated with 0.2 M Tris-HCI, pH 8.0), filtered through glass-wool and again cleared by centrifugation. In some cases the borate buffer was replaced by 0.1 M Tris-HCI, pH 7.0. All extraction procedures yielded approximately the same specific enzyme activity. The standard assay was used as described previously [21] for measuring phenylalanine ammonia-lyase activity. Protein was determined by the biuret method [281. Inhibition with Actinomycin D Two dark-grown cultures of stage 111, each containing 400 ml, were combined, mixed and divided into equal portions of 40ml. All cultures were then irradiated continuously according to the standard procedure [13,14,24]. The inhibitor was dissolved in sterile water (1.67 mg/ml) and added to the appropriate number of cultures 2.5 h after the onset of the irradiation. The final concentration of actinomycin D in each culture was 5 pg/ml [20]. The cells were harvested at the appropriate times as described above and assayed for the enzyme activity.

RESULTS Growth Curve and Specific Growth Stages The various growth stages of the cell cultures used for the present studies were chosen in order to allow a comparison of the most different curves possible for the induced changes in phenylalanine ammonia-lyase activity. The growth curve of a Petroselinum hortense cell culture is shown in Fig. 1A, in which arrows indicate the five selected growth stages. The different stages represent a number of distinct

139

phases during the growth cycle, including the initiation of the cell culture (stage I), a late period of exponential growth (stage IT), a linear growth period (stage III), a period of a progressively decelerating growth rate (stage IV) and an early period of the stationary phase (stage V). Muximum Level of Phenylalunine Ammonia-Lyase Activity The maximum specific activity of phenylalanine ammonia-lyase, which could be obtained within 15 h of continuous irradiation, was greatly dependent upon the growth stage of the cell culture. Fig. I A shows the growth curve of a culture and the curve for the changes in the specific enzyme activity obtained 15 h after the onset of induction. The latter curve includes two pronounced peaks, one at stage I and one at stage I11 of the growth cycle. In addition, Fig. 1 A depicts the changes in the conductivity of the medium during the growth of the culture. According to previous results [25],the sigmoidal curve for the conductivity changes allows the time, at which the exhaustion of nitrate from the medium occurs, to be determined. The broken line in Fig.1 A demonstrates that this time coincides with the second peak in the maximum inducible enzyme activity at stage 111 of the culture. Later experiments (see Fig.2 and Table 1) demonstrated that the peak in the enzyme activity occurred under certain conditions somewhat earlier or even considerably later than after 15 h. However, Fig. 2 shows that possible differences between the levels of the enzyme activity at 15 h and the actual peak position were small even in extreme cases and varied only within a range of about 10 at the most. The enzyme activity in Fig. 1 A was expressed on the basis of extractable cellular protein. Fig. 1 B shows that the amount of protein extracted per gram fresh weight of cells varied considerably during the growth cycle of the culture. No conclusive explanation can be given for this phenomenon with the present data available, even though a similar observation has been made with a Glycine max cell culture [29]. Part of the effect might be explained by a considerable decreased in the ratio of dry weight to fresh weight during late growth stages of a Petrosehum hortense cell culture, indicating a relative increase in the water content of the cells by more than 20"/, [13]. In principle, the curve shown in Fig. 1 A for the changes in the maximum inducible enzyme activity remained the same, when cell fresh weight instead of extractable cellular protein was used as a basis for the expression of the enzyme activity. As in Fig. 1 A, all of the data in this report concerning phcnylalanine aminonia-lyase are expressed as specific activities in order to correct for the variations in the concentration of cellular protein.

140

Regulation of Phenylalanine Ammonia-Lyase

Time Courses of Changes in the Enzyme Activity. after Induction Fig.2 shows the time courses of changes in the phenylalanine ammonia-lyase activity at three different stages during the growth cycle of a cell culture.

