196 Biochimica et Biophysica Acta, 582 (1979) 196--212

© Elsevier/North-Holland Biomedical Press

BBA 28765 SYNTHESIS AND REMOVAL OF PHENYLALANINE AMMONIA-LYASE ACTIVITY IN ILLUMINATED DISCS OF POTATO TUBER PARENCHYME C.J. LAMB a,., T.K. MERRITT b and V.S. BUTT 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 June 12th, 1978) Key words: Phenylalanine ammonia-lyase synthesis; Differentiation; (Solanum tuberosum, Parenchyme)

Summary (1) The synthesis and removal of phenylalanine ammonia-lyase (EC 4.3.1.5) in illuminated discs of potato ( S o l a n u m t u b e r o s u m cv King Edward) tuber tissue has been investigated by density labelling with deuterium (2H) from deuterium oxide (2H20) followed by centrifugation to equilibrium in a CsC1 density gradient. (2) Temporal changes in enzyme level have been described in terms of the equation ( d E / d t ) = k s - - k d E 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 k d is the rate constant for the removal of active enzyme. (3) The optimal concentration of 2H20 was determined by analysis of the relationship between :H20 concentration, development of enzyme activity and the magnitude of the increase in b u o y a n t density of phenylalanine ammonialyase. A concentration of 2H20 of about 40% (v/v) was found to be optimal, allowing achievement of maximal or near maximal increases in the b u o y a n t density of the enzyme without inhibition of the development of enzyme activity, thereby circumventing the major drawback of 2H:O as a source of density label. (4) The overlapping distribution profiles of enzyme activity after density gradient centrifugation were resolved by an iterative m e t h o d 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 con* To whom

correspondence

s h o u l d be a d d r e s s e d .

197 stant 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 level is caused by decay of both rate constants to relatively low levels. (6) The results are consistent with the hypothesis that rapid modulation of enzyme levels during tissue differentiation is achieved by simultaneous changes in the rate constants for both enzyme synthesis and for removal of active enzyme.

Introduction Phenylalanine ammonia-lyase (EC 4.3.1.5) catalyses the first reaction in the biosynthesis from phenylalanine of a wide variety of phenylpropanoid compounds including lignin, esters of hydroxycinnamic acids and flavonoids [1]. The enzyme is widely distributed in higher plants [2]. The level of phenylalanine ammonia-lyase activity is sensitive to the physiological status of the tissue and increases dramatically in response to a variety of stimuli including wounding [3,4], light [5--7] and ethylene [8,9]. The general pattern of the response of phenylalanine ammonia4yase to a stimulus is (a) a lag phase (usually 60 to 120 rain), (b) a phase of rapid increase in extractable activity, (c) a phase of decline in extractable activity (not always seen) and (d) a phase of constant enzyme level [5]. Temporal changes in enzyme levels can be described by the equation (dE/dt) = k s -- kdE

(1)

where (dE/dr) is rate of change of enzyme level per unit of tissue (E) with respect to time (t), k s is the rate constant for synthesis of the enzyme and k d is the rate constant for removal of active enzyme [10]. It has recently been sug• gested that rapid changes in enzyme levels, as exemplified by phenylalanine ammonia-lyase, might involve not only control over the rate of enzyme synthesis (change in ks) , b u t also control over the rate of removal of active enzyme (change in kd) [11]. The phase of rapid increase in phenylalanine ammonia-lyase activity has been intensively investigated in a number of systems [5--7] b u t less attention has been given to the balance between the rates of enzyme synthesis and removal of active enzyme throughout the entire time course [12--14]. In the present paper, we use the technique of density labelling with deuterium (2H) from deuterium oxide (2H20) to measure the rate constants for (a) synthesis of phenylalanine ammonia-lyase and (b) removal of active phenylalanine ammonia-lyase during the period of rapid changes in the activity of the enzyme in illuminated discs of potato (Solarium tuberosurn cv King Edward) tuber tissue [15]. Density labelling, unlike other techniques used to study the turnover of specific enzymes such as radio-labelling and immunoassay, provides a quantitative measure of the ratio of newly synthesised and, therefore, labelled or heavy enzyme to pre,existing and, therefore, unlabelled or light enzyme [15]. If the enzyme

