On the regulation of tyrosine transaminase, glutamatic dehydrogenase and aspartic transaminase in Tetrahymena

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Experimental

Cell Research 77 (1973) 335-345

ON THE REGULATION OF TYROSINE TRANSAMINASE, GLUTAMATIC DEHYDROGENASE AND ASPARTIC TRANSAMINASE IN TETRAHYMENA PAMELA

PORTER1

and J. J. BLUM

Department of Physiology and Pharmacology, Duke University Medical Center, Durham, N.C. 27710, VSA

SUMMARY

Aspartictransaminase, tyrosinetransaminase, lacticdehydrogenase, andglutamicdehydrogenase werestudiedin Tetrahymena pyriformis in order to gaina better understanding of the control of theentranceandexit of metabolicintermediates to andfrom themajorcarbohydratepathways. Glucosedecreased the activity of aspartictransaminase, tyrosine transaminase and glutamic dehydrogenase but not lactic dehydrogenase. Actinomycin D ( 6 and 12 pg/ml) blockedthe decrease in glutamicdehydrogenase and aspartictransaminase activity causedby glucose;12 ,ug/mlpartially preventedthe decrease in tyrosinetransaminase activity. Actinomycin D alone had little effect on enzymeactivity. Uracil incorporationinto RNA wasdoubledby 6 pg/ml actinomycinD, a concentrationwhich did not alter the RNA contentof the cells.At 12pg/ml this drugcauseda smalldecrease in RNA spec.act. Cyclobeximideat 10pg/ml,a concentration which inhibitedproteinsynthesisby 70%, causeda three-foldincreasein aspartictransaminase and a two-fold increasein glutamicdehydrogenase. In the presence of both cycloheximideand glucose,the drugeffect predominated. Thusboth actinomycinD andcycloheximideblockedthe glucose-induced decrease in enzymeactivity. Theseresultssuggestthat the levelsof aspartic transaminase, glutamicdehydrogenase, and probably tyrosine transaminase are regulatedat leastin part by a degradativecontrol system.

The ciliate Tetrahymena pyriformis can synthesize over 20 “/ of its body weight as glycogen from the amino acids and peptides of a proteose peptone medium [33]. The regulation of the conversion of amino acids to keto acids, which in a sense is the first step in glyconeogenesis, has received little attention in Tetrahymena although many studies have been done in higher animals [17]. For example, a high glucose diet decreased the activities of rat liver histidase, serine dehydratase, ornithine transaminase, mitochondrial aspartate transaminase, and cyto1 Presentaddress:Departmentof Medicine,University of California,La Jolla, Calif. 92037,USA. 22 - 721817

plasmic alanine transaminase and countered the induction of several of these enzymes by cortisol or glucagon [43]. Mavrides & D’Iorio [34] showed that glucose could reduce the level of tyrosine aminotransferase in Tetrahymena and suggestedthat this might be an instance of glucose repression in a protozoan. We were interested in learning more about the control of enzymes which allow substrates and products to enter and leave the main pathways of carbohydrate metabolism, and studied the effect of glucose and a variety of other substrateson the activities of tyrosine transaminase(Tyr TA), aspartic transaminase (Asp TA), glutamic dehydrogenase (GDH), and lactic dehydrogenase (LDH). We found Exptl Cell Res 77 t 1973)

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that glucose decreased the activities of Tyr TA, Asp TA, and GDH, but not that of LDH. Further investigation showed that actinomycin D prevented this effect of glucose and, contrary to expectations, increased the rate of incorporation of labelled uracil into the RNA of this cell. These observations suggested that the activities of some of these enzymes might be regulated at least in part by a degradative control system. A systematic study of the regulation of the activities of these enzymes was then undertaken, and is the subject of this paper.

dehydrogenase (1.4.1.2), lactic dehydrogenase (1.1.1. 27) aspartic transaminase (2.6.1.1.) and tyrosine transaminase (2.6.1.5) were assayed according to the methods of Balinsky et al. [l], Bergmeyer et al. [2,3] and Lin et al. [27], respectively. None of the conditions reported here changed the relationship between enzyme activity and substrate concentration. For further details, see Porter [45].