Time after onset of induction (h)

Fig. 2. Time courses of changes in phenylalanine ammonia-lyase activity. Cell cultures were diluted into fresh medium in a fermenter at stage I and propagated in the dark ( v),or continuously irradiated either in a fermenter at stage I1 (A) or in the appropriate number of small conical flasks at stage I V (0).The onset of induction was at time zero, and samples were harvested at the times indicated. Each value represents the mean from a duplicate experiment. The data shown in curve IV were taken from a previous report, but plotted on a different scale [24]

The changes in the enzyme activity were induced at stage I in the absence of light upon starting a new culture [I81 and at stages I1 and IV by the onset of continuous irradiation. The general shapes of all three curves in Fig.2 appear very similar, despite the different modes of induction and the different growth stages of the cultures. However, the curves differ considerably with respect to the positions of the peaks and the absolute amounts of enzyme activity. The precise data calculated for these and some other parameters are summarized in Table 1 (Expts 1, 3 and 7). In addition, Table 1 contains a list of the corresponding data from a number of similar experiments in which cell cultures of various other stages were used, and where the mode of induction included short light periods of only 0.5 h or 2.5 h. While the peak positions as well as the band widths at halfmaximum of the curves for changes in the enzyme activity varied within a range of about two-fold, the maximum specific activities differed by more than six-fold under the various conditions. By contrast, the values determined for the half-life periods during the decline in enzyme activity were all very similar, with an average around 10- 11 h. The level of the enzyme activity in non-induced cells was very low during all stages of the cell culture, ranging from about 1 -4 ykat/kg. Differences within this range, which represent the limits of the experimental error, were even obtained in separate experiments with cultures of the same age. Compared to the rapid large changes in the enzyme activity (e.g. as shown in Fig.2), the variations in the amount of extractable protein were relatively small. None of the data in Table 1, except the values

Table 1. Some characteristic data ,from time-course experiments for changes in the phenylalanine ammonia-lyase activity after induction at different growth stages of the cell cultures The growth stages were those indicated by the arrows in Fig. 1. The curves for the changes in activity were obtained in the same manner as shown in Fig.2. A dash indicates that values were not determined. The results of Expts 1, 3, and 7 were obtained by using the data of Fig.2, those of Expt 5 were calculated from previously reported data [15]. The apparent half-life periods were determined graphically from semi-logarithmic plots of the data for the changes in enzyme activity Expt no.

Growth stage of the cell culture

Mode of induction

Peak after the onset of induction

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App. half-life

Enzyme level prior to induction

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Fig.3. Demonstration of’ the lag period preceding the increase in phrnylalanine ummonia-lyase activity. Small cultures obtained from the same batch in each case were irradiated either for 15 min at stage 111 (A) or continuously at stage I11 (0)or stage V (0)and harvested at the times indicated. The onset of the induction was at time zero. Each value represcnts the mean from a duplicate experiment

for the enzyme activity in non-induced cells, was altered by more than about lo”/,, when the enzyme activity was expressed on the basis of cell fresh weight. The Lag Period

The increases in the phenylalanine ammonialyase activity have previously been shown to be preceded by lag periods of similar lenghts, regardless of whether irradiation or subculturing of the cells were the modes of induction [15,18-201. Fig.3 shows that the same lag period of about 2-2.5 h was observed, when cells from stage 111 or V were irradiated continuously, or when the light program for cells from stage I11 was reduced to an irradiation period of only 15 min. Time Courses of the Efficiency ojIrradiation

Curves demonstrating a progressive exhaustion of the enzyme-inducing efficiency of the irradiation at three different growth stages of the cell cultures are shown in Fig. 4. Cultures from stages 111, IV and V were irradiated for various periods of time, returned to darkness, and harvested 15 h (stages 111 and IV) or 27 h (stage V) after the onset of the induction. The