198 level is known at the beginning and end of a pulse of density label and the ratio of labelled to unlabelled enzyme at the end of the pulse is determined by analysis of the distribution of enzyme activity at equilibrium in a density gradient, then the rate constants ks and k d can be directly calculated. A considerable difficulty with the approach, and in general with density labelling experiments using 2H20 as a source of label, is that at high concentrations 2H20 has deleterious effects on plant metabolism including inhibition of respiration, photosynthesis and protein synthesis [ 17], inhibition or delay of developmental increases in enzyme activity [18--20] and in some cases inhibition of seedling germination [21]. The majority of density labelling studies have used concentrations of 2H20 sufficiently high (60--100% v/v) to inhibit the physiological process under investigation, presumably in the belief that optimal incorporation of 2H into proteins is achieved at high concentrations of 2H20. Clearly, if the concentration of 2H20 is sufficiently high that it significantly affects the change in enzyme activity during the pulse then the estimates of k s and k d obtained are meaningless with respect to the normal physiological state. This problem has been overcome by preliminary studies to determine the optimal concentration of 2H20. Analysis of the relationship, between 2H20 concentration, development of enzyme activity and the magnitude of the increase in b u o y a n t density of the enzyme revealed that a final 2H20 concentration of a b o u t 40% (v/v) was optimal in this system. At this concentration maximal or near maximal increases in the b u o y a n t density of the enzyme were obtained without inhibition or distortion of the change in enzyme level, thereby circumventing the major drawback of ~H20 as a source of density label. Materials and Methods

Preparation of plant material. Potato (S. tuberosum cv King Edward) tubers were purchased from the local market within 24 h of the experiment and were stored at 15°C in darkness. Only full turgid tubers weighing between 0.1 and 0.2 kg were used for experimentation. Discs were prepared as previously described [22], placed in Petri dishes (4 discs/dish), moistened with 5 ml H20 or ~H20 and incubated at 25°C under white light [23]. Exposure of plant material to 2H20. Before transfer to 2H20, any residual H20 on the surface of the discs was removed by blotting on filter paper. Four discs prepared from a fully turgid tuber have an initial fresh weight of 3.4 g of which 3.0 g (i.e. approx. 3.0 ml) is H20 [24]. The fresh weight of the discs does n o t increase significantly during incubation in H20 under illumination for 48 h. 2H20 in the incubation medium will rapidly equilibrate with tissue H20 [25]. Thus, exposure of four discs (containing 3.0 ml H20) to 3.0 ml 99.8% (v/v) 2H20 together with 2.0 ml H20 gives an effective 2H20 concentration of 37.5% (v/v) and, similarly, exposure to 5.0 ml 99.8% 2H20 gives an effective ~H20 concentration of 62.5%. Higher effective concentrations of 2H20 were achieved by a series of 15-min washings in 5.0 ml 99.8% 2H20 followed by incubation in 5.0 ml 2H20: one and two washings giving effective 2H20 concentrations of 86 and 95%, respectively. Preparation of extracts. Extracts were prepared in 0.1 M sodium borate buffer, pH 8.8, containing 2 mM 2-mercaptoethanol; cell debris were removed by

199 centrifugation at 500 × g for 5 min. The supernatant was collected and assayed for phenylalanine ammonia4yase activity. A second portion was stored at --80°C in plastic reaction vessels (1.5 ml capacity) until required. No enzyme activity was lost during storage for up to at least one m o n t h under these conditions and storage had no effect on the equilibrium distribution of enzyme activity in CsCI density gradients. When required, a 1.0 ml sample of the supernarant was gel-filtered through Sephadex G-25, the protein-containing fractions collected and portions used for equilibrium density gradient centrifugation or protein determination. Equilibrium density gradient centrifugation. Equilibrium density gradient centrifugation was carried out in an M.S.E. Superspeed 50 ultracentrifuge using an M.S.E. 10 X 10 ml fixed angle (20 °) rotor (catalogue no. 2410). Preparation of gradient tubes was adapted from the m e t h o d of ref. 18. The gradient originated from three layers, the lowest of 3.6 ml CsC1 solution (0.82 kgfl) onto which was carefully layered 3.6 ml of a second CsC1 solution (0.20 kg/1). The tubes were completed by careful addition of 1.7 ml gel-filtered extract (1.5-2.5 mg protein) containing f]-galactosidase (EC 3.2.1.23) purified from Eseherichia coli as an external marker enzyme, b u o y a n t density 1.300 kgfl in CsC1 [26]. In preliminary experiments to determine the optimal concentration of 2H20, 250 nkat of ~-galactosidase was used, in all subsequent experiments only 60 nkat was added. The rotor was operated for 40 h at 2°C and 110 000 X gay (rav= 5.61 cm). After centrifugation, a narrow gauge needle was lowered to the b o t t o m of the tube and four

t~J

I 0

L 20

I 40

I 60

Concenlration of 2H20

[ 80

I D0

(%)

Fig. 1. E f f e c t o f 2 H 2 0 c o n c e n t r a t i o n o n t h e d e v e l o p m e n t o f p h e n y l a l a n i n e a m m o n i a - l y a s e a c t i v i t y in i l l u m i n a t e d p o t a t o t u b e r discs. Discs o f p o t a t o t u b e r p a x e n c h y m e w e r e i n c u b a t e d f o r 9 h a t 2 5 ° C u n d e r c o n s t a n t i l l u m i n a t i o n in H 2 0 o r a range o f c o n c e n t r a t i o n s o f 2 H 2 0 . T h e c o n c e n t r a t i o n o f 2 H 2 0 (v/v) is c a l c u l a t e d a f t e r a l l o w i n g for d i l u t i o n o f a d d e d 2 H 2 0 b y tissue H 2 0 .