Protein determination Aliauots of sonic lvsates and the suuernatants of these lysates were precipitated with 5 X-(w/v) trichloroacetic acid (TCA) at 0°C. The oellet was dissolved in 0.2 N NadH, and the protein-content determined by the method of Lowry et al. [29]. The values obtained for the supernatant fraction were used to calculate the spec. act. of tyrosine transaminase; the protein content of the unfractionated sonic lysates were used for the other enzymes.

METHODS Growth

of cells

Tetrahymena pyriformis, strain HSM, were grown on 1 % oroteose pentone and 0.05 % liver extract in 0.02 k potassium phosphate buffer adjusted to pH 6.5 with NaOH. Cultures were routinely grown in a gyratory shaker bath (New Brunswick Scientific Co.) at 26°C in 500 ml flasks containina 100 ml of this medium. Cells were counted with a Coulter Counter, and growth was expressed as the ratio of cell number when the experiment was terminated (N,) to the number when the experiment was begun (NJ. Drugs and substrates were sterilized by filtration through sintered glass filters, and unless stated otherwise drugs or substrates were added approx. 16 h before the collection of the cells.

Preparation of sonicates Cultures were added to an equal volume of 0.08 M Trischloride, 36 m M NaCl, pH 7.5 at 0°C and the cells were collected by centrifugation at 250 g for 5 min. This and all subsequent steps were done at @-4°C. The pellet was resuspended in 25 mM imidazole, mM reduced glutathione, pH 6.8 (standard buffer) and again centrifuged at 250 g for 5 min. The pellet was suspended in standard buffer at a density of approx. 2 x lo8 cells/ml, and sonicates were made by treating this preparation with ultrasound twice for 30 set using a Branson Model LS-75 ultrasonic generator. This sonicate was used for all enzyme assays except for tyrosine transaminase in which the supernatant from a 1 h spin at 27 000 g was used. Enzymatic assays were completed within 8 h after sonication.

Glycogen determination Washed cells were treated with 95 % (v/v) ethanol and the pellet was collected by centrifugation at 250 g for 10 min. The uellet was susoended in 0.05 M acetate buffer at pH 4.5 and the-glycogen was converted to glucose by ,&glucoamylase. The glucose was then analysed by the glucose oxidase method [lo].

Incorporation of leucine and uracil Incorooration was initiated bv adding 15 ml of culture to 125 ml flasks containing-either 5.1 ml of a 0.14 mM solution containina 5 uCi iGleucine (U). The flasks were incubated with’ shaking at 25°C for 30 min. The uptake of radioactive material was terminated by adding 3 ml of the cells to 8 ml of ice-cold 10 % TCA or 0.5 % NaCl. The samples were washed 3 times and dissolved in hyamine. Counting an aliquot of the supernatant from the third wash showed this to be sufficient to remove the soluble counts. All samples were counted in a Packard scintillation counter in [2,5&s-2-(5-tert-butylbenzoxayolyl) thiophene-toluenel-ethanol (13 : 10 v/v} scintillation solution. Counts were corrected for quench by the use of an external standard. In some experiments the saline washed pellets were treated with 5 ml of 95 % ethanol and both the pellet and a portion of the supernatant counted in order to determine the labelling of intracellular uracil. RNA content was measured by a modified SchmidtThannhauser method [S]. Both cell counts and protein determinations were made in all experiments.

Enzyme assays

Reagents

Standard buffer was used for all assays except tyrosine transaminase in which 0.1 M Tris chloride at pH 8.2 was used. All reactions were run at 30°C except tyrosine transaminase which was run at 24°C. Glutamic

Chloramphenicol was obtained from Parke-Davis and cycloheximide from Sigma. Actinomycin D was generously supplied by Merck, Sharp and Dohme. All other chemicals were reagent grade.

