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Effects of lipotropic agents on the metabolism of Ehrlich ascites carcinoma cells and other tissues
514
BIOCHIMICA ET BIOPHYSICA ACTA
E F F E C T S O F L I P O T R O P I C A G E N T S ON T H E
METABOLISM OF
EHRLICH ASCITES CARCINOMA CELLS AND OTHER TISSUES R. M. JOHNSTONE AND J. H. QUASTEL McGill-Montreal General Hospital Research Institute, Montreal (Canada)
(Received July I5th, I96o)
SUMMARY Lipotropic substances, such as Amytal, pentothal, tribromethanol, chloretone and irzdole which exercise large inhibitory effects on brain respiration have equally large inhibitory effects on the respiration of Ehrlich ascites carcinoma cells. Indole and skatole, as well as tribromethanol, choretone and pentothal, inhibit the uptake of glycine into Ehrlich ascites cells under anaerobic conditions in presence of glucose, the extent of inhibition being greater than the inhibition of anaerobic glycolysis. Results with tribromethanol show that other tissues (mouse melanoma, mouse lymphoma, mouse spleen) are affected in a similar manner to Ehrlich ascites carcinoma cells. Tribromethanol does not increase the rate of effiux of glycine from the cells, so that the inhibition of amino acid uptake is due to a suppressing effect on the mechanism responsible for the influx. Tribromethanol also inhibits anaerobic glycolysis of ascites cell suspensions at concentrations having little or no effect on the rates of glycolysis of extracts of these cells. The concentration of the lipotropic agents required to inhibit the anaerobic uptake of glycine in ascites cells is of the same order as that required to obtain inhibitory effects on brain respiration. The results indicate that the transport systems of the cell are dependent for their activities on lipid components in the membrane structure, with which the lipotropic, or neurotropic, agents become associated. As a result of this association, transport rates of amino acids are affected.
INTRODUCTION It is already established that the transport of amino acids in the Ehrlich ascites cells is an energy dependent process 1-3. Moreover, it is known that the amino acids themselves, and related substances, will interfere with the transport of a given amino acid 1, 3-5, 20 in a variety of tissues. In a report presented by us to the National Cancer Institute of Canada in 1958 we showed that various barbiturates and anaesthetics depress respiration, and affect other aspects of metabolism, of tumours including Ehrlich ascites carcinoma cells. Since then it has been established *,? that Amytal inhibits the respiratory activities of Ehrlich ascites carcinoma cells. Abbreviations : DNA, desoxyribonucleic acid ; TCA, trichloracetic acid ; TBE, tribrornethanol ; (Avertin) ; ATP, adenosine triphosphate ; DPN, diphosphopyridine nucleotide. Biochim. Biophys. Acta, 46 (1961) 514---526
LIPOTROP1C AGENTS AND ASCITES CELL METABOLISM
515
Data will be presented in the present paper to demonstrate that substances, which have usually been associated with inhibition of metabolism in brain tissue, inhibit the transport of amino acids in tumour cells. It appears also that the inhibitions observed are not primarily the result of an inhibition of respiratory activity in the tumour tissues but are the results of changes associated with the cell membranes. MATERIALS AND METHODS
Preparations of tissues used Ehrlicb ascites cells were grown and prepared as described previously a. The cells were diluted with I I times their packed volume of Ringer medium composed as follows: NaC1 146 mM, KC1 6 mM, MgCI~ 1. 5 mM, KH2PO 4 1.5 mM. I ml of such a suspension was used in a final volume of 3 ml. The dry weight of the cells was usually 9.0 mg. Cell-free extracts of the Ehrlich ascites cells were prepared as described elsewhere s.
Chick embryo Five-day-old chick embryos were used. The whole embryo was excised, the membranes removed, weighed quickly, and added to flasks kept at o °. Tissue slices of other tissues were prepared using a Stadie-Riggs slicer. In most cases, slices of less than IOO mg and more than 50 mg were used.
Method of incubation In most experiments, the standard Warburg manometric apparatus was used for incubation. The incubation was carried out at 37 °. In some cases, 5o-ml Erlenmeyer flasks were used for incubation. For anaerobic incubations, the medium was composed of an isotonic Ringer of the same composition as given above together with 25 m M sodium bicarbonate. Glucose, when added, was present at a concentration of io mM. The gas phase was N,~-C02 (95:5). Glycolysis was measured by the carbon dioxide evolution from a bicarbonate buffer. For aerobic incubations, IO m M phosphate buffer at pH 7.4 was added to the Ringer medium, and the gas phase was pure oxygen unless otherwise specified in the text. o.15 ml of 20 °/o potash was present on a roll of filter paper in the centre well of the manometric vessel. As the filter paper had to be removed after incubation, and before the tissue was removed from the flasks, care was taken to avoid any potash dripping into the flasks. For this reason, a double thickness of filter paper was used, the potash added being sufficient to moisten the paper without danger of subsequent drip on removal of the filter paper.
Indole assays A method based on that by CIRIOTTI9 for DNA was adopted. The cells were washed with 7 ml ice-cold Ringer solution and the precipitated packed cell residue was extracted with 1.5 ml of 6 °/o TCA. After 30 min at room temperature, the precipitate was centrifuged and an aliquot of the clear supernatant, usually I ml, was used for the indole assay. 200 mg of DNA (from fish roe) were used for each milliliter of Biochim. Biophys. Acta, 46 (I96I) 514-526
516
R.M. JOHNSTONE, I. H. QUASTEL
TCA extract. The rest of the procedure was identical with that used by CIRIOTTI foI DNA 9. Indole from 5 to 50 Y was measured by this procedure in a final volume of 4 ml. The optical density was measured at 490 m/~ in a Beckman Model B Spectrophotometer. Recoveries were carried out with all experiments and were always quite satisfactory. A standard curve for indole is presented in Fig. I. 0.60 0.50
"~ 0.40 "6 .~. 0.30 0 0.20 0.10
o
18
2~
38
20
/.~g indole
5'0
Fig. 1. Variation of optical density with indole concentration. Ordinate, O.D. ; abscissa, #g indole.
Amino acid uptake and e~ux determinations Radioactive amino acids were obtained from Merck & Co., Montreal, Quebec. [IJ4C]glycine and DL-[IJ4C]alanine were used in this work. The amino acids were introduced into the main compartment of the Warburg flasks from the side arm after gassing and thermal equilibration. After a given incubation period, the flask contents were poured into centrifuge tubes containing 7 ml of ice-cold isotonic Ringer medium. The tubes were centrifuged, the supernatants poured off and recentrifuged without further addition of Ringer solution. The small residue of Ringer medium was removed carefully with a syringe and needle, and 3 ml of 95 % ethanol were added to the cells and quickly stirred. After 30 min, the tubes were centrifuged and an aliquot of the clear ethanol extract was plated on aluminium discs, dried and the radioactivity measured. The radioactivity was assayed with the aid of a Tracerlab scaler and of a Geiger Miiller mica end window probe. The uptake of amino acids was expressed as counts/min/mg dry wt. tissue. The efflux of radioactive amino acids was estimated similarly i.e. b y measurement of the radioactivity remaining in the cells. The procedure for extracting soluble amino acids from slices differed from that used for the Ehrlich ascites cells only in that the slices were homogenized in a Teflon homogenizer after addition of alcohol to the slice. Incorporation of radioactive amino acids into proteins was carried out according t o QUASTEL AND BICKIS]°.
Reagents used Indole was obtained from Nutritional Biochemical Corporation, trichlorethar~ol was obtained from the Mann Chemical Co., New York and tribromethanol from the Winthrop Laboratories, New York. 4-Bromindole, 6-bromindole and 2,3, dimethyl indole were kindly provided by Dr. R. K. BROWN, University of Alberta, Edmonton, Canada, Biochim. Biophys. Acta, 46 U96I) 514-526
LIPOTROPIC
AGENTS
AND
ASCITES
CELL
METABOLISM
517
Experimental The results presented in Table I show that the respiratory activities of Ehrlich ascites carcinoma cells are inhibited by a number of substances which are known especially to inhibit the respiration of brain tissue n-15,19, 21. Amytal, pentothal, tribromethanol and chloretone are all effective inhibitors of respiration in these cells at concentrations which have marked effects on brain tissue metabolism na, 12,14,19. Indole is also an effective inhibitor of respiration in Ehrlich ascites cells. The results in Table II show the sensitivity of these cells to indole. Substances related to indole, such as serotonin and indoleacetate, have little or no effect at comparable concentrations. To compare the sensitivity to indole of the Ehrlich ascites cells with normal cells, experiments were carried out with a variety of tissues. The typical results presented in Table III show that, of the tissues examined, only brain tissue is inhibited to the same extent as the Ehrlich ascites cells by indole. The sensitivity of brain respiration to indole and skatole is already known na. TABLE
I
E F F E C T S OF BARBITURATES AND TRIBROM- AND T R I C H L O R - E T H A N O L ON THE R E S P I R A T I O N OF E H R L I C H ASCITES TUMOUR CELLS
C o n d i t i o n s : R i n g e r - - p h o s p h a t e m e d i u m at p H 7 . 4 ; gas phase, o x y g e n ; i n c u b a t i o n time, 6 o m i n at 37 °. Additions
Ccncentration
Nil Amytal Amytal
Amytal Penthothal Tribromethanol Trichlorethanol
mM
002
-o.5 I 2 i 2 4
lO. 4 3-9 i .9 o.6 4.7 2.2 3-5
TABLE
II
E F F E C T S OF I N D O L E AND D E R I V A T I V E S ON T H E R E S P I R A T I O N OF E t J R L I C H ASCITES CELLS
I n c u b a t e d in R i n g e r - - p h o s p h a t e m e d i u m at p H 7.4. Gas phase, O ~ ; i n c u b a t i o n time, I h a t 3 7 °. Expt.