202 0,020

~ o-o~oi ¢, o

5-

c_

5

-~o.ooo[

j L_ 0

I 20

I 40

I 60

Concentration of 2H20

I 80

-0.004

] 100

(°/o)

Fig. 2, E f f e c t o f 2 H 2 0 c o n c e n t r a t i o n o n t h e i n c o r p o r a t i o n of 2 H i n t o p h e n y l a l a n i n e a n a m o n i a - l y a s e . Discs o f p o t a t o t u b e r p a r e n e h y m e w e r e i n c u b a t e d a t 2 5 ° C u n d e r c o n s t a n t i l l u m i n a t i o n in a r a n g e o f c o n c e n t r a t i o n s o f 2 H 2 0 . T h e c o n c e n t r a t i o n o f 2 H 2 0 (v/v) w a s c a l c u l a t e d a f t e r a l l o w i n g f o r d i l u t i o n o f a d d e d 2 H 2 0 b y tissue H 2 0 . A f t e r 9 h , p h e n y l a l a n i n e a m m o n i a - l y a s e w a s e x t r a c t e d a n d c e n t r i f u g e d t o equilib r i u m i n a CsC1 d e n s i t y g r a d i e n t . T h e e q u i l i b r i u m d i s t r i b u t i o n o f e n z y m e a c t i v i t y w a s a n a l y s e d as a f u n c t i o n o f b u o y a n t d e n s i t y c o r r e c t e d w i t h r e s p e c t t o t h e d i s t r i b u t i o n o f t h e e x t e r n a l m a r k e r e n z y m e fl-galact o s i d a s e . T h e i n c o r p o r a t i o n o f 2 H is m e a s u r e d b y (a) i n c r e a s e in b u o y a n t d e n s i t y o f p e a k e n z y m e a c t i v i t y (I i ) a n d (b) i n c r e a s e in b a n d w i d t h a t h a l f p e a k h e i g h t ( a A).

These studies, in which the level of enzyme activity is very low at the beginning of the labelling period, suggest that a 2H20 concentration of about 40% is optimal because adequate increases in buoyant density are observed without inhibition of the increase in enzyme activity. However, in the pulse density labelling experiments, it will generally be the case that significant levels of native enzyme are present at the beginning of the labelling period. Therefore, we have also examined the relationship between 2H20 concentration and incorporation of 2H into phenylalanine ammonia-lyase under conditions applicable to pulse labelling experiments. If discs are preincubated for 6 h in H20 before transfer to 2H20, then, at high concentrations of 2H20, paradoxically small increases in the buoyant density of phenylalanine ammonia-lyase are observed together with severe inhibition of the developmental increase in enzyme activity (Figs. 3 and 4). Maximal shifts are observed with 37.5% 2H20, at lower concentrations the increase in buoyant density is proportional to the concentration of 2H20. The low concentration of 2H20 needed for maximal buoyant density increase, and the small increases observed at high concentrations of 2H20 we ascribe to the interplay of two factors. First, the higher the concentration of 2H20 the greater the potential increase in buoyant density. Second, at high concentrations of 2H20, protein synthesis will be severely inhibited [17] leading to the observed inhibition of the increase in enzyme activity. Therefore, at high concentrations of 2H20, the population of phenylalanine ammonia-lyase is

203

1.0 :> 0.5

o

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,

1.32

l

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1.30 1.28 1.26 Buoyantdensity (kg/l)

1.24

Fig. 3. E q u i l i b r i u m d e n s i t y g r a d i e n t e c n t r i f u g a t i o n o f p h e n y l a l a n i n e a m m o n i a - l y a s e f o l l o w i n g d e l a y e d i n t r o d u c t i o n o f 2 H 2 0 , Discs o f p o t a t o t u b e r p a r e n c h y m e w e r e i n c u b a t e d a t 2 5 ° C u n d e r c o n s t a n t i l l u m i n a t i o n f o r 6 h in H 2 0 a n d t h e n i n c u b a t e d f o r a f u r t h e r 3 h in 2 H 2 0 a t c o n c e n t r a t i o n s o f 3 7 . 5 a n d 9 5 % . T h e c o n c e n t r a t i o n o f 2 H 2 0 (v/v) is c a l c u l a t e d a f t e r a l l o w i n g f o r d i l u t i o n o f a d d e d 2 H 2 0 b y tissue H 2 O , After 3 h exposure to 2H20, phenylalanine ammonia-lyase was extracted and centrifuged to equilibrium in a CsC1 d e n s i t y g r a d i e n t . T h e e q u i l i b r i u m d i s t r i b u t i o n o f e n z y m e a c t i v i t y is p r e s e n t e d as a f u n c t i o n o f b u o y a n t d e n s i t y c o r r e c t e d w i t h r e s p e c t t o t h e d i s t r i b u t i o n of t h e e x t e r n a l m a r k e r e n z y m e fl-galactosidase. Open symbols refer to 37.5% 2H20, closed symbols to 95% 2H20.