Exptl Cell Res 77 (1973)

Regulation of enzyme activity Tetrahpmena

RESULTS

337

Table 1. Effect of glucose at different culture ages

Effect of glucose Control Glucose “& Control The activities of glutamic dehydrogenase, aspartic transaminase, and tyrosine transcuhre aminase were decreased when 10 mM glucose Log GDH 146+7 84+2’ 57 was used to supplement the proteose peptone LDH 46k9 4813 104 Asp TA 10222 8216 80 medium (table 1). Lactic dehydrogenase Tyr TA 91 k8 76+1 83 activity, unlike the other activities, was not mg protein/ decreased and in several experiments inlo6 cells 1.03 3~0.06 1.21 * .02 118 creased slightly. Glucose resulted in de- Early stationary culture creased levels of aspartic transaminase, tyroGDH 1404 20 98 ’ 22a 70 LDH 280 1 50 350 - 80 125 sine transaminase, and glutamic dehydroTA 47 3 30 7a 64 genase in stationary phase cultures, where Asp Tyr TA 100 30 56 6’ 56 there was no change in cell number OI in mg protein/ the total amount of cellular protein. As lo6 cells 0.91 .02 0.91 ,~O.l 100 shown in table 1, the response of the enzymes Enzymatic activities are expressed as nmoles/min/mg to glucose was not very dependent on culture protein. The data are expressed as an average of 2 age although it appeared that the trans- or more expts - S.D. Cultures designated as log had a cell count of 250 000-330 000 cells/ml when collected aminases were slightly more sensitive to and a N,/N, of approx. 10; early stationary cultures were 600 000 cells/ml or higher and had a N,/Ni glucose in the older cultures. Even though of less than 1.7. The glucose concentration was 10 the glucose effect was largely independent mM in the other cultures. “p c:0.02. GDH, glutamic dehydrogenase; of culture age, the experiments presented in Abbreviations: LDH, lactic dehydrogenase; Asp TA, aspartic transtable 1 show that the activities of lactic aminase; Tyr TA, tyrosine transaminase. dehydrogenase and aspartic transaminase vary as a function of culture age. For this reason this experimental variable was carefully con- markedly increased within 2 h after glucose trolled in allexperiments. Throughout these ex- addition [45]. These results indicated that periments the ratio of mg protein to lo6 cells glucose did not directly affect the activities remained essentially constant; therefore none of any of these enzymes. These results on the effect of glucose on of the reported changes in enzyme activity are the result of changes in the protein tyrosine transaminase confirm the findings content of the cells. of Mavrides & D’lorio [34]. The effect of In a separate experiment the glucose con- glucose on LDH activity is somewhat smaller centration was varied from lo-30 mM than that observed by Levy & Wasmuth [26]. (data not shown), and there was no effect of the glucose concentration on the decrease Comparisonof substrates in activity of glutamic dehydrogenase, aspartic Lactate (5 mM), pyruvate (10 mM), glutatransaminase or tyrosine transaminase. At mate (3 mM), aspartate (2 mM), phenylthe highest concentration there was an alanine (5 mM), alanine (10 mM), tyrosine ethylester (0.5 mM), and glycerol (20 mM) increase in lactic dehydrogenase. A short exposure to glucose (2-4 h) did were without effect on the enzymes of this not change the activity of any of the enzymes study. Glycerol, however, significantly instudied, although glycogen content was creased glycogen content, an observation not Exptl

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Table 2. Effect of actinomycin D on the response to glucose % Control

Asp TA GDH LDH Tyr TA Glycogen mg protein/ lo6 cells

Experiment group

Act D

Glucose

Glucose + act D

A B A B A B A B A B

164f27@ 146511 93+3 117255 115&9 69+1 97+21 92+8 60flO 45&25

6128 69+8 62+11 6218 169+3 128&S 53fl 43+4 706+32 837193

106522 141&l 96+11 107*3 122&6 69FlO 46+2 73&4 632&92 605k 173

A B

105+1 llOf3

121+1 130+1

149+13 144*11

a Enzymatic activities were calculated on the basis of protein content, and then expressed as % control. In expt group A, actinomycin D (act D) concentration was 6 ,ug/ml; in B act D was 12 pug/ml and glucose concentration was 20 mM. The results are the average of 2 expts. Cultures in all experiments were in stationary phase (NfINi < 2).

hitherto reported for Tetrahymena to our our knowledge. Fructose, although less effective than glucose, did result in small decreases in glutamic dehydrogenase, aspartic transaminase, and tyrosine transaminase activities.

acting via receptors on the cell surface.) A study of the effect of adenine nucleotides on the glycogen content of Tetrahymena will be reported elsewhere [4].

