Periodic increases in enzyme activity in synchronized cultures of Chlamydomonas reinhardtii

BIOCHIMICA ET BIOPHYSICA ACTA

153

BBA 95662

PERIODIC INCREASES IN ENZYME ACTIVITY IN SYNCHRONIZED CULTURES OF CHLAMYDOMONAS REINHA RDTII

J O S E P H R. KATES" AND RAYMOND F. JONES

Department o[ Biological Sciences, State University o[ New York at Stony Brook, Stony Brook, Long Island, N.Y. (U.S.A.) (Received February 27th, 1967)

SUMMARY

During synchronized growth of Chlamydomonas reinhardtii on a I2-h light-I2-h dark cycle, net protein synthesis occurs approximately linearly in the light period but no appreciable increase is observed during the dark period of growth. Increases in activity of alanine dehydrogenase (L-alanine : NAD oxidoreductase, EC 1.4.i.I), glutamate dehydrogenase (L-glutamate : NAD (P) oxidoreductase, EC 1.4.i.3), phosphoenolpyruvate carboxylase (orthophosphate:oxaloacetate carboxylyase (phosphorylating), EC 4.I.I.3I), aspartate carbamoyltransferase (carbamoylphosphate :L-aspartate carbamoyltransferase, EC 2.I.3.2) and ornithine carbamoyltransferase (carbamoylphosphate: L-ornithine carbamoyl transferase, EC 2.1.3.3) occur within characteristic periods of time during the life cycle of the cell. The activity of alanine dehydrogenase and aspartate carbamoyltransferase increases primarily in the light period, whereas the activity of the other three enzymes increases during the dark period of growth.

INTRODUCTION

The periodic synthesis of enzymes observed in synchronously dividing cultures of bacterial, * and yeast3 indicates that enzymes double at characteristic times in the generation cycle of the cell thereby giving rise to a reproducible sequence of enzyme bursts. The investigations with yeast indicated that enzymes whose structural genes mapped closely together were synthesized at nearly the same time during the generation cycle. It was suggested, therefore, that the sequence of transcription of genes was linear along the length of each chromosome. The linear sequence of gene duplication has been established for Bacillus subtilis by YOSHIKAWA AND SUEOKA4, 5. Using synchronized cultures of this organism MASTERS AND PARDEE6 proposed a mathematical model to illustrate one of the possible mechanisms which could result in periodic autogenous enzyme synthesis in synchronous cultures of bacteria. In their model, gene duplication and co-repressor • Present address: Department of Chemistry, University of Colorado, Boulder, Colo. (U.S.A.).

Biochim. Biophys. Acta, 145 (1967) 153-158

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J . R . KATES, R. F. JONES

concentration due to increases in cell volume were considered as the major variables. Such a model accounts for periodic enzyme synthesis without the requirement that enzymes be synthesized at the time when their particular gene doubles, and does not necessarily imply that genes are transcribed sequentially according to their position on the chromosome. This model, however, does postulate the existence of cytoplasmic co-repressors. The purpose of the present investigation was to examine periodic enzyme synthesis in the unicellular green alga Chlamydomonas reinhardtii which can be readily synchronized by an alternating period of 12 h light and 12 h dark and which possesses a well defined and rather short period of gene duplication relative to the total duration of the cell generation cycle7,s. Variations in the activity of alanine dehydrogenase (L-alanine:NAD oxidoreductase, EC 1.4.1.1 ) and glutamate dehydrogenase (L-glutamate:NAD(P) oxidoreductase, EC 1.4.1.3) in synchronously dividing cells of C. reinhardtii maintained in continuous light were reported previously 9. It was anticipated that in the present system one could differentiate between gene doubling and increases in the synthesis or activity of the following enzymes: alanine dehydrogenase (L-alanine:NAD oxidoreductase, EC 1.4.I.I), glutamate dehydrogenase (L-glutamate :NAD (P) oxidoreductase, EC 1.4.1.3), phosphoenolpyruvate carboxylase (orthophosphate : oxaloacetate carboxy-lyase (phosphorylating), EC 4.1.1.31), aspartate carbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), and ornithine carbamoyltransferase (carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3).