0.010

100

5

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0065-~-- 10.0101

.>

LLI 0.000 I

0

I

1

I

I

20 40 60 I~O Concentrationof 2H20 (%)

J0 000~

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100

Fig. 4. E f f e c t of 2 H 2 0 c o n c e n t r a t i o n o n t h e d e v e l o p m e n t o f p h e n y l a l a n i n e a m m o n i a - l y a s e a c t i v i t y a n d t h e i n c o r p o r a t i o n o f 2 H f o l l o w i n g d e l a y e d i n t r o d u c t i o n o f 2 H 2 0 . Discs o f p o t a t o t u b e r p a r e n c h y m e w e r e i n c u b a t e d a t 2 5 ° C u n d e r c o n s t a n t i l l u m i n a t i o n f o r 6 h in H 2 0 a n d t h e n i n c u b a t e d f o r a f u r t h e r 3 h in H 2 0 o r a r a n g e o f c o n c e n t r a t i o n s o f 2 H 2 0 . T h e c o n c e n t r a t i o n of 2 H 2 0 (v/v) is c a l c u l a t e d a f t e r a l l o w i n g f o r d i l u t i o n of 2 H 2 0 b y tissue H 2 0 . A f t e r 3 h e x p o s u r e t o 2 H 2 0 , p h e n y l a l a n i n e a m m o n i a - l y a s e w a s e x t r a c t e d a n d s a m p l e s e i t h e r a s s a y e d f o r e n z y m e a c t i v i t y o r c e n t r i f u g e d t o e q u i l i b r i u m in CsCI d e n s i t y g r a d i e n t s . T h e e n z y m e a c t i v i t y o f c r u d e e x t r a c t s is e x p r e s s e d as % o f t h e a c t i v i t y o f e x t r a c t s f r o m c o n t r o l discs t r a n s f e r r e d t o H 2 0 i n s t e a d o f 2 H 2 0 (o ~). The equilibrium distribution of enzyme activity w a s a n a l y s e d as a f u n c t i o n o f b u o y a n t d e n s i t y c o r r e c t e d w i t h r e s p e c t t o t h e d i s t r i b u t i o n o f t h e e x t e r n a l m a r k e r e n z y m e /3-galactosidase. T h e i n c o r p o r a t i o n o f 2 H i n t o p h e n y l a l a n i n e a m m o n i a - l y a s e is m e a s u r e d b y (a) i n c r e a s e in b u o y a n t d e n s i t y o f p e a k e n z y m e a c t i v i t y ( e - o) a n d (b) i n c r e a s e i n b a n d w i d t h a t half peak height ( A ~ ) .

204 predominantly composed of molecules of native b u o y a n t density and correspondingly small increases in b u o y a n t density are observed. Accordingly, at all concentrations of 2H20 increases in the bandwidth of phenylalanine ammonia-lyase are observed, indicating in each case the presence of mixed populations of native, unlabelled and newly synthesised, labelled enzyme molecules (Fig. 4). We conclude that a 2H20 concentration of 37.5% is optimal for pulse density labelling experiments in this system: maximal shifts in b u o y a n t density are observed together with no inhibition of the increase in enzyme activity. Therefore, this concentration of 2H20 has been used in all subsequent experiments. Pulse density labelling experiments. The results presented in the previous section suggest that the initial increase in phenylalanine ammonia-lyase activity involved de novo synthesis of the enzyme and that activation of pre-existing enzyme was not quantitatively important. It can also be argued from the delayed transfer experiments that since significant amounts of native, pre-existing enzyme remain at the end of the labelling period, the removal of active enzyme cannot be proceeding at a very high rate at this stage. To obtain further information on the rate of enzyme synthesis (ks) and the rate of removal of active enzyme (kd), a series of 5-h pulses of 2H20 were administered at intervals throughout the entire time course. Thus, at each time point, t w o sets of discs were extracted: the first set having been transferred from H20 to 2H20 (37.5% final concentration) 5 h previously, the second set having been incubated continuously in H20. Both extracts were assayed for phenylalanine ammonia-lyase activity and the extract from discs pulsed with 2H20 was stored at --80 ° C until required for analysis by equilibrium density gradient centrifugation. The time course of the change in enzyme activity levels in H20 is shown in Fig. 5. There is a phase of rapid increase in activity between 5 and 15 h after disc preparation followed by a phase of rapid decline between 20 and 30 h and a subsequent stabilisation of the enzyme activity level at a b o u t 60% that of the maximum level. At no stage does prior transfer to 2H20 significantly affect the level of enzyme activity compared to the equivalent set of discs incubated continuously in H20. Analysis of extracts from pulse labelled discs by equilibrium density gradient centrifugation clearly demonstrates that de novo synthesis of the enzyme is occurring throughout the entire time course, even during the phase of decline in enzyme activity and the subsequent stabilisation of the enzyme level (Fig. 6). The data can be further analysed by using an integrated form of Eqn. 1 which leads to expressions for k d and ks: kd =