Cyclic AMP

To gain further insight into the cellular events involved in the response of GDH, LDH, Tyr TA, and Asp TA to glucose, the effect of inhibitors of RNA and protein synthesis was studied. Actinomycin D, which acts by inhibiting DNA-dependent RNA synthesis [48], was added to cultures in the presence and absence of glucose. Table 2 shows that actinomycin increased aspartic transaminase activity slightly at 6 and 12 ,ug/ml; both concentrations tested blocked the decrease in aspartic transaminase activity caused by glucose. Actinomycin D alone did not affect the activity of glutamic dehydrogenase, but prevented the decrease in activity which occurred in glucose grown cells. A low concentration of actinomycin (6 pg/ml) had no effect on

Cyclic AMP is known to block the glucose repression of p-glactosidase [38] and other enzymes in bacteria [l 11. Since Tetrahymena contain cyclic AMP (Toomey, R R & Gordee, R. Personal communication) experiments were designed to determine if cyclic AMP blocked the glucose in Tetrahymena. Cyclic AMP did not prevent the decrease in activity caused by glucose nor did it alter the enzyme activity in the absence of glucose. Glycogen content, however, was markedly increased by 4 mM cyclic AMP in 5 experiments (210 480 % of the control), thus excluding the possibility that cyclic AMP had not entered the cells. (This assumes that cyclic AMP and the other adenine compounds studied acted intracellularly as opposed to Exptl

Cell Res 77 (1973)

Effects of inhibitors of RNA and protein synthesison enzyme activities

Regulation

lactic dehydrogenase activity but some decrease occurred at 12 pg/ml. The variable increasein LDH caused by glucose was never observed in cultures treated with actinomycin D. Actinomycin D alone had no effect on tyrosine transaminase. At a concentration of 6 ,ug/ml actinomycin D did not interfere with the glucose effect, but 12 ,ug/ml actinomycin D partially blocked the effect of glucose on tyrosine transaminase. Thus although actinomycin D alone had little effect on enzyme activity, it largely prevented the effect of glucose on all four enzymes. Cycloheximide, known to inhibit protein synthesis in eukaryotic cells in general [16] and in Tetrahymena in particular [15], also had an unexpected effect on the enzymes studied. It nearly doubled the activity of glutamic dehydrogenase and nearly tripled the activity of aspartic transaminase (table 3). In the presence of both glucose and cycloheximide, the drug effect predominated. Tyrosine transaminase was not affected by cycloheximide alone, but cycloheximide nevertheless blocked the effect of glucose. Lactic dehydrogenase, unlike the other enzymes was decreased by cycloheximide; activity in the presence of cycloheximide and glucose was the same as with cycloheximide alone. Thus, cycloheximide, like actinomycin D, blocked the effect of glucose. However, cycloheximide, unlike actinomycin, caused an increase in glutamic dehydrogenase, and the increase in aspartic transaminase was considerably larger than with actinomycin D. When both cycloheximide and glucose were present, the drug effect predominated. The increase in glycogen content (table 3) shows that glucose entered the cycloheximide-treated cells and that there was no gross disturbance in the glucose metabolism of these cells. Fig. 1 shows that activity changed gradually over a period of about 18 h. This indicates no direct effect of cycloheximide on these enzymes,

of enzyme activity

Table 3. Effect

Tetrahymena

of cycloheximide

339

on the re-

sponse to glucose “0 Control CM (IO/cg/ml)

Glucose (20 mM)

-CM glucose --

GDH ASQ TA Tyr TA LDH Glycogen

118232 271+64 98214 57&7 14Oi60

13 + 17 63&4 4229 119+24 78Oh 150

205 t21 270-c-57 95 1-7 57 t5 880 L 280

mg protein/ IO8 cells

104L 12

133Z13

115 :6

-Enzymatic activity was calculated on the basis of protein content before being expressed as U; control; glycogen was calculated as pg/106 cells, then presented above as % control. The results were the average of 4 expts. Cultures were in stationary phase, N,/N, less than 1.6. CM, cycloheximide.