MATERIALS AND METHODS

Synchronized cultures of C. reinhardtii (strain 89-3-d ) were grown as described previously 7. In the present study the cells were grown on a large scale using two 12-1 algal culture vessels (as part of a continuous culture apparatus designed in collaboration with New Brunswick Scientific Co. Inc., New Jersey). Each vessel was maintained on a cycle of 12 h light and 12 h dark. The cell concentration in each vessel was readjusted to 2" lO6 cells per ml by dilution of the cultures with fresh sterile medium after the division burst, which occurred just prior to the onset of the light period. Under the conditions employed, each culture underwent approximately a 3.5-fold increase in cell number once every 24 h. All cells in the culture divided at least once in each cycle. Samples of 2 1 could be collected every 3 h of the vegetative life cycle and the remaining cells could be diluted and used to study the events in the next sequential cycle. Cell number was determined with a hemocytometer. Total protein was determined by the Folin phenol method 1°. DNA was estimated by the BURTOI~n modification of the diphenylamine colorimetric test for deoxyribosO 2. Cell-free extracts were prepared and the enzymes alanine dehydrogenase and glutamate dehydrogenase assayed as described by KATES AND JONES9. Aspartate transcarbamylase was assayed by the method of GERHARTAND PARDEE ls, and ornithine transcarbamylase was assayed according to the method of BARNETT AND COHEN14. Phosphoenolpyruvate carboxylase was assayed b y tile measurement of x4C02incorporation stimulated by phosphoenolpyruvate. The reaction mixture contained io/zmoles MgC12~ Biochim. t~iophys. Acta, 145 (1967) 153-158

ACTIVITY IN CHLAMYDOMONAS

ENZYME

155

0-7 #mole NADH, zoo #moles NaH14CO3 (spec. act. z C/mole), and 50 #moles phosphate buffer (pH 7.5) to make a total volume of 0.8 ml. Enough enzyme extract was added to fix zo s counts/min per rain at 25 °. The N A D H was added in order to convert the unstable oxaloacetate into malate via malate dehydrogenase which is present in excess in the enzyme preparation. The rate of fixation was linear for at least the first 5 rain. In these studies the reaction was stopped after 3 min by the addition of 1.2 ml of hot acetic acid-95 % ethanol (1:50, b y vol.). 1 ml of the suspension was transferred to a planchet and dried in an oven at 9 o°. The radioactivity was estimated with a Sharp low-background, wide-beta, gas-flow counter. RESULTS AND DISCUSSION

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Fig. I. I n c r e a s e in cell n u m b e r a n d t i m e course of D N A s y n t h e s i s d u r i n g s y n c h r o n i z e d v e g e t a t i v e g r o w t h of C. reinhardtii (strain 89-3-d). Fig. 2. N e t p r o t e i n p e r cell as a f u n c t i o n of t i m e d u r i n g t h e s y n c h r o n i z e d v e g e t a t i v e cycle of C. reinhardtii. T h e t r i a n g l e s a n d t h e circles r e p r e s e n t d a t a f r o m t h e first, a n d second, respectively, of two s e q u e n t i a l s y n c h r o n o u s cycles.

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Fig. 3. A c t i v i t y of a l a n i n e d e h y d r o g e n a s e as a f u n c t i o n of t i m e d u r i n g t h e s y n c h r o n i z e d veget a t i v e cycle of C. reinhardtii. A u n i t of e n z y m e a c t i v i t y is d e f i n e d as t h e a m o u n t of e n z y m e n e c e s s a r y to c a u s e a c h a n g e in a b s o r b a n c e a t 360 m/~ of o.ooI a b s o r b a n c e u n i t s p e r rain. T h e t r i a n g l e s a n d t h e circles r e p r e s e n t d a t a f r o m t h e first, a n d second, respectively, of two s e q u e n t i a l s y n c h r o n o u s cycles. Fig. 4. A c t i v i t y of g l u t a m a t e d e h y d r o g e n a s e as a f u n c t i o n of t i m e d u r i n g t h e s y n c h r o n i z e d vege t a t i v e life cycle of C. reinhardtii. A u n i t of e n z y m e a c t i v i t y is defined s i m i l a r l y to t h a t for a l a n i n e d e h y d r o g e n a s e (Fig. 3). T h e t r i a n g l e s a n d t h e circles r e p r e s e n t d a t a f r o m t h e first, a n d second, respectively, of two s e q u e n t i a l s y n c h r o n o u s cycles. Biochim. Biophys. Acta, 145 (1967) i 5 3 - i 5 8