A dditions
O0~
Nil
13.2 lO.8 8.8
0. 5 m M i n d o l e I m M indole 2 m M indole 4 m3//indole
4.9 1.2
Nil 2 2 4 2 4 3 4
mM mM mM mM mM mM m2~/~
lO. 4
indole serotonin* serotonin indole acetate indole acetate 3-hydroxy tyramine tryptophane
5.4 lO.5 9.5 11.8 9.6 lO. 7 9.3
* S e r o t o n i n w a s used as t h e creatinine s u l p h a t e salt.
Eicct, im. Bicphys. Acta, 4 6 ( 1 9 6 1 ) 5 1 4 - 5 2 6
518
R . M . JOHNSTONE, J. H. QUASTEL TABLE III EFFECTS
OF
INDOLE
I n c u b a t e d in R i n g e r - p h o s p h a t e
ON
THE
RESPIRATION
OF A N I M A L T I S S U E S
m e d i u m at p H 7.4 in o x y g e n ; temperature, 37 ° . Incubation time, I h.
Tissue
W h o l e chick e m b r y o
Additions
Nil 2 m M indole 4 m M indole
Mouse spleen slices
Nil
SaT m o u s e sarcoma slices
Glucose Glucose Glucose Glucose
(IO m3~r) + i m M indole + 2 m M indole + 4 m M indole
Nil I m M indole 2 m M indole 4 m M indole
R a t k i d n e y slices
6.6 5.3 2.0 7.2
i m M indole 2 m M indole 4 m M indole
R a t brain cortex slices
Q02
Nil P y r u v a t e (IO m M ) Indole 4 m M Indole 4 mM+ Pyruvate
7.5 5.I 3.O
I6.6 12.7 8.2 0.9 3.2 3.I 2.S 1.8 7,o I9.5 4.0 16.o
Effects of indole on the uptake of amino acids into Ehrlich ascites cells Aerobically, the presence of indole or of any other substance which depletes energy by inhibition of respiration, or by uncoupling, causes a marked inhibition of transport of amino acids. As it is known 8 that the uptake of amino acids is the same aerobically and anaerobically so long as glucose is present, most oI our investigations have been carried out under anaerobic conditions. The latter condition was preferred in order to ascertain whether the observed effects could be due to a direct effect on the transport mechanisms rather than to a depletion of the energy supply by inhibition of respiration, or by an uncoupling of oxidation from phosphorylation. The rate of glycolysis was estimated in most experiments to determine whether any of the substances examined had inhibitory effects on this process. The results of typical experiments given in Table IV show that indole has little effect on anaerobic glycolysis but inhibits considerably the effect of glucose on the uptake of alanine and glycine. The glucose-stimulated uptake of alanine is usually inhibited by 50 % with 2 m M indole. A similar inhibition of glycine uptake occurs in the presence of 2 m M indole. It was found that the uptake of amino acid is not decreased by indole below the level obtained in the absence of glucose. Therefore, the figure for the endogenous uptake was substracted in order to calculate the degree of inhibition. This procedure was considered to be justified as experiments have shown that the addition of indole at concentrations of 4 m M or less has little effect on the uptake of the amino acids anaerobically in the absence of glucose. Biochim. Biophys. Acla, 46 (I96I) 514-526
L I P O T R O P I C A G E N T S AND ASCITES CELL METABOLISM TABLE
519
IV
INFLUENCE OF INDOLE ON AMINO ACID UPTAKE IN EHRLICH ASCITES CELLS UNDER ANAEROBIC CONDITIONS E x p t . I , 3"3 m M L - a l a n i n e , 83" lO 8 c o u n t s / m i n p e r flask. E x p t . 2 EI-t4C]glycine 2 m M , 90" lO 3 c o u n t s ] r a i n / f l a s k . I n b o t h e x p e r i m e n t s : I n c u b a t e d i n N z - C O z (95:5) a t 37 °. I n c u b a t i o n t i m e , i h ; glucose concentration, io raM.
QN~ COa
Uptake counts~rain/rag dry wt. 6f tissue
alanine alanine + I mM indole alanine + 2 mM indole alanine + 4 mM indole
3. z 43 .0 43.0 40.o 38.0
360 775 635 550 37 °
Glycine Glycine + glucose Glycine + glucose + i mM indole Glycine + glucose + 2 mM indole
-51.o 51.o 51.o
300 144o 111o 935
Expt.
Additions
Alanine Glucose Glucose Glucose Glucose
+ + + +
TABLE
V
EFFECTS OF VARIATION OF AMINO ACID CONCENTRATION ON GLYCINE UPTAKE BY EHRLICH ASCITES CARCINOMA CELLS UNDER ANAEROBIC CONDITIONS G a s p h a s e , N 2 - C O 2 ( 9 5 : 5 ) . I n c u b a t i o n , 4 ° m i n a t 37 °. G l y c i n e r a d i o a c t i v i t y , 2200 c o u n t s / m i n / / t m o l e . Uptake counts~rain~rag dry wt. cells Glucose absent
Glycine c6ncn. (ram)
Glucose present (~ro raM)
Indole absent
I ndole (z raM) present
Indole absent
lndole (2 raM) present
Percentage inhibitionof glucose stimulated uptake
7.8 40.5 67.5
7.2 45.0 83. 5
50.5 183-o 240.0
32.7 126.5 18o.o
40 42 44
0.5 2.5 5.0
TABLE
VI
UPTAKE OF INDOLE BY EHRLICH ASEITES CELLS ANAEROBICALLY G a s p h a s e , N ~ - C O 2 ( 9 5 : 5 ) ; g l u c o s e c o n c e n t r a t i o n , IO m M ; i n c u b a t i o n t i m e I h 37 °. V a l u e s g i v e n a r e t h e a v e r a g e of t h r e e s e p a r a t e d e t e r m i n a t i o n s . Intracellular indole concentration E xtracellular indole concentration (raM)
I 2 4 8
Glucose absent
Glucose ( x o rnM) present
~ o. 5 < o. 5 1.8 7.o
mM mM mM mM
mM mM mM mM
B i o c h i m . B i o p h y s . A c t a , 46 (1961) 5 1 4 - 5 2 6
520
R. M. J O H N S T O N E ,
J . H. Q U A S T E L
Experiments were carried out with different glycine concentrations, at a constant indole concentration, to determine whether indole inhibits the glucose-stimulated uptake by competing with the amino acid for uptake. The results of typical experiments shown in Table V indicate that the extent of inhibition of glycine uptake by 2 m M indole is independent of the glycine concentration up to 5 mM. The uptake of indole itself was also examined to determine whether the lack of effect in the absence of glucose could be correlated with a decreased uptake of indole itself. The data in Table VI show that there is no concentrative uptake of indole in the cell either in the presence or the absence of glucose. The results also show that the indole concentration in the cell, even in the presence of glucose, is less than that of the medium. It was, therefore, concluded that indole inhibits the process of uptake by a mechanism other than competition between amino acid and indole, and that the inhibition does not involve the accumulation of indole in the cell.
Effects of some lipotropic substances on anaerobic uptake of glycine and anaerobic glycolysis To determine whether indole behaves in a unique manner on the uptake of glycine by the Ehrlich ascites cells, or whether other substances known to inhibit respiratory activity in brain behave in a similar way, the effects of lipotropic substances such as TBE, trichlorethanol, chloretone, pentanol, octanol and skatole were examined. The results of typical experiments given in Table VII show that the higher TABLE VII EFFECTS OF TRIBROMETHANOL, CHLORETONE, SKATOLE, PENTANOL AND OCTANOL ON ANAEROBIC GLYCOL¥SIS AND GLYCINE UPTAKE IN EHRLICH ASCITES CELLS [I-14C]glycine concentration 2 mM= lO s c o u n t s / r a i n p e r f l a s k . G l u c o s e c o n c e n t r a t i o n i o r a M . All reagents except octanol and pentanol are 4 mM. Octanol, o.5 ml of water saturated solution. P e n t a n o l , i o r a M . G a s p h a s e , N . 2 - C O t ( 9 5 : 5 ) . I n c u b a t i o n t i m e , i h a t 37 °. Additions
Id COz/h
Nil Glucose Tribromethanol + glucose Skatole + glucose Chloretone + glucose Pentanol + glucose Octanol + glucose
4o.o 465.o 35o.o 645.0 470.0 485.0 5o2.o
% inhibition
--24 + 4° Nil Nil Nil
Uptake dry wt. counts[rain[rag
% inhibition
lO9.O I I IO.O 3oo.o 420.0 730.0 965.0 114o.o
--8o 69 38 14 Nil
alcohols have no effect on uptake of glycine at the concentrations examined. (Ethanol up to concentrations of 0.5 M had no effect on the uptake of glycine under anaerobic conditions.) Stimulations of glycolysis of approximately 40 % were usually observed with skatole at 2-4 mM. Recently we have observed that other substituted indoles such as 4- or 6-bromindole and 2,3 dimethyl indole also stimulate the anaerobic glycolysis. However, it is apparent from the results presented in Table VII, that inhibitions of glycine uptake with skatole are observed in spite of the increased glycolysis. Tribromethanol and chloretone also had a pronounced effect on the uptake of glycine. Trichlorethanol was found to be much less effective than tribromethanol at the equivalent concentrations. Thus at 4 mM, trichorethanol inhibited the glucoseB i o c h i m . B i o p h y s . Acta, 4 6 (I961) 5 1 4 - 5 2 6
LIPOTROPIC
521
AGENTS AND ASCITES CELL METABOLISM
stimulated uptake of glycine under anaerobic conditions by 15-2o % and tribromethanolinhibited it by about 7 ° %. Chloretone and tribromethanol, at concentrations above 4 mM, usually inhibit anaerobic glycolysis as well as uptake of glycine. With increasing inhibitions of glycolysis, the uptake of amino acids is correspondingly depressed. However, the extent of inhibition of glycolysis is usually considerably less than that of the inhibition of uptake. Moreover, at concentrations of TBE, which have no effect on glycolysis, significant inhibitions of uptake are still observed (Table VIII). TABLE
VIII
EFFECTS OF TRIBROMETHANOL AND CHLORETONE ON ANAEROBIC GLYCOLYSIS, UPTAKE AND INCORPORATION OF [I-14CJGLYCINE IN EHRLICH ASCITES CELLS
Conditions: R i n g e r - - o . o 2 5 io
mM.