and again suggeststhat the changesin enzyme activities observed are due to alterations in the net balance between synthesis and degradation. To check the possibility that cycloheximide or actinomycin D acted by changing the levels of certain small molecules which could regulate enzyme activity, sonicates from control cultures, from cultures grown with 6 pg/ml actinomycin and from cultures grown with 1O/pug/ml cycloheximide were assayed for aspsrtate transaminase and for glutamic dehydrogenase activities separately and mixed in all combinations. In each case the activities were additive, suggesting that if small molecule regulators were present, their levels were not altered by growth in the presence of either drug. Three of the enzymes of this study, lactic dehydrogenase, glutamic dehydrogenase, and aspartate transaminase, are localized in mitochondria [46]. Because chlordmphenicoi selectively inhibits mitochondrial protein synthesis whereas cycloheximide is specific for the inhibition of cytoplasmic protein synthesis [2X], a comparison of these two Expfl

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Table 4. Effect of chloramphenicol response to glucose

on the 280 240

% Control 0.3 mM Chloramphenicol GDH Asp TA LDH Tyr TA mg protein/ 106 cells

140f20 lOOf 80&10

70flO 98i6

Asp T

20 mM Glucose 80f10 60+10 120410

Glucose Chloramphenicol

GDt

lOOk 10 50*20

40+10

90+20 so+4

140+_10

120+_10

Cells were in stationary phase (NIIN, ~2). Enzyme activity was calculated on the basis of protein content and then expressed as % control. These results are the average of 3 expts.

LDh

4

8

12

16

20

1. Abscissa: time after addition of cycloheximide (hours); ordinate: % control. Time-dependent effects of cycloheximide. Enzyme activity and incorporation of leucine and uracil unto TCA-insoluble material were measured following the addition of 10 pug/ml cycloheximide. See Methods for experimental details. Counts were divided by cell number and then expressed as % control. Enzymatic activity was calculated on the basis of protein content and then expressed as % control. Drug treatment did not affect the protein content of the cells. Fig.

drugs was made to determine which site of protein synthesis was involved in the response to glucose. A concentration of 0.3 mM chloramphenicol was used because this concentration completely inhibits the growth of Tetrahymena [20]. Table 5 shows that at 0.3 mM chloramphenicol, lactic dehydrogenase and tyrosine transaminase activities are slightly lower than in the control cultures while glutamic dehydrogenase activity was slightly higher. Chloramphenicol did not appreciably interfere with the effect of glucose on any of the enzymes. Therefore, chloramphenicol was clearly different from cycloheximide in that it had little effect on the activity of the enzymes by itself and did not significantly affect the response to glucose. Effects of actinomycin D and cycloheximide on RNA and protein synthesis

Because of the remarkable effects of actinomycin D and cycloheximide in blocking the effect of glucose on the 4 enzymes of this study and especially because the activity of two enzymes was increased by cycloheximide, it was important to establish whether or not these drugs were acting in the expected Exptl

Cell Res 77 (1973)

manner

on RNA

and protein

synthesis in

Tetrahymena.

The effect of actinomycin D on uracil incorporation is given in table 5. The conditions used for these experiments were identical to those used in the experiments where actinomycin largely blocked the effect of glucose. RNA spec. act. was determined by measuring the radioactivity of TCA-washed cells from cultures labelled with uracil for 30 min and dividing this count by the RNA content from a sample of the same culture. It was expected that actinomycin D would decrease uracil incorporation, but it was found that actinomycin D at 6 pug/ml increased the spec. act. of RNA in both control and glucose grown cultures. At 12 ,ug/ml actinomycin D, the RNA spec. act. was less than that of the control. Three other experiments were done on cultures grown without glucose. In each