In E0 -- ln( Y1 • Ef ) t

(2)

ks =

Yh • Ef • kd 1 -- exp(--tZd • t)

(3)

where Ef is the enzyme level at the end of the labelling period, E0 is the enzyme level at the beginning of the labelling period, YI is the fractional a m o u n t of pre-existing, unlabelled, light enzyme at the end of the labelling period, Yh is the fractional a m o u n t of newly synthesised, labelled, heavy

205

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5c

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0

10

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20

30

40

(c)

2 01

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[

0

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20 Time (h)

30

40

F i g . 5 . S y n t h e s i s and r e m o v a l o f p h e n y l a l a n l n e a m m o n i a - l y a s e a c t i v i t y in i l l u m i n a t e d discs o f p o t a t o t u b e r p a r e n c h y m e . (a) D e v e l o p m e n t o f e n z y m e a c t i v i t y ; ( b ) r a t e c o n s t a n t for e n z y m e s y n t h e s i s ; ( c ) r a t e c o n s t a n t for r e m o v a l o f active e n z y m e . R a t e c o n s t a n t s are d e r i v e d by analysis o f the d i s t r i b u t i o n o f e n z y m e activity at e q u i l i b r i u m in CsCl d e n s i t y gradients ( F i g . 6 ) f o l l o w i n g pulse labelling w i t h 2 H f r o m 2 H 2 0 (Table I). Discs w e r e i n c u b a t e d at 2 5 ° C .

enzyme at the end of the labelling period and t is the length of the labelling period. Details of the derivation of Eqns. 2 and 3 are given in the Appendix. E~ is measured directly, Y1 and Yh are estimated from the equilibrium distribution of enzyme activity in the density gradient when compared to the distributions with Y1 = 1, Yh = 0 and Y1 = 0, Yh = 1: pure light and heavy enzyme, respectively (Fig. 6). Y1 and Yh were estimated by computation using an iterative method of best fit or by probit analysis, the values of these parameters for each pulse determined by the two methods are compared in Table I. The t w o methods give broadly similar estimates although there was a tendency for probit analysis to give slightly higher Yh/Y1 ratios. In subsequent calculations to determine ks and hd the values obtained by computation were used. 2H from 2H20 is n o t directly incorporated into protein, the label must first be taken up by the tissue and incorporated into amino acids by various metabolic processes. The uptake of 2H by the tissue will be rapid [25] but subsequent incorporation into amino acids will n o t be so rapid and there will be a dead

206

1.0-

(a)

(15 0.C 1.0 (b) _

I

{

0 . 5 ~ ~ 0.0

I

5

o.5~-

(10--

7 1.32 1.30 1.28 1.26 7g

l'0I(f)

0,0

I

L32 1.30 1.28 1.26 Buoyantdensity(kg/[)

Fig. 6. Pulse d e n s i t y l a b e l l i n g o f p h e n y l a l a n i n e a m m o n i a - l y a s e i n i l l u m i n a t e d discs o f p o t a t o t u b e r p a r e n c h y m e . T h e d i s t r i b u t i o n o f e n z y m e a c t i v i t y a t e q u i l i b r i u m in CsC1 d e n s i t y g r a d i e n t s is p r e s e n t e d as a f u n c t i o n o f b u o y a n t d e n s i t y (o---------o) c o r r e c t e d w i t h r e s p e c t t o t h e d i s t r i b u t i o n o f t h e e x t e r n a l m a r k e r e n z y m e fl-galactosidase f o r e n z y m e e x t r a c t e d f r o m discs t h a t h a d b e e n i n c u b a t e d (a) 9 h in H2 O, (b) 24 h in 2 H 2 0 , (c) 9 h in H 2 0 a n d 2 4 h in 2 H 2 0 . S a m p l e s d t o k w e r e i n 2 H 2 0 f o r 5 h a f t e r p r e - i n c u b a t i o n in H 2 0 f o r t h e f o l l o w i n g t i m e s (d) 0 h , (e) 5 h0 (f) 1 0 h , (g) 1 5 h , (h) 2 0 h , (i) 2 3 h , (j) 2 9 h , (k) 3 4 h. P r o b i t p l o t s o f t h e e q u i l i b r i u m d i s t r i b u t i o n o f e n z y m e a c t i v i t y are also p r e s e n t e d (e e ) . Discs w e r e incubated at 25bC. The concentration of 2H20 was 37.5% after allowing for dilution of added 2H20 by tissue H 2 0 .