I56

j . R . KATES, R. F. JONES

Fig. I illustrates the increase in cell number during the two cycles of growth from which samples were harvested for enzyme analysis. It should be noted that the cells divide to produce four daughter cells in each cycle. The period of DNA synthesis is also represented in Fig. I. The amount of protein per cell during the vegetative life cycle is shown in Fig. 2. It is seen that net protein synthesis occurs approximately linearly in the light period but no appreciable increase is observed during the dark period. There is a 3.2-fold increase in total protein, on the average, in each of the two cycles. Since the average cell number increment was 3.5, it is seen that balanced growth is occurring with respect to protein synthesis. The activity of alanine dehydrogenase (Fig. 3) appears to increase primarily in the first 6 h of the light period. Some increase in activity of this enzyme is observed in the last 6 h of the light period, but little, if any, increase is observed during the dark period. Conversely, glutamate dehydrogenase (Fig. 4) increases in activity only during the last 6 h of the dark period. Both enzymes increase in activity by a factor of approx. 4.5.

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Fig. 5. Activity of p h o s p h o e n o l p y r u v a t e carboxylase as a function of time during the synchronized vegetative cycle of C. reinhardtii. A u n i t of enzyme activity is defined as the a m o u n t of enzyme required to incorporate iooo counts of NaHt4CO~ per rain at 25 ° under the conditions of the assay procedure. The triangles a n d the circles represent d a t a from the first, and second, respectively, of two sequential synchronous cycles. Fig. 6. Activity of aspaxtate t r a n s c a r b a m y l a s e as a function of time during the synchronized vegetative cycle of C. reinhardtii. A u n i t of enzyme activity is defined as the a m o u n t of enzyme necessary to produce o . i / , m o l e of c a r b a m y l a s p a r t a t e per h. The triangles and the circles represent d a t a from the first, and second, respectively, of two sequential synchronous cycles.

Phosphoenolpyruvate carboxylase (Fig. 5) shows a pattern of increase in activity similar to that observed for glutamate dehydrogenase. Similarly, aspartate transcarbamylase activity increases only during the latter half of the dark period (Fig. 6). On the other hand ornithine transcarbamylase activity begins to increase at the end of the dark period and appears to stop increasing 3 h prior to the end of the light period (Fig. 7). Phosphoenolpyruvate carboxylase, aspartate transcarbamylase and ornithine transcarbamylase increase in activity by factors of 2. 9, 2.2 and 3.6 per cycle, respectively. The increases in enzymatic activities which were observed in these experiments m a y well represent the de novo synthesis of enzyme molecules. No evidence for the existence of readily diffusible inhibitors of enzyme activity was found by mixing l?iochim. Biophys. Acla, 145 (1967) 153-158

ENZYME ACTIVITY IN CHLAMYDOMONAS

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Fig. 7. A c t i v i t y of o r n i t h i n e t r a n s c a r b a m y l a s e as a f u n c t i o n of t i m e d u r i n g t h e s y n c h r o n i z e d v e g e t a t i v e life cycle of C. reinhardtii. A u n i t of e n z y m e a c t i v i t y is defined as t h e a m o u n t of e n z y m e r e q u i r e d to c a t a l y z e t h e f o r m a t i o n of i / , m o l e of citrulline p e r h. T h e t r i a n g l e s a n d t h e circles r e p r e s e n t d a t a f r o m t h e first, a n d second, respectively, of two s e q u e n t i a l s y n c h r o n o u s cycles.