M N a H C O 3 a t p H 7.5. Gas phase, N 2 - C O 2 (95 : 5), glucose concentration, [ I - 1 4 C ] g l y c i n e , 2 m M lO 5 counts/min per flask. Incubation t i m e , 6 o min (at 3 7 ° ) . r,7
Additions
Nil Glucose Glucose Glucose Glucose Glucose Glucose Glucose
Q~
+ TBE + TBE + TBE
(i m M ) (2 m M ) (8 r a M ) + chloretone (i m M ) + chloretone (2 m M ) + chloretone (4 m M )
*"
3.8 48.o 45.8 44.5 4°.0 44.5 44.0 41.o
uptake counts[rain/rag dry wt. 222 1700 1275 i I Io 535 141o 124o 985
TABLE
% inhibition
Incorporation into proteins counts/rain/rag protein
--29 4° 79 19 31 48
0.6 8 i. 5 77.0 68.5 15.6 82.0 81.o 37.0
% inhibition
--5 16 8i Nil
Nil 54
IX
EFFECTS OF TRIBROMETHANOL ON ANAEROBIC GLYCOLYSIS OF EXTRACTS AND INTACT CELL PREPARATIONS OF EHRLICH ASCITES CELLS
Incubation medium: Buffer, o . o 2 8 M N a H C O 3 ; gas phase, N z - C O ~ ( 9 5 : 5 ) - Cell e x t r a c t s were fortified with o. 3 m M A T P , o . i m M of D P N a n d 4 ° m M nicotinamide. Glucose concentration, IO m M . I n c u b a t i o n t i m e , 6 o min at 37 °. pl COs/h Additions
Nil
Glucose Glucose + T B E (12 m M )
Cells
Extracts
15.o 187.0 20. 3
60.0 226.o 217.o
The degree of inhibition of glycolysis by a given concentration of tribromethanol varies widely with different preparations of cells. The variation may, to some extent, depend on the number of viable cells in the preparations, as experiments have shown that the percentage inhibition of glycolysis depends on the cell concentration. Greater inhibitions of glycolysis are obtained with less dense cell populations. Although the anaerobic glycolysis of intact cells is inhibited by TBE, the glycolytic activity of cell-free preparations is unimpaired even in the presence of 12 mM" TBE. The results in Table IX show that high concentrations of TBE have little effect on glycolysis in cell-free preparations but cause considerable inhibition with intact cell suspensions. Biochim.
B i o p h y s . Acta, 4 6 (1961) 5 1 4 - 5 2 6
522
R . M . JOHNSTONE, J. H. QUASTEL
Effects of TBE on glycine uptake with other tissues The effects of tribromethanol on the uptake of amino acids in several other turnouts and normal tissues have been examined. The results given in Table X show that in three tissues besides Ehrlich ascites cells, a significant inhibition of uptake of glycine is observed. Anaerobic glycolysis is affected to a much smaller extent. With guinea pig-brain slices, T B E had no effect on glycolysis at concentrations less than IO mM. The anaerobic uptake of glycine in brain slices is very small, little more than would be expected on the basis of passive diffusion, and no effects of tribromethanol on this process under anaerobic conditions were observed.
Effect of tribromethanol on the rate of glycine uptake Experiments were carried out to discover whether an inhibition of the rate of uptake by T B E takes place immediately or gradually. The results in Table X I show TABLE X EFFECT
OF TBE
ON
GLUCOSE-STIMULATED
GLYCINE
UPTAKE
IN
ANIMAL
TISSUES
I n c u b a t i o n m e d i u m : Ringer-o.o25 M N a H C O s ; p H , 7.5; gas phase, N f C O 2 (95:5); glucose concentration, i o m M , [I -aac~ glycine concentration, 2 raM, 105 counts/min/flask; i n c u b a t i o n time, 60 rain at 37 °. Tissue
Uptake of glycine % Inhibition of counts/rain~rag dry weight glucose stimulated uptake
,4dditions
Mouse m e l a n o m a
Nil Glucose Glucose + T B E (2 m M ) Glucose + T B E (4 m M )
65.o 12o.o 58.5 65.0
--ioo ioo
Mouse l y m p h o m a
Nil Glucose Glucose + T B E (2 m M ) Glucose + T B E (4 mM)
248.0 5o5.o 290.0 256.0
--84 97
Mouse spleen
Nil Glucose Glucose + T B E (4 m M )
187.o
--
230.0
--
I47.o
too
Nil Glucose Glucose + T B E (2 m M ) Glucose + T B E (4 m M )
220.0 169o.o i i io.o 780.0
--39 62
Ehrlich ascites t u r n o u t cells
XI
TABLE EFFECT
OF
TRIBROMETHANOL
ON
ON EHRLICH
RATE
OF
ASCITES
UPTAKE
OF
[1-14C]GLYCINE
CELLS
1Ringer---o.o25 M NaHCO8; gas phase, N2-CO z (95:5); [I-14C]glycine concentration, 2 m M ; lO 5 c o u n t s / m i n / f l a s k ; glucose concentration, i o m M in all flasks; t e m p e r a t u r e , 37 °. Time (rain)
io 20 3° 4°
Glycine concentration counts/minlmg dry wt. Control
TBE (4 raM)
I97 ° 2670 316o 3300
715 lO9O 143o 161o
B i o c h i m . B i o p h y s . Acta, 46 (I961) 514--526
LIPOTROPIC AGENTS AND ASCITES CELL METABOLISM
523
t h a t in the presence of tribromethanol the rate of uptake is immediately decreased and approaches the steady state level more slowly than the control. Moreover, in presence of TBE, the steady state level is considerably below that of the control. As a decreased rate of uptake m a y be the result of (a) a diminished rate of uptake or (b) an irtcreased rate of efflux, the effect of tribromethanol on the efflux of glycine from cells packed with glycine was also examined. For these experiments, the cells were preincubated for 40 min with radioactive glycine, and then centrifuged and washed quickly at o °. The packed cells were resuspended in a small quantity of cold Ringer medium (about I ml) and added to two flasks which contained no glycine but one of which contained 4 m M tribromethanol. The results show that tribromethanol decreases the rate of glycine efflux from the cells (Table XlI). Similar results were obtained with indole. Thus it is apparent that the decreased rate of uptake observed in .the presence of tribromethanol is unlikely to be the result of a higher efflux rate, but is rather the result of an inhibition of the uptake of amino acids. TABLE XlI EFFECT OF TRIBROMETHANOL ON RATE OF EFFLUX OF GLYCINE To m e a s u r e efflux, cells were p r e i n c u b a t e d w i t h 2 m M [I-14Clglycine. T h e y were t h e n c e n t r i f u g e d a n d w a s h e d once. A f t e r r e s u s p e n d i n g t h e cells in fresh R i n g e r m e d i u m , e q u a l v o l u m e s of cells were a d d e d to t w o flasks, one of w h i c h c o n t a i n e d 4 m M t r i b r o m e t h a n o l . S a m p l e s w e re t a k e n a t g i v e n i n t e r v a l s . M e d i u m , o . o i M p h o s p h a t e buffer, p H 7.4. Gas phase, a i r ; t e m p e r a t u r e , 37 °. Time
o 2 6 lO 15 25 60
Glyci~e concentration counts/rain/rag dry wt. Control
TBE (4 raM)
835 580 460 415 34 ° 232 15o
835 700 575 54 ° 485 44 ° 256
Effects of barbiturates Both pentothal and Amytal were found to effect marked decreases in respiration of the Ehrlich ascites cell (Table I). As might be expected, in view of these large inhibitions of respiration, the uptake of glycine into the cell is diminished. The diminution of the uptake of glycine usually corresponds to the inhibition of respiration. As with TBE, the glycolytic activity of Ehrlich ascites cells m a y also be inhibited b y Amytal and pentothal. With Amytal and pentothal, as well as with TBE, the extent of inhibition of anaerobic glycolysis for a given concentration of inhibitor varies with different preparations of cells. However, it is possible to show that under anaerobic conditions the uptake of glycine is inhibited b y low concentrations (1-1.5 mM) of pentothal to a much greater extent than glycolysis in inhibited. The data in Table X l I I show the results of experiments with pentothal and Amytal. I t is quite apparent that pentothal inhibits the uptake of glycine b y these cells to a greater extent than it inhibits glycolysis, while with Amytal the inhibition of uptake is about the same as the inhibition of glycolysis. B i o c h i m . B i o p h y s . A c t a , 46 (1961) 514-526
524
R.M.