Regulation of enzyme activity Tetrahymena 341

experiment incorporation was doubled in cells exposed to 6 pg/ml actinomycin D. The average RNA spec. act. for the 5 experiments was 240+ 30% of the control. The pellets from the saline-washed cells were also counted, and the results were the same as for TCA-washed cells. These data clearly show that the spec. act. of the RNA was greater than the control at 6 pg/ml, but less than the control at 12 ,ug/ml. RNA content was not appreciably changed by the addition of glucose or actinomycin. The data in table 5 also show that the effect of actinomycin on uracil incorporation was similar in the presence or absence of glucose. Although the increased spec. act. of RNA was probably not due to increased uptake of uracil by the cell, this possibility was investigated. Cells that had been exposed to labelled uracil were washed 3 times with saline and then 95% ethanol was added to the packed cells. The ethanol-washed cells were centrifuged and both the supernatant and pellet counted. The pellet contained uracil that had been incorporated into macromolecules; the supernatant contained free uracil and uracillabelled nucleotides. The data show that the free uracil counts of the actinomycin Dtreated cells were not significantly greater than those of the control. Therefore, increased uptake of labelled uracil into an expanding pool of intracellular uracil-containing nucleotides was not responsible for the increase in RNA spec. act. The effect of actinomycin D on protein synthesis was also examined, using leucine as a marker. Leucine incorporation into acidinsoluble material was 60 & 2 “/ of the control at 6 lug/ml actinomycin D and 13 t- 3 % at 12,~g/ml. The inhibitory effect of actinomycin D on leucine incorporation was not altered by the presence of glucose. In the presence of 10 pg/ml cycloheximide, leucine incorporation into trichloroacetic acid

Table 5. Effect of actinomycin

D on uracil

incorporation RNA content ::ts Control

6 m/ml act D

12iuglml

act D mM glucose 20 mM glucose,

RNA spec. act. CPM/m RNA

Free uracil
103-1-4

52+1--

9oi14

113&10

124

96-1-2 .~

3612

70

126~21

68123

56

108 -‘r 1

9812

76

50+_26

58

20

6 ,%/ml

act D mM glucose, 12 jLg/ml act D

20

95 t15

Incubations were carried out as described in Methods. The data are the average of 2 expts except the ‘free uracil’ data which are the result of 1 expt. ‘Free uracil was measured as explained in the methods section by washing cells in NaCI, then treating with 95 “b ethanol and counting the supernatant.

pellets was 30 & 10 “/ of the control and uracil incorporation was 502 20 ?A of the control. The increase in enzyme activity in cycloheximide-treated cells was therefore, not due to an aberrant effect of the drug on protein synthesis. The inhibition of protein synthesis by cycloheximide reported here is somewhat less than that reported in cultures synchronized by heat treatment 18, 151. In the experiments shown in fig. 1, the incorporation of uracil and leucine were measured at three times following the addition of cycloheximide. Both leucine and uracil incorporation were significantly inhibited 1 h after the drug was added and remain.ed inhibited. As mentioned earlier, the time course of the change in enzymatic activity was much longer. The inhibitory effect of cycloheximide on leucine incorporation therefore precedes its enhancing effect on enzyme activity. Previous work on Tetrahymena had shown that 0.3-1.5 mM chloramphenicol resulted Exptl

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in smaller mitochondria with intact outer membranes but degenerated cristae structure [52]. Manson et al. [32] had shown that a 1 h exposure to 1.5 mM chloramphenicol caused no detectable inhibition of leucine incorporation. After an 8 h incubation of isolated mitochondria, there was a 50 % decrease in the spec. act. of mitochondrial membrane enzymes, but an increase in the spec. act. of soluble mitochondrial protein. Thus, chloramphenicol has specific effects on mitochondria in Tetrahymena as well as on mitochondria in other cells [28]. A comparison of the effect of chloramphenicol and cycloheximide on leucine incorporation showed that chloramphenicol at 2.3 mM inhibited the incorporation of leucine into trichloracetic acid-insoluble material somewhat more effectively than cycloheximide. The cpm/mg protein were 22 + 2 % of the control with chloramphenicol as compared to 30-J 10 % with cycloheximide. Therefore, even though chloramphenicol and cycloheximide had different effects on enzyme activity and on the effect of glucose both chloramphenicol and cycloheximide significantly inhibited protein synthesis under conditions used for the glucose studies. This inhibition of leucine incorporation by chloramphenicol is greater than that reported by Crocket et al. [9] and is greater than would be expected if only mitochondrial protein synthesis were affected. A comparison of these results and those of Mason et al. [32] indicates that the length of exposure to chloramphenicol is critical in determining the extent of inhibition of leucine incorporation. Perhaps microsomal protein synthesis is inhibited as a secondary result of the effect of chloramphenicol on mitochondria as has teen reported in yeast [54]. Uracil incorporation into washed trichloroacetic acid pellets was increased by chloramphenicol to 240+ 10% of the control. Exptl