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Fig.4. Time C O U ~ S ~ofS the efficiency of ir.radialion a t three dijf&renl growth srages. The appropriate number of duplicates of small cultures from the same batch were irradiated for the periods of time indicated, beginning at timc zero, and then returned to darkncs3, except for the last set of cultures in each experirncnt. The cells were harvested 15 h (stage I11 and stage IV) or 27 h (stage V) aftcr thc onset of the induction, as indicated by the arrows. The valucs givcn for half-maximal induction with light (HM,) were calculated from the data by Lineweaver-Burk plots. The reason for a possible large experimental error associated with the HM, value at stage V is discussed in the text

shapes of the curves are similar to those obtained for saturation-type kinetics. Hence, double-reciprocal plots of the data shown in Fig.4 resulted in straight lines from which the lengths of the light periods required for the induction of half-maximal enzyme activity at the respective peak position (HM, values) were calculated. Values of about 2.7,2.9, and 10- 20 h were obtained for the three different stages, 111, IV, and V, respectively. A similar curve for cultures at

142

Regulation of Phenylalanine Ammonia-Lyase

stage I11 has been reported in a case where the experiment had been terminated after 13 h rather than 15 h ~241. Five separate experiments with cultures from stage I11 were carried out altogether in order to estimate the accuracy of the HM, value calculated from the data of Fig.4 at this stage. The resulting data varied within a range from 1.4 h to 3.4 h, with a mean value of 2.6 f 0.4 h (standard deviation). A much higher experimental error is associated with the value determined for the cell culture from stage V. Unlike the situation at stages 111 and IV, the position of the peak in the enzyme activity relative to the time of the onset of induction at stage V varied greatly within the range from about 12-24 h, depending upon the length of the light period. It must be considered that the lowest values in Fig. 4, panel V, are too low by up to about loo%, since the respective cells were harvested several hours beyond the time at which the peak in the enzyme activity occurred. Thus, the HM, value, which was calculated from the experimental data to be about 20 h, would actually be only about 10 h. However, the general tendency towards higher HM, values for cell cultures progressing in age from stages 111 through V is clearly demonstrated by the results presented in Fig. 4. Inhibitory Ejfect of Actinomycin D A strong inhibition by low doses of actinomycin D has been reported for the light-induced increase in phenylalanine ammonia-lyase activity [20]. Fig. 5 depicts the time courses of the changes in enzyme activity during the first 7 h of incubation of cells under continuous irradiation in the presence and in the absence of actinoinycin D. The inhibitor ( 5 pg/ml) was added to one set of probes 2.5 h after the onset of the induction. Initially the enzyme activity increased even in the presence of actinomycin D at the same rate as in the control experiment. However, starting approximately 1.5-2 h after the addition of the inhibitor, the rate of increase was greatly reduced. Rate Constants of Enzyme Synthesis and Degrudation According to the mathematical model derived by Schimke [6], the zero-order rate constant of enzyme synthesis is described by the equation k,

=

dE/dt

+ k,E,

(1)

and the first-order rate constant of enzyme degradation is described by the equation

when E is the enzyme activity changing with time (f).

Time after onset of induction (h)

Fig. - 5. Inhibition of the 1i.ght-induc.ed increase in nhenv) lanine ammonia-lyase activity by actinomjcin D. Duplicate cultures wcre harvested at the times indicated, and the time courses of changes in the enzyme activity in the absence (0)and in the presence ( 0 ) of actinomycin D were compared. The inhibitor was added to an appropriate number of cultures 2.5 h after the onset of continuous irradiation, as indicated by the arrow. As reported previously [20], the employed inhibitor concentration of 5 pg/ml caused an over-all inhibition of the order of 80% within 7 h of incubation, when added simultaneously with the onset of the induction .