IOHNSTONE, TABLE
J. H. QUASTEL XIII
PERCENTAGE INHIBITIONS BY PENTOTHAL AND AMYTAL OF ANAEROBIC GLYCOLYSIS, UPTAKE OF [I-14C~GLYCrNE AND INCORPORATION INTO PROTEINS OF [I-14C]GLYCINE IN EHRLICH ASCITES CARCINOMA CELLS IN PRESENCE OF GLUCOSE Conditions: Incubation glucose concentration
m e d i u m c o n t a i n s o . 0 2 5 M N a H C O a ; p H , 7.5 ; g a s p h a s e , N ~ - C O 2 ( 9 5 : 5 ) ; IO m.~:r; [ I - 1 4 C ] g l y c i n e c o n c e n t r a t i o n , 2 m M ; incubation time, I h; t e m p e r a t u r e , 37 ° .
Amytal Pentothal
2 mM 3mM i mM 2m3I
Anaerobic glycolysis
uptake o] glycine
Incorporation of glycine into proteins
6 38 13 38
i8 27 5o 61
12 44 23 31
Effects of lipotropic drugs on incorporation of [IJ4CIglycine into proteins Several of the inhibitors examined also affect the incorporation of Ci-14C]glycine into the proteins. For example, the results in Table V I I I show that T B E at a concentration of 8 m M causes an 81 °/o inhibition of incorporation into proteins when the inhibition of glycolysis is much less. I t is significant that, at the lower concentrations, the inhibitions of incorporation disappear, but the inhibition of uptake is still marked. Similar results were obtained with chloretone. With the barbiturates, the inhibition of incorporation is usually proportional to the inhibition of glycolysis (Table X l I I ) . DISCUSSION
As it is already established that, in a variety of tissues 2°, 22, the transport of nutrients such as phosphate is, amino acids I and sugars 17, into cells is energy, dependent, a decrease of the energy supply, as a result of an inhibition of respiration, m a y obviously reduce such transport. In the present experiments, conditions have been chosen so that the effects of lipotropic substances on the uptake of amino acids m a y be dissociated from their effects on respiratory ATP formation. With Ehrlich ascites tumour cells, under anaerobic conditions in the presence of glucose, sufficient glycolytic energy is produced to obtain an intracelhilar level of amino acid equivalent to that under aerobic conditions s. By examining, the effects of lipotropic agents under these conditions, it was possible to assess the relative effects of these agents on the rate of glycolysis, and hence on the rate of ATP production, and on the amino acid transport system. The results obtained show that a variety of lipotropic substances can, under anaerobic conditions in presence of glucose, markedly depress the amount, as well as the rate, of glycine and alanine uptake, without affecting the ATP production. Thus, irtdole, tribromethanol, chloretone and pentothal all inhibit glycine uptake to a greater extent than they inhibit the rate of glycolysis. The inhibition by these lipotropic or neurotropic drugs of anaerobic amino acid uptake is not peculiar to the Ehrlich ascites cells. Amino acid uptake b y mouse melanoma, mouse lymphoma and mouse spleen slices is inhibited in the presence of B i o c h i m . B i o p h y s . A c t a , 4 6 (1961) 5J 4 - 5 2 6
LIPOTROPIC AGENTS AND ASC1TES CELL METABOLISM
525
2-4 m M tribromethanol. The extent of glucose-stimulated glycine uptake with the slice preparations is considerably less than that observed with the Ehrlich ascites cells. The percentage inhibition b y tribromethanol appears greater with slices than with the Ehrlich ascites cells, but this m a y be due in part to the general lower level of uptake. For example, in the experiments quoted in Table X, the glucose-stimulated glycine uptake with mouse melanoma was 55 counts/min/mg dry wt. of tissue whereas that with Ehrlich ascites cells is 147o counts/min/mg dry wt. of tissue. The results suggest that the inhibition of amino acid uptake in Ehrlich ascites cells is associated with the lipotropic properties of the inhibitor. Thus, trichlorethanol is a b o u t three times more soluble in water than is the tribromo derivative, and is about three times less inhibitory, mole for mole, than tribromethanol on the uptake of glycine. Similarly, with the indole derivatives, skatole and indole are potent inhibitors of uptake at concentrations of 2- 4 m M whereas the less lipotropic derivatives, such as serotonin and indoleacetic acid, are not inhibitory at these concentrations. In fact, CHRISTENSEN et al. TM have shown that indoleacetate stimulates uptake of amino acids. It is, therefore, reasonable to conclude that the transport mechanism of these ceils is associated with a lipid component of the membrane and that these lipotropic drugs interfere with transport by association with the lipid components. Additional evidence to suggest that the activity of these inhibitory substances is associated with their effects on the cell membrane was obtained by examining the effects of tribromethanol on glycolysis in cell free and in whole cell preparations. Whereas 4 m M tribromethanol usually inhibits anaerobic glycolysis in intact cell suspensions, concentrations up to 12 m M have no effect on glycolysis in cell free preparations. It seemed possible that these substances might inhibit the uptake of glycine by increasing cell*permeability in such a way that the rate of efflux is increased more than the rate of influx of the amino acid. However, experiments with indole and tribromethanol have shown that the rate of efflux m a y be actually decreased in presence of these substances. I t must, therefore, be concluded that, at the concentrations used and under the present experimental conditions, these substances act primarily by interfering with the influx of amino acids into the cells. The concentrations of the lipotropic agents required to inhibit the anaerobic uptake of amino acids is Usually of the same order as that required to obtain inhibitory effects on brain respiration. For example, 2-3 m M chloretone has been shown to inhibit K*-stimulated brain respiration by approximately 40-50 °Jo (see refs. 14, 15). In the present experiments with chloretone, 30-40 % inhibition of uptake of glycine under anaerobic conditions was observed with 2-4 m M chloretone. Tribromethanol inhibits unstimulated rat brain respiration by over 30 % at the anaesthetic concentration (in the rat) of I m M 19. In the present experiments, in vitro, 1- 4 m M T B E produced strong inhibitory effects on the glycine uptake in Ehrlich ascites cells under anaerobic conditions in presence of glucose. It is apparent from this, and results presented earlier, that the amino acid transport system, particularly that in Ehrlich ascites cells, and the metabolism of brain tissue are inhibited to approximately the same extents b y similar concentrations of these lipotropic agents. Moreover, the respiratory activity of the Ehrlich ascites cells is also inhibited b y the substances in question, the extent of inhibition b y indole, chloretone, triBiochim. Biophys. Acta, 46 (r96I) 514-526
526
R . M . JOHNSTONE, J. H. QUASTEL
bromethanol, Amytal and pentothal being of the same order as that observed with brain tissue. These results suggest that respiratory mechanisms and the transport systems of the whole cell are dependent for their activities on lipid components in the membrane structures, with which the lipotropic, or neurotropic, agents become associated, thereby causing a diminution of these activities. ACKNOWLEDGEMENTS
We are grateful to the National Cancer Institute of Canada for financial assistance and for a Fellowship to one of us (R.M.J.) during the period of this work. REFERENCES 1 H. N. CHRISTENSEN AND T. R. RIGGS, J. Biol. Chem., 194 (1952) 57. 2 E. HRINZ AND H. A. MARIANI, J. Biol. Chem., 228 (1957) 97. 3 R. M. JOHNSTONE AND P. G. SCHOLEFIELD, Cancer Research, 19 (1959) 114o. 4 G. WISEMAN AND F. H. GHADIALLY, Brit. J. Cancer, 9 (1955) 480. 5 L. M. BIRT AND F. J. R. HIRD, Biochem. J., 7° (1958 ) 286. 6 B. CHANCE AND B. HESS, J. Biol. Chem., 234 (1959) 34o4 . v C. t3. WENNER, J. Biol. Chem., 234 (1959) 2472. s R. M. JOHNSTONE, Can. J. Biochem. and Physiol., 37 (1959) 589 • 8 G. CIRIOTTI, J. Biol. Chem., 198 (1952) 297. 10 j . H. QOASTEL AND I. J. BicKis, Nature, 183 (1959) 281. 11 j. H. QUASTEL AND A. H. M. WHEATLEY, Proe. Roy. Soc. (London~ B., 112 (1932) 60. n a j . H. QUASTEL AND A. H. M. WHEATLEY, Biochem. J., 27 (1933) 16o9; 28 (1934) 1521. 12 M. JOWETT AND J. H. QUASTEL, Biochem. J., 31 (1937) 565; 31 (1937) I I O I . la M. MICHAELIS AND J. H. QUASTEL, Biochem. J., 35 (I941) 918. 14 j . j . GHOSH AND J. H. QUASTEL, Nature, I74 (1954) 28. 15 O. LINDAN, J. H. QUASTEL AND S. SVED, Can. J. Biochem. and Physiol., 35 (1957) 1135. ae E. J. HARRIS, Transport and A ccumulaticn in Biological Systems, B u t t e r w o r t h ' s Scientific P u b l i c a t i o n s , L o n d o n , 1956. 17 W. DARLINGTON AND J. H. QUASTEL, Arch. Biochem. Biophys., 43 (1953) 194. is H. N. CHRISTENSEN, T. R. RIGGS AND B. A. COYNE, J. Biol. Chem., 209 (1954) 413 • 18 M. JOWETT, J. Physiol. (London), 92 (1938) 322. 20 A. TENENHOUSE AND J. H. QUASTEL, Can. J. Biochem. and Physiol., 38 (196o) 1311. 21 j . H. QUASTEL, Physiol. Rev., 19 (1939) 135; Am. Rev. Biochem., 8 (1939) 435. 22 j. H. QUASTEL, Amer. J. Clin. Nutrition, 8 (196o) 137.