enzyme extracts from different stages of the life cycle and measurement of resulting activity. Experiments are currently in progress to determine, by the use of inhibitors, whether or not protein and RNA synthesis are required for the observed increases in enzymatic activities. It appears, therefore, that the synthesis of particular enzymes, as measured by increases in enzymatic activity, occurs within characteristic periods of time during the life cycle of the cell. If enzyme synthesis is occurring, different enzymes m a y have markedly different periods of synthesis. It should be noted that those enzymes increasing in activity during the dark period do so in the absence of appreciable net protein synthesis. In the cases of alanine dehydrogenase and glutamate dehydrogenase, the pattern of synthesis shown here for a light-dark synchronous cycle was similar to that found for synchronously dividing cells maintained in continuous light 9. Since the rate of ornithine transcarbamylase synthesis is not apparently affected by the darkto-light transition, it is unlikely, therefore, that the observed bursts in enzyme activity are a direct consequence of the environmental cycle. The magnitude of the increment in most of the enzyme activities per synchronized cycle is roughly of the same order of magnitude as the increase in cell number per cycle. However, deviations in the latter relationship between enzyme activity and cell division do not necessarily mean that enzyme molecules are not synthesized proportionately to cell number increment in our system. Other factors m a y affect the activity of the enzymes in question besides the number of enzyme molecules present (e.g. allosteric factors, enzyme inactivation during extraction, etc.). Under the conditions employed in the present experiments DNA synthesis in Chlamydomonas begins in the last hour of the light period and ends at the end of the fourth hour of the dark period. Thus DNA synthesis occurs in less than one quarter of the life cycle. None of the enzymes which were studied were observed to increase significantly during the period of DNA synthesis. Initiation of the increase of alanine dehydrogenase activity was observed 12 h before the onset of DNA synthesis. The initiation of increase in activity of ornithine transcarbamylase occurred 4 h after the end of DNA synthesis. Thus for the latter two enzymes it has been possible to distinguish between the time of gene doubling and the increase in enzyme activity. Biochim. Biophys. Acta, 145 (1967) I 5 3 - I 5 8

I58

J . R . KATES, R. F. JONES

The present findings in Chlamydomonas, therefore, support the conclusion of

KUEMPEL, MASTERS AND PARDEE1 that the bursts in the synthesis of autogenous enzymes do not necessarily correspond to the time of gene doubling. The observation that the five enzymes which were studied were not synthesized extensively during the period of D N A synthesis and cell division does not imply that other enzymes are not made during this time.

ACKNOWLEDGEMENTS

This investigation was supported by contract AT(3o-I ) 3475 with the Atomic Energy Commission. Mr. J. R. KATES was a recipient of a National Institute of Health Predoctoral Fellowship.

REFERENCES i P. L. I{UEMPEL, M. MASTERS AND A. B. 1DARDEE, Bioch(m. Biophys. Res. Commun., 18 (1965) 858. 2 M. MASTERS, P. L. KUEMPEL AN~ A. }3. PARSEE, Biochem. Biophys. Res. Commun., 15 (1964)38. 3 J. GORMAN, P. TARUO, M. LABERGE AND H. HALVORSON, Biochem. Biophys. Res. Commun., 15 (1964) 43. 4 H. YOSHIKAWA AND N, SUEOKA, Proc. Natl. Acad. Sci. U.S., 49 (1963) 559. 5 H. YOSHIKAWA AND ~ . SOEOKA, Proc. Natl. Acad. Sci. U.S., 49 (1963) 8o6. 6 M. MASTERS AND A. n . PARDEE, Proc. Natl. Acad. Sci. U.S., 54 (1965) 54. 7 J. R. KATES AND R. F. JONES, J. Cellular Comp. Physiol., 63 (1964) 157. 8 J. R. KATES, K. S. CHIANG AND R. F. JONES, Exptl. Cell Res., in t h e p r e s s . 9 J. R. HATES AND R. F. JONES, Biochim. Biophys. Acta, 86 (1964) 438. io O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265 . 11 K. BURTON, J. Biochem., 62 (1955) 315 • 12 Z. DlSCHE AND K. SCHWARTS, Mikrochim. Acta, 2 (1937) 13. 13 J. C. GERHART AND A. B. PARDEE, J. Biol. Chem., 237 (1962) 891. 14 G. I-I. BARNETT AND P. P. COHEN, J. Biol. Chem., 229 (1957) 337.

Biochim. Biophys. Acta, 145 (1967) 153-158