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Chloramphenicol has been reported to stimulate RNA synthesis in E. coli by causing an accumulation of aminoacyl-tRNA [21], but whether this is the molecular mechanism responsible for the increase in Tetrahymena is not known. DISCUSSION A comparison of control mechanisms between eukaryotes and prokaryotes is of interest because of the striking differences already known tetween these two groups. (See Margulis [31] for a detailed discussion.) The response of the enzymes of this study to glucose is unlike catabolite repression in bacteria in that it is not affected by cyclic AMP [38]. In E. coli within minutes after the addition of glucose enzyme synthesis has essentially stopped [40]. In Tetrahymena, however, there was no effect of glucose on enzyme activity 2-4 h after its addition. If the rate of enzyme synthesis had been greatly repressed in logarithmically growing cultures of Tetrahymena, the activity per cell would have decreased markedly during exponential multiplication. Since the response to glucose in stationary cultures was equal to or greater than that in logarithmically growing cultures, some process other than division must be responsible for the decreased activity in glucose grown ceils. In exponentially growing bacterial cultures there is no appreciable protein turnover [19, 23, 491. This is clearly not the case in mammalian tissues [44, 51, 531. Therefore, in eukaryotes, unlike prokaryotes, the amount of an enzyme is due to a balance of synthesis and degradation. (For a recent review, see Schimke & Doyle [50].) The finding that in Tetrahymena the activities of aspartic transaminase and glutamic dehydrogenase were increased by a concentration of cycloheximide that caused 70 % inhibition of protein synthesis cannot be explained by the transcriptional control

Regulation of enzyme activity Tetrahymena systems devised for bacteria. These results instead suggest that enzyme degradation as well as synthesis is regulated in Tetrahymena. If the degradative enzymes (e.g., proteinases) themselves turned over more rapidly than any of the four enzymes investigated then cycloheximide would cause a rapid loss of degradative capacity. Since some protein synthesis continues (30% of the control in the experiments reported here) there could be a net increase in the amount of, say, glutamic dehydrogenase, even though its rate of synthesis had been reduced. Haider & Segal [18] have recently investigated the alanine aminotransferase and arginase inactivating system in rat liver. They found a good correlation between the in vivo rate of inactivation of these enzymes and the rate at which they were degraded in vitro by disrupted lysosomes. They calculate that the lysosomes strain cell constitutents from approx. 8 times their own volume per hour. Tetrahymena have proteinases in their lysosomes, and indeed, release some of them into the medium at an appreciable rate [12, 361. However, the effects of cycloheximide and actinomycin D on lysosomal function are not known. If these drugs were found to decrease the effective lysosomal proteinase activity, the argument that these drugs decrease the turnover of glutamic dehydrogenase and aspartic transaminase could be made. Although there are many reports on the effect of actinomycin D on Tetrahymena, none are directly comparable to those reported here because the experiments were done on either synchronized cultures or on cultures in the logarithmic phase of growth, whereas the present experiments were done on early stationary phase cells in order to avoid the complication of variation in growth rate between control and drug-treated cultures rate between control and drug-treated cultures. Sensitivity to actinomycin D varies