Biochim. Biophys. Acta, 46 (1961) 514-526
BIOCHIMICA ET BIOPHYSICA ACTA
E F F E C T S O F L I P O T R O P I C A G E N T S ON T H E
METABOLISM OF
EHRLICH ASCITES CARCINOMA CELLS AND OTHER TISSUES R. M. JOHNSTONE AND J. H. QUASTEL McGill-Montreal General Hospital Research Institute, Montreal (Canada)
(Received July I5th, I96o)
SUMMARY Lipotropic substances, such as Amytal, pentothal, tribromethanol, chloretone and irzdole which exercise large inhibitory effects on brain respiration have equally large inhibitory effects on the respiration of Ehrlich ascites carcinoma cells. Indole and skatole, as well as tribromethanol, choretone and pentothal, inhibit the uptake of glycine into Ehrlich ascites cells under anaerobic conditions in presence of glucose, the extent of inhibition being greater than the inhibition of anaerobic glycolysis. Results with tribromethanol show that other tissues (mouse melanoma, mouse lymphoma, mouse spleen) are affected in a similar manner to Ehrlich ascites carcinoma cells. Tribromethanol does not increase the rate of effiux of glycine from the cells, so that the inhibition of amino acid uptake is due to a suppressing effect on the mechanism responsible for the influx. Tribromethanol also inhibits anaerobic glycolysis of ascites cell suspensions at concentrations having little or no effect on the rates of glycolysis of extracts of these cells. The concentration of the lipotropic agents required to inhibit the anaerobic uptake of glycine in ascites cells is of the same order as that required to obtain inhibitory effects on brain respiration. The results indicate that the transport systems of the cell are dependent for their activities on lipid components in the membrane structure, with which the lipotropic, or neurotropic, agents become associated. As a result of this association, transport rates of amino acids are affected.
INTRODUCTION It is already established that the transport of amino acids in the Ehrlich ascites cells is an energy dependent process 1-3. Moreover, it is known that the amino acids themselves, and related substances, will interfere with the transport of a given amino acid 1, 3-5, 20 in a variety of tissues. In a report presented by us to the National Cancer Institute of Canada in 1958 we showed that various barbiturates and anaesthetics depress respiration, and affect other aspects of metabolism, of tumours including Ehrlich ascites carcinoma cells. Since then it has been established *,? that Amytal inhibits the respiratory activities of Ehrlich ascites carcinoma cells. Abbreviations : DNA, desoxyribonucleic acid ; TCA, trichloracetic acid ; TBE, tribrornethanol ; (Avertin) ; ATP, adenosine triphosphate ; DPN, diphosphopyridine nucleotide. Biochim. Biophys. Acta, 46 (1961) 514---526
LIPOTROP1C AGENTS AND ASCITES CELL METABOLISM
515
Data will be presented in the present paper to demonstrate that substances, which have usually been associated with inhibition of metabolism in brain tissue, inhibit the transport of amino acids in tumour cells. It appears also that the inhibitions observed are not primarily the result of an inhibition of respiratory activity in the tumour tissues but are the results of changes associated with the cell membranes. MATERIALS AND METHODS
Preparations of tissues used Ehrlicb ascites cells were grown and prepared as described previously a. The cells were diluted with I I times their packed volume of Ringer medium composed as follows: NaC1 146 mM, KC1 6 mM, MgCI~ 1. 5 mM, KH2PO 4 1.5 mM. I ml of such a suspension was used in a final volume of 3 ml. The dry weight of the cells was usually 9.0 mg. Cell-free extracts of the Ehrlich ascites cells were prepared as described elsewhere s.
Chick embryo Five-day-old chick embryos were used. The whole embryo was excised, the membranes removed, weighed quickly, and added to flasks kept at o °. Tissue slices of other tissues were prepared using a Stadie-Riggs slicer. In most cases, slices of less than IOO mg and more than 50 mg were used.
Method of incubation In most experiments, the standard Warburg manometric apparatus was used for incubation. The incubation was carried out at 37 °. In some cases, 5o-ml Erlenmeyer flasks were used for incubation. For anaerobic incubations, the medium was composed of an isotonic Ringer of the same composition as given above together with 25 m M sodium bicarbonate. Glucose, when added, was present at a concentration of io mM. The gas phase was N,~-C02 (95:5). Glycolysis was measured by the carbon dioxide evolution from a bicarbonate buffer. For aerobic incubations, IO m M phosphate buffer at pH 7.4 was added to the Ringer medium, and the gas phase was pure oxygen unless otherwise specified in the text. o.15 ml of 20 °/o potash was present on a roll of filter paper in the centre well of the manometric vessel. As the filter paper had to be removed after incubation, and before the tissue was removed from the flasks, care was taken to avoid any potash dripping into the flasks. For this reason, a double thickness of filter paper was used, the potash added being sufficient to moisten the paper without danger of subsequent drip on removal of the filter paper.
Indole assays A method based on that by CIRIOTTI9 for DNA was adopted. The cells were washed with 7 ml ice-cold Ringer solution and the precipitated packed cell residue was extracted with 1.5 ml of 6 °/o TCA. After 30 min at room temperature, the precipitate was centrifuged and an aliquot of the clear supernatant, usually I ml, was used for the indole assay. 200 mg of DNA (from fish roe) were used for each milliliter of Biochim. Biophys. Acta, 46 (I96I) 514-526
516
R.M. JOHNSTONE, I. H. QUASTEL
TCA extract. The rest of the procedure was identical with that used by CIRIOTTI foI DNA 9. Indole from 5 to 50 Y was measured by this procedure in a final volume of 4 ml. The optical density was measured at 490 m/~ in a Beckman Model B Spectrophotometer. Recoveries were carried out with all experiments and were always quite satisfactory. A standard curve for indole is presented in Fig. I. 0.60 0.50
"~ 0.40 "6 .~. 0.30 0 0.20 0.10
o
18
2~
38
20
/.~g indole
5'0
Fig. 1. Variation of optical density with indole concentration. Ordinate, O.D. ; abscissa, #g indole.
Amino acid uptake and e~ux determinations Radioactive amino acids were obtained from Merck & Co., Montreal, Quebec. [IJ4C]glycine and DL-[IJ4C]alanine were used in this work. The amino acids were introduced into the main compartment of the Warburg flasks from the side arm after gassing and thermal equilibration. After a given incubation period, the flask contents were poured into centrifuge tubes containing 7 ml of ice-cold isotonic Ringer medium. The tubes were centrifuged, the supernatants poured off and recentrifuged without further addition of Ringer solution. The small residue of Ringer medium was removed carefully with a syringe and needle, and 3 ml of 95 % ethanol were added to the cells and quickly stirred. After 30 min, the tubes were centrifuged and an aliquot of the clear ethanol extract was plated on aluminium discs, dried and the radioactivity measured. The radioactivity was assayed with the aid of a Tracerlab scaler and of a Geiger Miiller mica end window probe. The uptake of amino acids was expressed as counts/min/mg dry wt. tissue. The efflux of radioactive amino acids was estimated similarly i.e. b y measurement of the radioactivity remaining in the cells. The procedure for extracting soluble amino acids from slices differed from that used for the Ehrlich ascites cells only in that the slices were homogenized in a Teflon homogenizer after addition of alcohol to the slice. Incorporation of radioactive amino acids into proteins was carried out according t o QUASTEL AND BICKIS]°.
Reagents used Indole was obtained from Nutritional Biochemical Corporation, trichlorethar~ol was obtained from the Mann Chemical Co., New York and tribromethanol from the Winthrop Laboratories, New York. 4-Bromindole, 6-bromindole and 2,3, dimethyl indole were kindly provided by Dr. R. K. BROWN, University of Alberta, Edmonton, Canada, Biochim. Biophys. Acta, 46 U96I) 514-526
LIPOTROPIC
AGENTS
AND
ASCITES
CELL
METABOLISM
517
Experimental The results presented in Table I show that the respiratory activities of Ehrlich ascites carcinoma cells are inhibited by a number of substances which are known especially to inhibit the respiration of brain tissue n-15,19, 21. Amytal, pentothal, tribromethanol and chloretone are all effective inhibitors of respiration in these cells at concentrations which have marked effects on brain tissue metabolism na, 12,14,19. Indole is also an effective inhibitor of respiration in Ehrlich ascites cells. The results in Table II show the sensitivity of these cells to indole. Substances related to indole, such as serotonin and indoleacetate, have little or no effect at comparable concentrations. To compare the sensitivity to indole of the Ehrlich ascites cells with normal cells, experiments were carried out with a variety of tissues. The typical results presented in Table III show that, of the tissues examined, only brain tissue is inhibited to the same extent as the Ehrlich ascites cells by indole. The sensitivity of brain respiration to indole and skatole is already known na. TABLE
I
E F F E C T S OF BARBITURATES AND TRIBROM- AND T R I C H L O R - E T H A N O L ON THE R E S P I R A T I O N OF E H R L I C H ASCITES TUMOUR CELLS
C o n d i t i o n s : R i n g e r - - p h o s p h a t e m e d i u m at p H 7 . 4 ; gas phase, o x y g e n ; i n c u b a t i o n time, 6 o m i n at 37 °. Additions
Ccncentration
Nil Amytal Amytal
Amytal Penthothal Tribromethanol Trichlorethanol
mM
002
-o.5 I 2 i 2 4
lO. 4 3-9 i .9 o.6 4.7 2.2 3-5
TABLE
II
E F F E C T S OF I N D O L E AND D E R I V A T I V E S ON T H E R E S P I R A T I O N OF E t J R L I C H ASCITES CELLS
I n c u b a t e d in R i n g e r - - p h o s p h a t e m e d i u m at p H 7.4. Gas phase, O ~ ; i n c u b a t i o n time, I h a t 3 7 °. Expt.