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The application of these equations to the present system seems to be justified, since the large exponential decreases subsequent to the peaks in the enzyme activity suggest that a possible slow rate of enzyme synthesis, even during the decline, would be negligible for the purpose of the following calculations. Using the experimentally determined apparent half-life of about 10 h for the enzyme at stages 111 and IV, and assuming that this value remained approximately constant throughout all conditions of experimentation, including the non-induced state, a constant value of k , = 0.069 h-' was calculated from Eqn (2). This value and the data shown in Fig.6 for the changes in the enzyme activity (broken lines) allowed the changes in the rate constants for phenylalanine ammonialyase synthesis to be calculated, using Eqn (1). The results are illustrated by the solid lines in Fig.6 for two cases of induction, either by irradiation for 2.5 h at stage I11 (Fig.6A) or by continuous irradiation at stage 1V (Fig. 6B). The level of enzyme activity and the k , value for the non-induced state were subtracted from the curves shown in Fig. 6, which depicts only the light-induced changes. The k , value for the non-induced state was calculated to be about 0.17 pkat x kg-' x h-', using

K . Hahlbrock

143

,

Time after onset of irradiation (h)

Fig. 6. Calculated changes of the rate constant.s for phenylalanine ammonia-Iyase .synthesis eitlzer ( A ) after 2.5 h of irradiation of cultures ,fiorn stage III, or ( R ) under continuous irradiation of cultures from stage IV. The original curves for the changes in phenylalanine ammonia-lyase activity during the first 26 h after the onset of induction (----) were taken from [15] (A) and Fig.2 (B), and were corrected for the non-induced level of the enzyme activity during the lag period. The solid curves were calculated from the enzymc activities (represented by the broken curves) as described in the text. Dotted portions of the curves indicate arcas with which high experimental errors are associated. The hatched area in panel A represents the irradiation period, the shaded area is proposed to indicate the period of time during which the rate of provision of thc synthesizing activity for phenylalanine ammonia-lyase is increased above the non-induced level

a mean value of 2.4 pkat/kg for the enzyme activity prior to induction (see Table 1). The exponential decline of the solid curve in Fig.6A, beginning about 5 h after the onset of the light period, allows an apparent half-life of about 3.6 h for the enzyme-synthesizing activity to be calculated. On the other hand, the corresponding curve in Fig. 6 B demonstrates that, under the conditions of continuous irradiation, the decline of the enzymesynthesizing activity is not exponential. The shape of this latter curve seems to indicate the co-occurrence of significant rates of both synthesis and degradation during most of the decline.

Fig. I. Theoretically defived time couyses Of the putuiiw ttccumuluiion rates ,for the en;yme-s.~nthesizing activity in the absence of inactivation either ( A ) after irradiationfor 2.5 h at stage 111, or i R ) under continuous irradiation at stage I V . The curves were ohtaincd by correcting the solid curves of Fig. 6 A and Fig. 6 B respectively, a1 intervals of 0.6 h for a decay of 10 'i: of the enzyme-synthesizing activity. This decay rate follows from thc apparent half-life of about 3.6 h, as determined from the data of Fig.6A. Thc curves were shifted by 2.5 h to the left o n the abscissa in order to correct for the lag period preceding the increase in enzyme activity. (0) The data already shown in Fig.4, panel IV, which were replotted on a 100:" basis relative to the two curves. (0)The data from Table 1 for the maximum enzyme activities obtained after short-term irradiation for 0.5 h and 2.5 h respectively. In this case the latter valuc was expressed as 1007;. The relative scale was chosen i n order to correct for variations in the absolute amounts of the eniyme activity, which were frequently encountered in separate experiments (see text for details). The maximum values calculated for curves A and B were20.4 pkat x kg-' x h-' and 39.4 pkat x kg-' x h - ' rcspectively

Rate of Formation o f Enzyme-Synthesizing Activity Using the estimated value of 3.6 h for the half-life of the enzyme-synthesizing activity, the solid curves of Fig.6 were corrected for the decay as described in the legend of Fig.7. The resulting curves A and B (Fig.7) are assumed to represent the changes in the enzyme-synthesizing activity, which would have been measured in the absence of its inactivation. As already pointed out above, the half-life of the sythesizing activity could be calculated only from the solid curve in Fig.6A, but not from the corresponding curve in Fig.6B. However, the fact that plateaus were obtained for both curves in Fig.7 suggests that the application of the half-life period of 3.6 h as derived from Fig.6A might be justified in both cases. The data of Fig. 4, panel IV, were plotted in Fig. 7 (open symbols) on a 100% basis in relation to the two curves. This illustration allows a direct comparison of the theoretically derived curve B and the experimentally determined data for the time course of the progressively exhausting efficiency of the irradiation