207 TABLE I S Y N T H E S I S A N D R E M O V A L O F P H E N Y L A L A N I N E A M M O N I A - L Y A S E IN I L L U M I N A T E D D I S C S OF P O T A T O T U B E R P A R E N C H Y M E Discs w e r e p r e - i n c u b a t e d in H 2 0 at 2 5 ° C u n d e r c o n s t a n t i l l u m i n a t i o n and t h e n transferred t o 2 H 2 0 for 5 h b e f o r e e x t r a c t i o n o f e n z y m e a c t i v i t y . T h e e n z y m e w a s c e n t r i f u g e d t o e q u i l i b r i u m in a CsCl d e n s i t y gradient and the e q u i l i b r i u m d i s t r i b u t i o n o f e n z y m e a c t i v i t y w a s r e s o l v e d in t e r m s o f t h e f r a c t i o n a l c o n t r i b u t i o n o f t w o s p e c i e s : pure light e n z y m e (Y1) and pure h e a v y e n z y m e ( Y h ) using an iterative m e t h o d o f b e s t fit. E q u i v a l e n t e s t i m a t e s o f Y1 and Yh b y p r o b i t analysis are given in p a r e n t h e s e s . E f w a s d i r e c t l y assayed. T h e e f f e c t i v e pulse w a s 3.5 h and values o f E 0 w e r e e s t i m a t e d by t h e a p p r o p r i a t e i n t e r p o l a t i o n (Fig. 5). V a l u e s o f k d a n d k s w e r e c a l c u l a t e d a c c o r d i n g t o the e q u a t i o n s : In E 0 - - ln(Y1Ef) kd =

t YhEfkd

k s -

1 -- exp(--kdt )

using the values o f Y1 and Yh o b t a i n e d b y the iterative m e t h o d . Time (h)

5 10 15 20 25 28 34 39

Enzyme activity (pkat/disc)

Resolution of enzyme population

Final (Ef)

Initial (Eo)

Labelled (Yh)

Unlabelled (YI)

10 48 108 112 92 80 68 68

6 22 70 116 106 94 72 68

0.38 0.52 0.35 0.46 0.35 0.20 0.24 0.18

0.62 0.48 0.65 0.54 0.65 0.80 0.76 0.82

(0.46) (0.59) (0.36) (0.50) (0.37) (0.27) (0.27) (0.23)

kd (h-1)

(0.54) (0.41) (0.64) (0.50) (0.63) (0.73) (0.73) (0.77)

--0.010 --0.009 0.000 0.185 0.167 0.097 0.092 0.055

ks ( p k a t / d i s c per h )

1.1 7.2 10.8 20.0 12.2 5.4 5.2 3.8

time between the administration of label to the tissue and the availability of labelled amino acids for the synthesis of heavy phenylalanine ammonia-lyase. The dead time was estimated by preincubation of discs in H20 for either 5 or 24 h followed by transfer to 2H20. After various periods of exposure to 2H20, sets of discs were extracted and the incorporation of 2H into the enzyme measured by equilibrium density gradient centrifugation. In both cases a shift in buoyant density was first observed 2.25 h after treatment with 2H20 and by extrapolation of the data to zero shift in buoyant density a dead time of 1.5 h was obtained (Figs. 7 and 8) which was independent of the length of preincubation in H20. It follows that the effective length of each density label pulse (t) was 5 minus 1.5 h, i.e. 3.5 h. Moreover, the enzyme level at the effective beginning of each pulse (E0) can n o w be estimated by interpolation of the time course for changes in enzyme level (Fig. 5 and Table I). In the first three pulses, E0 = Y1 • E~, that is any enzyme preexisting at the effective start of a pulse remains at the end of the pulse, implying the absence of significant removal of active enzyme. Subsequently, E0 is more than Yi'Ef, implying removal of active enzyme. Analysis of the data in terms of Eqn. 1 reveals that the value of ks increases linearly over the first 20 h and subsequently drops rapidly to a relatively low level (Fig. 5 and Table I). In contrast, the value of k d remains zero in

208

Of (a)

0.0

?

/

Q5

J (t)

(b)

°I

"-'",

L 0.5

~ °5I ~0.0

I

0.0

"J 1,32

E

1.30

1.2B

1.26

1.0 Buoyant density

0.5

(kg/I)

(g)

~ 0.006

0.0

^

I

I

~0.004 z3 c__

1.0 (d)