A dditions
O0~
Nil
13.2 lO.8 8.8
0. 5 m M i n d o l e I m M indole 2 m M indole 4 m3//indole
4.9 1.2
Nil 2 2 4 2 4 3 4
mM mM mM mM mM mM m2~/~
lO. 4
indole serotonin* serotonin indole acetate indole acetate 3-hydroxy tyramine tryptophane
5.4 lO.5 9.5 11.8 9.6 lO. 7 9.3
* S e r o t o n i n w a s used as t h e creatinine s u l p h a t e salt.
Eicct, im. Bicphys. Acta, 4 6 ( 1 9 6 1 ) 5 1 4 - 5 2 6
518
R . M . JOHNSTONE, J. H. QUASTEL TABLE III EFFECTS
OF
INDOLE
I n c u b a t e d in R i n g e r - p h o s p h a t e
ON
THE
RESPIRATION
OF A N I M A L T I S S U E S
m e d i u m at p H 7.4 in o x y g e n ; temperature, 37 ° . Incubation time, I h.
Tissue
W h o l e chick e m b r y o
Additions
Nil 2 m M indole 4 m M indole
Mouse spleen slices
Nil
SaT m o u s e sarcoma slices
Glucose Glucose Glucose Glucose
(IO m3~r) + i m M indole + 2 m M indole + 4 m M indole
Nil I m M indole 2 m M indole 4 m M indole
R a t k i d n e y slices
6.6 5.3 2.0 7.2
i m M indole 2 m M indole 4 m M indole
R a t brain cortex slices
Q02
Nil P y r u v a t e (IO m M ) Indole 4 m M Indole 4 mM+ Pyruvate
7.5 5.I 3.O
I6.6 12.7 8.2 0.9 3.2 3.I 2.S 1.8 7,o I9.5 4.0 16.o
Effects of indole on the uptake of amino acids into Ehrlich ascites cells Aerobically, the presence of indole or of any other substance which depletes energy by inhibition of respiration, or by uncoupling, causes a marked inhibition of transport of amino acids. As it is known 8 that the uptake of amino acids is the same aerobically and anaerobically so long as glucose is present, most oI our investigations have been carried out under anaerobic conditions. The latter condition was preferred in order to ascertain whether the observed effects could be due to a direct effect on the transport mechanisms rather than to a depletion of the energy supply by inhibition of respiration, or by an uncoupling of oxidation from phosphorylation. The rate of glycolysis was estimated in most experiments to determine whether any of the substances examined had inhibitory effects on this process. The results of typical experiments given in Table IV show that indole has little effect on anaerobic glycolysis but inhibits considerably the effect of glucose on the uptake of alanine and glycine. The glucose-stimulated uptake of alanine is usually inhibited by 50 % with 2 m M indole. A similar inhibition of glycine uptake occurs in the presence of 2 m M indole. It was found that the uptake of amino acid is not decreased by indole below the level obtained in the absence of glucose. Therefore, the figure for the endogenous uptake was substracted in order to calculate the degree of inhibition. This procedure was considered to be justified as experiments have shown that the addition of indole at concentrations of 4 m M or less has little effect on the uptake of the amino acids anaerobically in the absence of glucose. Biochim. Biophys. Acla, 46 (I96I) 514-526
L I P O T R O P I C A G E N T S AND ASCITES CELL METABOLISM TABLE
519
IV
INFLUENCE OF INDOLE ON AMINO ACID UPTAKE IN EHRLICH ASCITES CELLS UNDER ANAEROBIC CONDITIONS E x p t . I , 3"3 m M L - a l a n i n e , 83" lO 8 c o u n t s / m i n p e r flask. E x p t . 2 EI-t4C]glycine 2 m M , 90" lO 3 c o u n t s ] r a i n / f l a s k . I n b o t h e x p e r i m e n t s : I n c u b a t e d i n N z - C O z (95:5) a t 37 °. I n c u b a t i o n t i m e , i h ; glucose concentration, io raM.
QN~ COa
Uptake counts~rain/rag dry wt. 6f tissue
alanine alanine + I mM indole alanine + 2 mM indole alanine + 4 mM indole
3. z 43 .0 43.0 40.o 38.0
360 775 635 550 37 °
Glycine Glycine + glucose Glycine + glucose + i mM indole Glycine + glucose + 2 mM indole
-51.o 51.o 51.o
300 144o 111o 935
Expt.
Additions
Alanine Glucose Glucose Glucose Glucose
+ + + +
TABLE
V
EFFECTS OF VARIATION OF AMINO ACID CONCENTRATION ON GLYCINE UPTAKE BY EHRLICH ASCITES CARCINOMA CELLS UNDER ANAEROBIC CONDITIONS G a s p h a s e , N 2 - C O 2 ( 9 5 : 5 ) . I n c u b a t i o n , 4 ° m i n a t 37 °. G l y c i n e r a d i o a c t i v i t y , 2200 c o u n t s / m i n / / t m o l e . Uptake counts~rain~rag dry wt. cells Glucose absent
Glycine c6ncn. (ram)
Glucose present (~ro raM)
Indole absent
I ndole (z raM) present
Indole absent
lndole (2 raM) present
Percentage inhibitionof glucose stimulated uptake
7.8 40.5 67.5
7.2 45.0 83. 5
50.5 183-o 240.0
32.7 126.5 18o.o
40 42 44
0.5 2.5 5.0
TABLE
VI
UPTAKE OF INDOLE BY EHRLICH ASEITES CELLS ANAEROBICALLY G a s p h a s e , N ~ - C O 2 ( 9 5 : 5 ) ; g l u c o s e c o n c e n t r a t i o n , IO m M ; i n c u b a t i o n t i m e I h 37 °. V a l u e s g i v e n a r e t h e a v e r a g e of t h r e e s e p a r a t e d e t e r m i n a t i o n s . Intracellular indole concentration E xtracellular indole concentration (raM)
I 2 4 8
Glucose absent
Glucose ( x o rnM) present
~ o. 5 < o. 5 1.8 7.o
mM mM mM mM
mM mM mM mM
B i o c h i m . B i o p h y s . A c t a , 46 (1961) 5 1 4 - 5 2 6
520
R. M. J O H N S T O N E ,
J . H. Q U A S T E L
Experiments were carried out with different glycine concentrations, at a constant indole concentration, to determine whether indole inhibits the glucose-stimulated uptake by competing with the amino acid for uptake. The results of typical experiments shown in Table V indicate that the extent of inhibition of glycine uptake by 2 m M indole is independent of the glycine concentration up to 5 mM. The uptake of indole itself was also examined to determine whether the lack of effect in the absence of glucose could be correlated with a decreased uptake of indole itself. The data in Table VI show that there is no concentrative uptake of indole in the cell either in the presence or the absence of glucose. The results also show that the indole concentration in the cell, even in the presence of glucose, is less than that of the medium. It was, therefore, concluded that indole inhibits the process of uptake by a mechanism other than competition between amino acid and indole, and that the inhibition does not involve the accumulation of indole in the cell.
Effects of some lipotropic substances on anaerobic uptake of glycine and anaerobic glycolysis To determine whether indole behaves in a unique manner on the uptake of glycine by the Ehrlich ascites cells, or whether other substances known to inhibit respiratory activity in brain behave in a similar way, the effects of lipotropic substances such as TBE, trichlorethanol, chloretone, pentanol, octanol and skatole were examined. The results of typical experiments given in Table VII show that the higher TABLE VII EFFECTS OF TRIBROMETHANOL, CHLORETONE, SKATOLE, PENTANOL AND OCTANOL ON ANAEROBIC GLYCOL¥SIS AND GLYCINE UPTAKE IN EHRLICH ASCITES CELLS [I-14C]glycine concentration 2 mM= lO s c o u n t s / r a i n p e r f l a s k . G l u c o s e c o n c e n t r a t i o n i o r a M . All reagents except octanol and pentanol are 4 mM. Octanol, o.5 ml of water saturated solution. P e n t a n o l , i o r a M . G a s p h a s e , N . 2 - C O t ( 9 5 : 5 ) . I n c u b a t i o n t i m e , i h a t 37 °. Additions
Id COz/h
Nil Glucose Tribromethanol + glucose Skatole + glucose Chloretone + glucose Pentanol + glucose Octanol + glucose
4o.o 465.o 35o.o 645.0 470.0 485.0 5o2.o
% inhibition
--24 + 4° Nil Nil Nil
Uptake dry wt. counts[rain[rag
% inhibition
lO9.O I I IO.O 3oo.o 420.0 730.0 965.0 114o.o
--8o 69 38 14 Nil
alcohols have no effect on uptake of glycine at the concentrations examined. (Ethanol up to concentrations of 0.5 M had no effect on the uptake of glycine under anaerobic conditions.) Stimulations of glycolysis of approximately 40 % were usually observed with skatole at 2-4 mM. Recently we have observed that other substituted indoles such as 4- or 6-bromindole and 2,3 dimethyl indole also stimulate the anaerobic glycolysis. However, it is apparent from the results presented in Table VII, that inhibitions of glycine uptake with skatole are observed in spite of the increased glycolysis. Tribromethanol and chloretone also had a pronounced effect on the uptake of glycine. Trichlorethanol was found to be much less effective than tribromethanol at the equivalent concentrations. Thus at 4 mM, trichorethanol inhibited the glucoseB i o c h i m . B i o p h y s . Acta, 4 6 (I961) 5 1 4 - 5 2 6
LIPOTROPIC
521
AGENTS AND ASCITES CELL METABOLISM
stimulated uptake of glycine under anaerobic conditions by 15-2o % and tribromethanolinhibited it by about 7 ° %. Chloretone and tribromethanol, at concentrations above 4 mM, usually inhibit anaerobic glycolysis as well as uptake of glycine. With increasing inhibitions of glycolysis, the uptake of amino acids is correspondingly depressed. However, the extent of inhibition of glycolysis is usually considerably less than that of the inhibition of uptake. Moreover, at concentrations of TBE, which have no effect on glycolysis, significant inhibitions of uptake are still observed (Table VIII). TABLE
VIII
EFFECTS OF TRIBROMETHANOL AND CHLORETONE ON ANAEROBIC GLYCOLYSIS, UPTAKE AND INCORPORATION OF [I-14CJGLYCINE IN EHRLICH ASCITES CELLS
Conditions: R i n g e r - - o . o 2 5 io
mM.