144

at stage IV of the cell culture. A similar comparison with curve A was made by plotting the data of Table 1 for the maximal enzyme activities obtained after short-term irradiation for 0.5 h and 2.5 h at stage I11 (closed symbols in Fig. 7) on the same scale.

DISCUSSION Variations in the Extent ofthe Enzyme Induction The data of Fig.3A confirm and extend earlier results, which have demonstrated the occurrence of an early [21] and a late peak [13,21] in the inducible level of phenylalanine ammonia-lyase activity. There exists a striking similarity between the positions of the second peak in the light-induced enzyme activity in the Petroselinum hortense cell culture and of similarly large, endogeneously induced peaks in this enzyme activity at the corresponding growth stages of Glycine mux [27,29] and Acer pseudoplatanus cell cultures [30]. Despite the different modes of induction, the position of these peaks coincide in all three cell cultures with the time of exhaustion of the nitrogeneous nutrient, nitrate, from the medium (see Fig. 1 and [27,30]). The large seemingly random variations in the maximum specific enzyme activity shown in Table 1 appear to contradict the results of Fig. 1A. However, while samples of cells from the same batch culture were used for the experiments of Fig. 1, the various data of Table 1 were obtained using separate cultures over a period of more than 2 years. For instance, values ranging from 44- 106 pkat/kg were observed for the maximum levels of specific enzyme activity in the 3 cultures of stage V used for Expts 8 and 9 of Table 1 and V of Fig.4. Similar inconsistencies have been reported by Wellmann [16] for Petroselinum hortense cell cultures from stage 111. Most likely these variations observed for separate cultures from the same growth stage are at least partly due to the steep slopes of the peaks for the inducible enzyme activity and the experimental error associated with an accurate determination of the individual growth stages defined in Fig. 1. The Shapes of the Curves j o r Changes in the Enzyme Activity No significant variation has ever been detected with respect to the position of the peak in the enzyme activity relative to the time of the onset of induction, provided that identical conditions were applied at a given growth stage of a cell culture. Likewise, no detectable variation in the half-life, the lag period and the peak width at half-maximum has been observed in such a case. Thus, the basic shapes of the

Regulation of Phenylalanine Ammonia-Lyase

curves (e.g. those shown in Fig.2) were always identical for a certain growth stage, whereas the scales for specific enzyme activity on the ordinate varied considerably. On the other hand, the present results and those recently reported elsewhere [18,19] seem to indicate that the small differences of 2 - 3 h between the positions of the peaks observed after induction under varying conditions at stages I and I11 (see Table 1) are significant. Peaks at 12- 13 h occurred when dilution of the cells at stage I or short light periods at stage I11 were the modes of induction (Expts 1,4, and 5 in Table 1). Peaks at 15 - 16 h were obtained under continuous irradiation of cells from either stage (Expts 2 and 6 in Table 1 and [13,14]). Continuous irradiation at stages IV, V or 11, in this order, caused a progressive delay of the peak of up to about 12 h relative to the position of the peak under continuous irradiation at stage 111. An important aspect seems to be that this delay was associated with a corresponding increase in the HM, values, at least at stages 111-V. Hypothetical Mechanism,for the Regulation o j Phenylalun ine Ammonia-Lyase Activity An attempt to develop a general model, which would readily explain the various curves for the changes in the enzyme activity, is based on the experimental results presented here and on the following earlier observations. a) Labelling experiments in vivo have demonstrated an increased rate of enzyme synthesis only during the period of increasing activity [15,22] and a low, but detectable rate of synthesis in the non-induced state [22]. b) The results of experiments in vivo with inhibitors of transcription and translation indicated that specific RNA as well as protein synthesis were involved in the light-induced stimulation of the phenylalanine ammonia-lyase activity [20]. A stimulation of RNA synthesis seemed to commence immediately upon induction, while an increased rate of protein synthesis was not observed before the end of the lag period which precedes the increase in enzyme activity [20]. c) A period of time strikingly similar to the length of this lag period was required until the inhibitory effect of actinomycin D on the increase in the enzyme activity became apparent [20]. In a differently designed experiment using the same inhibitor (Fig. 5 in this communication), further support was lent to the assumption [20] that the stimulation of specific (presumably messenger) RNA synthesis required approximately 2-2.5 h to become effective in an increased rate of enzyme synthesis. Taking all the results together, the following hypothetical model is proposed for the molecular mechanisms involved in the regulation of phenyl-