~0,002

0.5 0 1

2

3

4

Length of exposure to 2H20 (h) O.C

1

M

1.32

1.30

1.28

1.26

Buoyant density (kg/I) Fig. 7. M e a s u r e m e n t o f t h e d e a d t i m e f o r t h e i n c o r p o r a t i o n of 2 H f r o m 2 H 2 0 i n t o p h e n y l a l a n i n e a m m o n i a - l y a s e in i l l u m i n a t e d discs o f P o t a t o t u b e r p a r e n e h y m e p r e i n e u b a t e d 6 h i n H 2 0 . A f t e r p r e i n c u b a t i o n discs w e r e t r a n s f e r r e d t o 2 H 2 0 f o r (a) 0 h , (b) 0 . 7 5 h , (c) 1 . 5 h , (d) 2 . 2 5 h , (e) 3 . 0 h , (f) 4 . 0 h b e f o r e e x t r a c t i o n of e n z y m e a c t i v i t y a n d e s t i m a t i o n o f the i n c o r p o r a t i o n of 2 H i n t o p h e n y l a l a n i n e a m m o n i a l y a s e b y a n a l y s i s o f t h e e q u i l i b r i u m d i s t r i b u t i o n o f e n z y m e a c t i v i t y i n CsC1 d e n s i t y g r a d i e n t s . In b t o f t h e d a s h e d l i n e s i n d i c a t e t h e e q u i l i b r i u m d i s t r i b u t i o n o f n a t i v e e n z y m e . In g t h e i n c r e a s e in b u o y a n t d e n s i t y o f p e a k e n z y m e a c t i v i t y is p l o t t e d as a f u n c t i o n o f t h e l e n g t h o f e x p o s u r e t o 2 H 2 0 . Discs w e r e i n c u b a t e d a t 2 5 ° C . T h e c o n c e n t r a t i o n of 2 H 2 0 w a s 3 7 . 5 % a f t e r a l l o w i n g f o r d i l u t i o n o f a d d e d 2 H 2 0 b y tissue H 2 0 .

the first 15 h, but subsequently, there is an abrupt increase in gradual decay to a relatively low level (Fig. 5).

kd

followed by a

Discussion The pulse density labelling experiments described in this paper offer a direct method for the estimation of the rate constants for enzyme synthesis and for removal of active enzyme. A number of conditions and assumptions should be satisfied for successful implementation of the method. First, the density label,

209

(a)

05

0£

I

I 0

>, 1 Or-

^

1.0

/0o%

0.5

0£

# F I

1.30

1.32

1.28

Buoyant density

,

1.26 ( kg/I )

1.24

(t)

0.5]

o.o02 1.32

1.30

1.2

Buoyant density

1.26 (kg/[)

1.24. ~

0

1

2

3

Length of exposure to 2H20

4

(h)

Fig. 8. M e a s u r e m e n t o f the dead t i m e for the i n c o r p o r a t i o n o f 2 H f r o m 2 H 2 0 i n t o p h e n y l a l a n i n e anam o n i a - t y a s e in i l l u m i n a t e d discs of p o t a t o t u b e r p a x e n c h y m e p r e - i n c u b a t e d 24 h in H20. A f t e r pre-inccub a t i o n discs w e r e transferred t o 2 H 2 0 for (a) 0 h, (b) 1.5 h, (c) 2.25 h, (d) 3.0 h, (e) 4.0 h b e f o r e e x t r a c tion o f e n z y m e a c t i v i t y and e s t i m a t i o n o f the i n c o r p o r a t i o n of 2 H i n t o p h e n y l a l a n i n e a m m o n i a - l y a s e b y analysis o f the e q u i l i b r i u m d i s t r i b u t i o n o f e n z y m e a c t i v i t y in CsC1 d e n s i t y gradients. In b to e the dashed lines i n d i c a t e the e q u i l i b r i u m d i s t r i b u t i o n o f native e n z y m e . In f the increase in b u o y a n t d e n s i t y o f peak e n z y m e activity is p l o t t e d as a f u n c t i o n o f the l e n g t h o f e x p o s u r e t o 2H20. All c o n d i t i o n s w e r e as d e s c r i b e d in Fig. 7.

in this case 2H from 2H20, must n o t affect the changes in enzyme level under investigation. A study of the relationship between concentration of 2H20, development of enzyme activity and the magnitude of the increase in buoyant density of the enzyme under study demonstrated that a 2H20 concentration of about 40% (v/v) was optimal. At this concentration maximal or near maximal increases in buoyant density were observed without inhibition or distortion of the physiological change in enzyme level. Second, the rate of labelling of the enzyme must be limited by its rate of turnover and n o t by the rate of turnover of the amino acid pools used for protein synthesis. Sufficiently rapid labelling of amino acids can easily be demonstrated by a bandwidth increase of the enzyme profile [ 2 7 ] . In the present study, bandwidth increases as measured by a reduction in the slope of probit plots were observed with each pulse labelling (Fig. 6) thereby demonstrating a rapid rate of turnover of the amino acid pools relative to the turnover of the enzyme. This conclusion is n o t incompatible with the observation of a dead time of 1.5 h following application of 2H20. The dead time represents the period taken for the front of the deuterium pulse to enter the cell, give rise to deuterated metabolites and for these metab-