M N a H C O 3 a t p H 7.5. Gas phase, N 2 - C O 2 (95 : 5), glucose concentration, [ I - 1 4 C ] g l y c i n e , 2 m M lO 5 counts/min per flask. Incubation t i m e , 6 o min (at 3 7 ° ) . r,7
Additions
Nil Glucose Glucose Glucose Glucose Glucose Glucose Glucose
Q~
+ TBE + TBE + TBE
(i m M ) (2 m M ) (8 r a M ) + chloretone (i m M ) + chloretone (2 m M ) + chloretone (4 m M )
*"
3.8 48.o 45.8 44.5 4°.0 44.5 44.0 41.o
uptake counts[rain/rag dry wt. 222 1700 1275 i I Io 535 141o 124o 985
TABLE
% inhibition
Incorporation into proteins counts/rain/rag protein
--29 4° 79 19 31 48
0.6 8 i. 5 77.0 68.5 15.6 82.0 81.o 37.0
% inhibition
--5 16 8i Nil
Nil 54
IX
EFFECTS OF TRIBROMETHANOL ON ANAEROBIC GLYCOLYSIS OF EXTRACTS AND INTACT CELL PREPARATIONS OF EHRLICH ASCITES CELLS
Incubation medium: Buffer, o . o 2 8 M N a H C O 3 ; gas phase, N z - C O ~ ( 9 5 : 5 ) - Cell e x t r a c t s were fortified with o. 3 m M A T P , o . i m M of D P N a n d 4 ° m M nicotinamide. Glucose concentration, IO m M . I n c u b a t i o n t i m e , 6 o min at 37 °. pl COs/h Additions
Nil
Glucose Glucose + T B E (12 m M )
Cells
Extracts
15.o 187.0 20. 3
60.0 226.o 217.o
The degree of inhibition of glycolysis by a given concentration of tribromethanol varies widely with different preparations of cells. The variation may, to some extent, depend on the number of viable cells in the preparations, as experiments have shown that the percentage inhibition of glycolysis depends on the cell concentration. Greater inhibitions of glycolysis are obtained with less dense cell populations. Although the anaerobic glycolysis of intact cells is inhibited by TBE, the glycolytic activity of cell-free preparations is unimpaired even in the presence of 12 mM" TBE. The results in Table IX show that high concentrations of TBE have little effect on glycolysis in cell-free preparations but cause considerable inhibition with intact cell suspensions. Biochim.
B i o p h y s . Acta, 4 6 (1961) 5 1 4 - 5 2 6
522
R . M . JOHNSTONE, J. H. QUASTEL
Effects of TBE on glycine uptake with other tissues The effects of tribromethanol on the uptake of amino acids in several other turnouts and normal tissues have been examined. The results given in Table X show that in three tissues besides Ehrlich ascites cells, a significant inhibition of uptake of glycine is observed. Anaerobic glycolysis is affected to a much smaller extent. With guinea pig-brain slices, T B E had no effect on glycolysis at concentrations less than IO mM. The anaerobic uptake of glycine in brain slices is very small, little more than would be expected on the basis of passive diffusion, and no effects of tribromethanol on this process under anaerobic conditions were observed.
Effect of tribromethanol on the rate of glycine uptake Experiments were carried out to discover whether an inhibition of the rate of uptake by T B E takes place immediately or gradually. The results in Table X I show TABLE X EFFECT
OF TBE
ON
GLUCOSE-STIMULATED
GLYCINE
UPTAKE
IN
ANIMAL
TISSUES
I n c u b a t i o n m e d i u m : Ringer-o.o25 M N a H C O s ; p H , 7.5; gas phase, N f C O 2 (95:5); glucose concentration, i o m M , [I -aac~ glycine concentration, 2 raM, 105 counts/min/flask; i n c u b a t i o n time, 60 rain at 37 °. Tissue
Uptake of glycine % Inhibition of counts/rain~rag dry weight glucose stimulated uptake
,4dditions
Mouse m e l a n o m a
Nil Glucose Glucose + T B E (2 m M ) Glucose + T B E (4 m M )
65.o 12o.o 58.5 65.0
--ioo ioo
Mouse l y m p h o m a
Nil Glucose Glucose + T B E (2 m M ) Glucose + T B E (4 mM)
248.0 5o5.o 290.0 256.0
--84 97
Mouse spleen
Nil Glucose Glucose + T B E (4 m M )
187.o
--
230.0
--
I47.o
too
Nil Glucose Glucose + T B E (2 m M ) Glucose + T B E (4 m M )
220.0 169o.o i i io.o 780.0
--39 62
Ehrlich ascites t u r n o u t cells
XI
TABLE EFFECT
OF
TRIBROMETHANOL
ON
ON EHRLICH
RATE
OF
ASCITES
UPTAKE
OF
[1-14C]GLYCINE
CELLS
1Ringer---o.o25 M NaHCO8; gas phase, N2-CO z (95:5); [I-14C]glycine concentration, 2 m M ; lO 5 c o u n t s / m i n / f l a s k ; glucose concentration, i o m M in all flasks; t e m p e r a t u r e , 37 °. Time (rain)
io 20 3° 4°
Glycine concentration counts/minlmg dry wt. Control
TBE (4 raM)
I97 ° 2670 316o 3300
715 lO9O 143o 161o
B i o c h i m . B i o p h y s . Acta, 46 (I961) 514--526
LIPOTROPIC AGENTS AND ASCITES CELL METABOLISM
523
t h a t in the presence of tribromethanol the rate of uptake is immediately decreased and approaches the steady state level more slowly than the control. Moreover, in presence of TBE, the steady state level is considerably below that of the control. As a decreased rate of uptake m a y be the result of (a) a diminished rate of uptake or (b) an irtcreased rate of efflux, the effect of tribromethanol on the efflux of glycine from cells packed with glycine was also examined. For these experiments, the cells were preincubated for 40 min with radioactive glycine, and then centrifuged and washed quickly at o °. The packed cells were resuspended in a small quantity of cold Ringer medium (about I ml) and added to two flasks which contained no glycine but one of which contained 4 m M tribromethanol. The results show that tribromethanol decreases the rate of glycine efflux from the cells (Table XlI). Similar results were obtained with indole. Thus it is apparent that the decreased rate of uptake observed in .the presence of tribromethanol is unlikely to be the result of a higher efflux rate, but is rather the result of an inhibition of the uptake of amino acids. TABLE XlI EFFECT OF TRIBROMETHANOL ON RATE OF EFFLUX OF GLYCINE To m e a s u r e efflux, cells were p r e i n c u b a t e d w i t h 2 m M [I-14Clglycine. T h e y were t h e n c e n t r i f u g e d a n d w a s h e d once. A f t e r r e s u s p e n d i n g t h e cells in fresh R i n g e r m e d i u m , e q u a l v o l u m e s of cells were a d d e d to t w o flasks, one of w h i c h c o n t a i n e d 4 m M t r i b r o m e t h a n o l . S a m p l e s w e re t a k e n a t g i v e n i n t e r v a l s . M e d i u m , o . o i M p h o s p h a t e buffer, p H 7.4. Gas phase, a i r ; t e m p e r a t u r e , 37 °. Time
o 2 6 lO 15 25 60
Glyci~e concentration counts/rain/rag dry wt. Control
TBE (4 raM)
835 580 460 415 34 ° 232 15o
835 700 575 54 ° 485 44 ° 256
Effects of barbiturates Both pentothal and Amytal were found to effect marked decreases in respiration of the Ehrlich ascites cell (Table I). As might be expected, in view of these large inhibitions of respiration, the uptake of glycine into the cell is diminished. The diminution of the uptake of glycine usually corresponds to the inhibition of respiration. As with TBE, the glycolytic activity of Ehrlich ascites cells m a y also be inhibited b y Amytal and pentothal. With Amytal and pentothal, as well as with TBE, the extent of inhibition of anaerobic glycolysis for a given concentration of inhibitor varies with different preparations of cells. However, it is possible to show that under anaerobic conditions the uptake of glycine is inhibited b y low concentrations (1-1.5 mM) of pentothal to a much greater extent than glycolysis in inhibited. The data in Table X l I I show the results of experiments with pentothal and Amytal. I t is quite apparent that pentothal inhibits the uptake of glycine b y these cells to a greater extent than it inhibits glycolysis, while with Amytal the inhibition of uptake is about the same as the inhibition of glycolysis. B i o c h i m . B i o p h y s . A c t a , 46 (1961) 514-526
524
R.M.