K. Hahlbrock

alanine ammonia-lyase activity in cell suspension cultures of Petvoselinum hortense. An approximately constant level of the enzyme activity in the non-induced state of a culture results from a balance between constant rates of synthesis and degradation. Upon induction, this balance is disturbed for a limited period of time by an increased rate of enzyme synthesis (see [15] for a more detailed discussion). Under continuous irradiation the lirnitation is described by hyperboliccurvesfor the time courses of changes in the light efficiency. In the case of a shortterm irradiation the duration of the inducing effect can be even more rigorously defined and is identical with the length of the light period, albeit delayed by approximately 2-2.5 h. This latter period of time is equivalent to the lag period occurring between the onset of the induction and the increase in the enzyme activity. Upon irradiation of a culture for 2.5 h, for instance (Fig. 6A), the rate of synthesis of the enzyme increases after a lag of about 2-2.5 h, but only during a subsequent period of 2.5 h. The enhanced capacity for the synthesis of the enzyme (probably the elevated level of messenger RNA) then decreases with an estimated half-life of about 3.6 h. This hypothetical mechanism is consistent with the results shown in Fig.6 and 7. It is assumed that the solid curves in Fig.6 represent the changes in the proposed elevated levels of messenger RNA [15,20], while the curves of Fig.7 would represent the rates of accumulation of this RNA in the absence of its degradation. The latter curves seem to be either identical with, or at least similar to, those for the time courses of the efficiency of the irradiation under the respective conditions. This assumption is supported in particular (a) by the coincidence of the curves and the respective experimentally derived data shown in Fig.7 and (b) by the coincidence of the lengths of the irradiation period (hatched area in Fig.6A), the subsequent period preceding the exponential decay of the solid curve (shaded area in Fig. 6A) and, consequently, the period of time during which curve A in Fig.7 increases. Such a direct, quantitative relationship between light and enzyme activity would be consistent with the recent reports by Wellmann [16,17] on the ultraviolet-dose dependency of phenylalanine ammonialyase induction in this system. In principal the present hypothetical mechanism corresponds to that discussed by Yagil [S] for the case in which both messenger RNA and its translational product are unstable, with the further property of this system that, under the present conditions of induction, the inducibility is limited to a period of only a few hours.

145 This work was supported by the Deutsche Forschungsgemeinschyft (SFB 46) and Fonds der Chrmisc,hen Industvie. The excellent technical assistancc of Mrs E. Kuhlcn and valuable discussions with E. Schafer, P. Schopfer, and E. Wcllmann are gratefully acknowledged.

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J. Koch and A. Bruhn, Institut fur Biologische Chemie der Universitiit Hohenheim, Garbenstrane 30. D-Stuttgart-IIohenheim, Federal Rcpublic of Germany

Regulation of phenylalanine ammonia-lyase activity in cell-suspension cultures of Petroselinum hortense. Apparent rates of enzyme synthesis and degradation.

The time courses for induced changes in the phenylalanine ammonia-lyase activity at five different stages during the growth cycle of cell-suspension c...
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