343

with the method of synchrony [14, 24, 25, 371 and the composition of the medium [ 131. Furthermore, the sensitivity of synthesis of different types of RNA is known to vary [6, 39, 41, 421. Cleffman [7] reported that 3 ,ug/ml inhibited RNA synthesis 90’::,, while Christenson [6] found that 10 ,ug/ml strongly inhibited ribosomal RNA synthesis but caused a weak inhibition of synthesis of other types of RNA. Moner [35], however, found that uridine incorporation was increased in heat synchronized cultures when actinomycin was added at either 0 or 10 min following the heat treatment. In the present experiments 6 pg/ml actinomycin D increased W-uracil incorporation over twofold, but without changing the level of RNA per cell. This requires either that there must have been a corresponding increase in degradation of RNA or that the uracil was being incorporated primarily into a class of RNA present in small amount but with a high turnover rate and that the synthesis of this class of RNA must have been increased by the actinomycin. It has recently been reported that Tetrahymena contain a rapidly labelled heterodisperse RNA which has a high turnover rate [47] and that synthesis of this type of RNA is less sensitive to actinomycin D than the synthesis of ribosomal RNA [6]. Because this heterodisperse RNA comprises a small part of the total RNA, there could be an increased incorporation of label into this fraction while the total amount of RNA per cell remained constant. It is, however, generally agreed that actinomycin acts by binding to DNA and thereby preventing transcription of RNA [22]. No study on the mechanism of action of actinomycin D has ever indicated an increase of transcription. However, the results presented in table 5 and those of Moner 1351 show that under certain conditions the incorporation of uracil into RNA is increased. Exptl

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A possible explanation, consistent with the interpretation already suggested to explain the effect of cycloheximide on GDH and Asp TA activity, is that degradation of RNA is regulated in a manner analogous to the degradation of enzymes. If the synthesis of ribonuclease (known to be present in the lysosomes of Tetrahymena [36]) was very sensitive to inhibition by actinomycin D then the degradation of a fast turnover class of RNA such as the heterodisperse RNA discussed above could be temporarily reduced to a greater extent than its synthesis. The effect of actinomycin D in blocking the glucose-caused reduction in activity of GDH, Tyr TA, and Asp TA is probably best explained by a mechanism similar to that proposed for the effect of cycloheximide. At every concentration tested, actinomycin D blocked leucine incorporation into protein. If glucose acted (as assumed above) by increasing the amount or activity of the degradative system and actinomycin prevented this increase, then glucose would be rendered ineffective by the actinomycin. The results reported here, in which cycloheximide and actinomycin D block the decrease in activity of several enzymes, resemble the results obtained in rat thymocytes, where these two drugs prevent or reverse the cortisol-induced synthesis of a proteininhibitor of transport and phosphorylation [30]. In that system Makman et al. [30] have postulated that cortisol induces the synthesis of a messenger RNA coding for the inhibitory protein(s) with more rapid turnover rate than that of the average messenger RNA. It should be emphasized that not all inhibitors of protein synthesis inhibit the response to glucose. Chloramphenicol, which inhibited protein synthesis by 80 % had little effect on the glucose response. Presumably the chloramphenicol inhibited only mitochondrial protein synthesis initially and this, Exptl

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in turn, led to a general inhibition of cytoplasmic protein synthesis which differed in its characteristics from the direct inhibitory effects of cycloheximide and actinomycin D. The data presented here are not sufficient to prove that the control of activity is by a degradative system. An alternative explanation is that the changes reported here are due to differential changes in the synthesis of different cellular proteins. However, since cycloheximide has repeatedly been shown to inhibit protein synthesis and never to our knowledge been shown to increase it, we found a degradative control system to be an attractive hypothesis to explain the somewhat unusual group of findings reported in this paper. It should be clear that much additional work would be required to prove this hyptothesis. Measurements of turnover rates (using immunochemical techniques) and studies on the susceptibilities of these enzymes to the proteases of Tetrahymena would be the minimum initial steps in such a study. The control of lactic dehydrogenase activity is not understood at this time. The differences between the response of LDH and the other three enzymes of this study to glucose and to cycloheximide suggests that the regulation of LDH activity is somewhat different than that of the other three. Aspartic transaminase, tyrosine transaminase, and glutamic dehydrogenase appear to be regulated by a degradative system. Since these enzymes are not under coordinate control [45], it is likely that degradative control is common in Tetrahymena. For this reason investigation of the control of other enzymes in this organism would be of considerable interest.

This work was supported by grants from NIH (5 Rol HD01269) and NSF (GB-25032). Dr Blum is the recipient of a Research Career Development Award (SK3 GM0341) from NIH. Dr Porter was supported by a USPHS traineeship.

Regulation of enzyme activity Tetrahymena

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Received May 15, 1972 Revised version received September 1, 1972.

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