IOHNSTONE, TABLE
J. H. QUASTEL XIII
PERCENTAGE INHIBITIONS BY PENTOTHAL AND AMYTAL OF ANAEROBIC GLYCOLYSIS, UPTAKE OF [I-14C~GLYCrNE AND INCORPORATION INTO PROTEINS OF [I-14C]GLYCINE IN EHRLICH ASCITES CARCINOMA CELLS IN PRESENCE OF GLUCOSE Conditions: Incubation glucose concentration
m e d i u m c o n t a i n s o . 0 2 5 M N a H C O a ; p H , 7.5 ; g a s p h a s e , N ~ - C O 2 ( 9 5 : 5 ) ; IO m.~:r; [ I - 1 4 C ] g l y c i n e c o n c e n t r a t i o n , 2 m M ; incubation time, I h; t e m p e r a t u r e , 37 ° .
Amytal Pentothal
2 mM 3mM i mM 2m3I
Anaerobic glycolysis
uptake o] glycine
Incorporation of glycine into proteins
6 38 13 38
i8 27 5o 61
12 44 23 31
Effects of lipotropic drugs on incorporation of [IJ4CIglycine into proteins Several of the inhibitors examined also affect the incorporation of Ci-14C]glycine into the proteins. For example, the results in Table V I I I show that T B E at a concentration of 8 m M causes an 81 °/o inhibition of incorporation into proteins when the inhibition of glycolysis is much less. I t is significant that, at the lower concentrations, the inhibitions of incorporation disappear, but the inhibition of uptake is still marked. Similar results were obtained with chloretone. With the barbiturates, the inhibition of incorporation is usually proportional to the inhibition of glycolysis (Table X l I I ) . DISCUSSION
As it is already established that, in a variety of tissues 2°, 22, the transport of nutrients such as phosphate is, amino acids I and sugars 17, into cells is energy, dependent, a decrease of the energy supply, as a result of an inhibition of respiration, m a y obviously reduce such transport. In the present experiments, conditions have been chosen so that the effects of lipotropic substances on the uptake of amino acids m a y be dissociated from their effects on respiratory ATP formation. With Ehrlich ascites tumour cells, under anaerobic conditions in the presence of glucose, sufficient glycolytic energy is produced to obtain an intracelhilar level of amino acid equivalent to that under aerobic conditions s. By examining, the effects of lipotropic agents under these conditions, it was possible to assess the relative effects of these agents on the rate of glycolysis, and hence on the rate of ATP production, and on the amino acid transport system. The results obtained show that a variety of lipotropic substances can, under anaerobic conditions in presence of glucose, markedly depress the amount, as well as the rate, of glycine and alanine uptake, without affecting the ATP production. Thus, irtdole, tribromethanol, chloretone and pentothal all inhibit glycine uptake to a greater extent than they inhibit the rate of glycolysis. The inhibition by these lipotropic or neurotropic drugs of anaerobic amino acid uptake is not peculiar to the Ehrlich ascites cells. Amino acid uptake b y mouse melanoma, mouse lymphoma and mouse spleen slices is inhibited in the presence of B i o c h i m . B i o p h y s . A c t a , 4 6 (1961) 5J 4 - 5 2 6
LIPOTROPIC AGENTS AND ASC1TES CELL METABOLISM
525
2-4 m M tribromethanol. The extent of glucose-stimulated glycine uptake with the slice preparations is considerably less than that observed with the Ehrlich ascites cells. The percentage inhibition b y tribromethanol appears greater with slices than with the Ehrlich ascites cells, but this m a y be due in part to the general lower level of uptake. For example, in the experiments quoted in Table X, the glucose-stimulated glycine uptake with mouse melanoma was 55 counts/min/mg dry wt. of tissue whereas that with Ehrlich ascites cells is 147o counts/min/mg dry wt. of tissue. The results suggest that the inhibition of amino acid uptake in Ehrlich ascites cells is associated with the lipotropic properties of the inhibitor. Thus, trichlorethanol is a b o u t three times more soluble in water than is the tribromo derivative, and is about three times less inhibitory, mole for mole, than tribromethanol on the uptake of glycine. Similarly, with the indole derivatives, skatole and indole are potent inhibitors of uptake at concentrations of 2- 4 m M whereas the less lipotropic derivatives, such as serotonin and indoleacetic acid, are not inhibitory at these concentrations. In fact, CHRISTENSEN et al. TM have shown that indoleacetate stimulates uptake of amino acids. It is, therefore, reasonable to conclude that the transport mechanism of these ceils is associated with a lipid component of the membrane and that these lipotropic drugs interfere with transport by association with the lipid components. Additional evidence to suggest that the activity of these inhibitory substances is associated with their effects on the cell membrane was obtained by examining the effects of tribromethanol on glycolysis in cell free and in whole cell preparations. Whereas 4 m M tribromethanol usually inhibits anaerobic glycolysis in intact cell suspensions, concentrations up to 12 m M have no effect on glycolysis in cell free preparations. It seemed possible that these substances might inhibit the uptake of glycine by increasing cell*permeability in such a way that the rate of efflux is increased more than the rate of influx of the amino acid. However, experiments with indole and tribromethanol have shown that the rate of efflux m a y be actually decreased in presence of these substances. I t must, therefore, be concluded that, at the concentrations used and under the present experimental conditions, these substances act primarily by interfering with the influx of amino acids into the cells. The concentrations of the lipotropic agents required to inhibit the anaerobic uptake of amino acids is Usually of the same order as that required to obtain inhibitory effects on brain respiration. For example, 2-3 m M chloretone has been shown to inhibit K*-stimulated brain respiration by approximately 40-50 °Jo (see refs. 14, 15). In the present experiments with chloretone, 30-40 % inhibition of uptake of glycine under anaerobic conditions was observed with 2-4 m M chloretone. Tribromethanol inhibits unstimulated rat brain respiration by over 30 % at the anaesthetic concentration (in the rat) of I m M 19. In the present experiments, in vitro, 1- 4 m M T B E produced strong inhibitory effects on the glycine uptake in Ehrlich ascites cells under anaerobic conditions in presence of glucose. It is apparent from this, and results presented earlier, that the amino acid transport system, particularly that in Ehrlich ascites cells, and the metabolism of brain tissue are inhibited to approximately the same extents b y similar concentrations of these lipotropic agents. Moreover, the respiratory activity of the Ehrlich ascites cells is also inhibited b y the substances in question, the extent of inhibition b y indole, chloretone, triBiochim. Biophys. Acta, 46 (r96I) 514-526
526
R . M . JOHNSTONE, J. H. QUASTEL
bromethanol, Amytal and pentothal being of the same order as that observed with brain tissue. These results suggest that respiratory mechanisms and the transport systems of the whole cell are dependent for their activities on lipid components in the membrane structures, with which the lipotropic, or neurotropic, agents become associated, thereby causing a diminution of these activities. ACKNOWLEDGEMENTS
We are grateful to the National Cancer Institute of Canada for financial assistance and for a Fellowship to one of us (R.M.J.) during the period of this work. REFERENCES 1 H. N. CHRISTENSEN AND T. R. RIGGS, J. Biol. Chem., 194 (1952) 57. 2 E. HRINZ AND H. A. MARIANI, J. Biol. Chem., 228 (1957) 97. 3 R. M. JOHNSTONE AND P. G. SCHOLEFIELD, Cancer Research, 19 (1959) 114o. 4 G. WISEMAN AND F. H. GHADIALLY, Brit. J. Cancer, 9 (1955) 480. 5 L. M. BIRT AND F. J. R. HIRD, Biochem. J., 7° (1958 ) 286. 6 B. CHANCE AND B. HESS, J. Biol. Chem., 234 (1959) 34o4 . v C. t3. WENNER, J. Biol. Chem., 234 (1959) 2472. s R. M. JOHNSTONE, Can. J. Biochem. and Physiol., 37 (1959) 589 • 8 G. CIRIOTTI, J. Biol. Chem., 198 (1952) 297. 10 j . H. QOASTEL AND I. J. BicKis, Nature, 183 (1959) 281. 11 j. H. QUASTEL AND A. H. M. WHEATLEY, Proe. Roy. Soc. (London~ B., 112 (1932) 60. n a j . H. QUASTEL AND A. H. M. WHEATLEY, Biochem. J., 27 (1933) 16o9; 28 (1934) 1521. 12 M. JOWETT AND J. H. QUASTEL, Biochem. J., 31 (1937) 565; 31 (1937) I I O I . la M. MICHAELIS AND J. H. QUASTEL, Biochem. J., 35 (I941) 918. 14 j . j . GHOSH AND J. H. QUASTEL, Nature, I74 (1954) 28. 15 O. LINDAN, J. H. QUASTEL AND S. SVED, Can. J. Biochem. and Physiol., 35 (1957) 1135. ae E. J. HARRIS, Transport and A ccumulaticn in Biological Systems, B u t t e r w o r t h ' s Scientific P u b l i c a t i o n s , L o n d o n , 1956. 17 W. DARLINGTON AND J. H. QUASTEL, Arch. Biochem. Biophys., 43 (1953) 194. is H. N. CHRISTENSEN, T. R. RIGGS AND B. A. COYNE, J. Biol. Chem., 209 (1954) 413 • 18 M. JOWETT, J. Physiol. (London), 92 (1938) 322. 20 A. TENENHOUSE AND J. H. QUASTEL, Can. J. Biochem. and Physiol., 38 (196o) 1311. 21 j . H. QUASTEL, Physiol. Rev., 19 (1939) 135; Am. Rev. Biochem., 8 (1939) 435. 22 j. H. QUASTEL, Amer. J. Clin. Nutrition, 8 (196o) 137.
Biochim. Biophys. Acta, 46 (1961) 514-526