210 olites to be elaborated to deuterated amino acids. The bandwidth measurements demonstrate that once the pulse of deuterium has reached the amino acids, the rate of turnover of the amino acid pools is sufficiently rapid for the deuterated amino acids to quickly replace the unlabelled amino acids. Third, the final specific activity of label in the amino acid pools must n o t change t h r o u g h o u t the time course under study. The saturation level of the increase in enzyme b u o y a n t density is a function not of the turnover of the enzyme but of the specific activity of label in the amino acid pools. In the present study, labelling of phenylalanine ammonia-lyase reaches the same saturation level on prolonged exposure to 2H20 whether label is applied immediately after disc preparation or after 24 h preincubation on H20 (Fig. 6). Therefore, we conclude that following administration of label at different times after disc preparation the final specific activities of the amino acid pools are the same. Fourth, the enzyme should not have completely turned over during the labelling period. This condition is clearly satisfied in the present study: significant amounts of unlabelled, pre-existing enzyme are found after each pulse of label (Fig. 6 and Table I). Fifth, integration of Eqn. 1 carries the assumption that ks and kd are constant throughout the duration of the labelling period. Ideally, the effective length of the labelling period should be as short as possible. However, practical considerations place a lower limit on the length of the pulse. Analysis of the distribution of enzyme activity in density gradients in terms of the contribution of two species of different mean b u o y a n t densities is most accurate when the two species are present in equal amounts. If one c o m p o n e n t predominates then analysis will lead to large relative errors in the estimation of the contribution of the minor component. Therefore if the pulse is very short relative to the time needed to reach equal amounts of labelled and unlabelled enzyme then large errors in Yh can be expected leading to correspondingly large errors in the estimate of k s. In the present study an effective pulse labelling period has been selected which is a compromise between the short ptflse desirable on theoretical grounds and the practical requirement of a pulse long enough to give adequate labelling. Thus in all cases except one the value of Yh ~> 0.2 and in the majority of cases 0.3 < Yh < 0.7. It is unlikely that tes and k d remained exactly constant during each pulse. However, the pulses were short compared to the duration of each of the three phases of the development of phenylalanine ammonia-lyase activity (rapid increase, rapid decrease, stationary). Therefore, the values of k s and k d derived from Eqns. 2 and 3 give a clear picture of the changes in the balance between enzyme synthesis and removal of active enzyme t h r o u g h o u t the time course. The initial rapid increase in phenylalanine ammonia-lyase activity 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 level is caused by decay of both rate constants to relatively low levels. The changes in rate constants for enzyme synthesis and removal of active enzyme follow exactly the pattern predicted by Zucker [15] on the basis of the effects of cycloheximide on the changes in enzyme level. Clearly these

211

results are entirely consistent with the hypothesis that rapid modulation of enzyme levels during tissue differentiation is achieved by simultaneous changes in the rates of both enzyme synthesis and removal [11]. It is worth noting that the rate constants k s and kd are both composite. For example, included in k s are rate constants governing polypeptide biosynthesis and the turnover of any compulsory, inactive polypeptide precursors of the active enzyme. Similarly, in the present case the methods used do n o t distinguish between removal of active enzyme by inactivation or by degradation. The value of kd is the sum of the rate constants for any such processes. The mechanism(s) of the removal of active enzyme are currently under study. Appendix Temporal changes in enzyme levels can be described by the equation: (dE/dt) = k s - - k d E (1) 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 ko is the rate constant for removal of active enzyme. In general k~, k d and E will be functions of t, but if small increments of t are considered, as is the case in pulse labelling experiments, k s and ko can be assumed to be constant and integration of Eqn. 1 gives: Ef

=

exp(--h~t)

{ks e x p ( k d t ) + C] ha

(2)

where EI is the enzyme level at the end of the pulse and C is a constant of integration. Setting t = 0 gives: (3)

C = Eo -- (ks/kd)

where E0 is the enzyme level at the beginning of the pulse. Substituting for C in Eqn. 2 gives: ks Ef = k-d" { 1 -- exp(--kdt)} + E0 • exp(--kdt)

(4)

The last term of Eqn. 4 refers to the removal of pre-existing, unlabelled enzyme and the first term refers to the production and removal of newly synthesised, labelled enzyme. Therefore Y1 " E f

= E 0 •

(5)

exp(--kot)

and Yh " Ef

= (ks/kd)

• (J

--

exp(--kdt)}

(6)

where YI is the fraction of Ef that is unlabelled and Yh is the fraction of Ef that is labelled. Eqns. 5 and 6 can simultaneously be solved for kd and ks: kd=lnE0--1n

Yt"Ef

t (Yh " Ef • hal) ks = 1 -- exp(--hdt)

(7) (8)

212

In pulse density labelling experiments, Y, and Yh are determined by analysis of the equilibrium distribution of enzyme activity in a density gradient and E,, and E, are the enzyme levels at the beginning and end respectively of the pulse of duration t. All these parameters are experimentally observable and h, and k, can therefore be directly obtained. Acknowledgements We are grateful to Dr. J. Aitchison of Pembroke College, Oxford for performing the computer analysis and for helpful discussions. C.J.L. gratefully thanks Oxford University for an ICI Research Fellowship and the Queen’s College, Oxford for a Browne Research Fellowship. References 1

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