- Email: [email protected]
Glutamine metabolism by animal cells growing in a synthetic medium
Experimenfal
Cell Reseurch
GLUTAMINE
27, 307-316
METABOLISM BY ANIMAL CELLS GROWING A SYNTHETIC MEDIUM1 P. A. KITOS,
Department
307
(196:‘)
R. SINCLAIR*
IN
and C. WAYMOUTH
of Biochemistry, Unioersity of Kansas, Lawvence, Kansas, and Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine, U.S.rl. Received
September
25, 1961
A
NUTRIENT medium for the continuous propagation of animal cells in vitro must supply the raw materials needed for maintenance and replication. Several recipes for synthetic media that satisfy the minimum requirements for survival and growth have been designed [A, 11, 13, 21,221. In almost every instance, regardless of cell line, glutamine is included as a principal constituent. \Vhether it is essential for growth remains to be established [2, 3, 211. In synthetic medium MD 705/l the growth of NCTC clone 929 (strain L) mouse cells is stimulated by a concentration of glutamine greater than that used in diet hlL 838/l [T] (Table I). The latter medium contains a supplement of peptone. The extent to which the extra glutamine is used for energy and structural purposes has not been demonstrated. However certain well demonstrated functions such as the donation of its gamma-amide nitrogen to appropriate acceptors in the biosynthesis of aminosugars, purines, and some amino acids [5,6, 8, 12, 11, 191implicate it as an important unit in biosynthetic processes. The carbon skeleton of glutamine is susceptible to metabolic dissimilation by a pathway similar to that for glutamic acid. This paper describes the fate of the carbon skeleton of glutamine during the growth of L cells in synthetic and semi-synthetic media.
MATERIALS
AND
METHODS
Subline D 705 mouse L cells were cultivated through 50 or 60 serial passagesin synthetic medium MD 705/l. This medium differs from MB 752/l reported by Waymouth [21] in that it contains the trace minerals iron, copper, manganese,zinc, molybdenum, and cobalt and that it contains only half as much magnesium sulfate. Thus
the principal
difference
between
the two media
is that
MD 705/l
has added
1 This investigation was supported in part by ACS institutional grant to the University of Kansas and by .4CS grants P-87A and IN-19 and PHS grant CRT-5013 to the Roscoe B. Jackson Memorial Laboratory. 2 Present address:
Department
of Zoology,
The
University
of Edinburgh, Experimental
Scotland. Cell Research
27
308
P.
;I.
1Cito.s.
K.
Sindtrir
cu7tl
C.
I\‘clymouth
trace elements. The inoculum for each esperimcnt was grown in a single (:arrcl LX<..‘, flask (approximate area of growth surface: 10 cm’). The cells were harvested X days after planting during which time there was a single change of nutrient. The cells were
mg/ I (JO ml common to IUD X/l and ntL 83X/l
Ingredient
600.0 15.0 30.0 8.0 224.0 500.0 1.75 2.5 1.5 15.0 -I .3 m,.a 6.5 6.0
NaCl KC1
Na,HPO, KH,PO, NaHCO, Glucose Xscorbic acid Hypoxanthine Glutathione L-Glutamic acid L-Threonine L-Arginine * HCI L-Valine L-Aspartic acid Glycine L-Proline L-Leucine L-hlethionine L-Phenylalanine L-Tyrosine L-Tryptophan L-Isoleucine L-Cystine
.i.O
-5.0 5.0 5.0 C5.0 4.0 4.0 2.5 1.5
mg/IIlo Ingredient CaC1, . 2H,O CatNO,), * 4H,O iugc1,. 6~~0 MgSO, . SH,O FeSO, CuSO, * 5H,O JInSO, . H,O ZnSO, * 7H,O (NH,),hlo,O,, . 4H,O COCI, * 6H,O Choline . HCl Thiamine * HCI Calcium pantothenate Riboflavin Pyridoxine . HCI Folic acid Biotiu m-Inositol nicotinamide vitamin B,, Peptone Glutamine Cysteine * HCl L-l.ysine * HCI L-Histidine * HCl
I\ID
705/l 12.U (J 24.0 10.0 0.026 0.025 0.008 0.015 0.012 0.011 25.0 1.0 0.1 (1.1 0.1 0.05 0.002 0.1 0.1 0.02 0 35.0 9.0 24.0 15.0
ml 111. 838/l 0 ‘0 (I
20 I) 0 0 0 0 0 0 0.5 0.05 0.05 0.05 0.0 I 0.0 I I) 0 (1 500.1 j.ll i.5 16.0 r.:,
removed from the glass surface by gentle scraping with a capillary pipette and clumps of cells were broken up by repeated squirting through the tip. The cells were suspended in 8 ml of MD 705/l and were transferred to a T 60 flask (approximate area of growth surface: 60 cm*). The vessel was stoppered and incubated at 37°C for 4 days. The cells attached normally to the glass and by the fourth day developed as an uniform layer over the bottom of the flask. By means of a capillary pipette the used culture medium was removed and discarded. To the cell layer was added 7 ml of fresh MD 705/l which contained 2.8 mg Experimentul
Cell
Reseurch
27
Glutamine
metabolism
309
of uniformly labeled %-glutamine (Schwarz Laboratories, Inc.) of specific activity 1.96 pc/mg. The resulting concentration of glutamine in the medium was S/7 that stipulated in Table I for MD 705/l. The vessel was stoppered with a tightly fitting rubber vial seal and returned to the incubator for 72 hr. At the end of the incubation period the flask was inverted without opening it and slanted to permit drainage and complete separation of the medium from the cell layer. To the medium was added by hypodermic needle 2 ml of IO per cent perchloric acid. The gaseous system was flushed for 2 hr by passing-in carbon dioxide free air and bubbling the exhaust gases through 1 N NaOH in a scrubbing tower. The carbon dioxide was trapped in the alkali as carbonate and was recovered quantitatively as the barium salt by precipitation with barium chloride. Then the reaction vessel was opened and the acidic medium removed and retained for analysis. The cell layer was washed with two 2 ml portions of fresh non-radioactive MD 705/l and retained for analysis. Samples were prepared for radioactive counting as follows: The barium carbonate precipitate was washed twice with 50 ml of water and was resuspended in 50 ml of 70 per cent ethyl alcohol. Infinitely thin barium carbonate plates were prepared directly on 1.5 inch aluminum planchets. The radioactivity was determined using a Nuclear thin-window gas-flow Geiger counter. When practicable, large enough counts were collected to assure less than 5 per cent standard error of counting. Replicate samples were counted routinely. All other liquid samples were prepared for counting by direct plating. Acidic and alkaline samples were neutralized before being applied to the planchet. Free amino acids in the medium were converted to their dinitrophenyl (DNP) derivatives and resolved by paper chromatography [IS]. -4 1.0 ml sample of the acidified medium was brought to pH 8.0 by the addition of 0.5 N NaOH. Unchanged fluorodinitrobenzene was extracted with ether and the pH of the aqueous layer brought to zero by the addition of 1 N HCl. The DNP-amino acids were extracted in five washings of ether and, after evaporating the solvent, taken up in acetone and spotted near one corner of a sheet of chromatography paper (Whatman No. 1, 184 x 22)“). The first dimension separation was made with a mixture of toluene, chloroethanol, and pyridine (10: 6: 3), both the resolving solution and the paper being equilibrated with 0.8 N ammonia. The paper was dried and 1.5 AI phosphate buffer (1.0 Af NaH,PO,, 0.5 Al Na,HPO,) was used for resolution in the second dimension. The developed chromatograms were exposed to no-screen X-ray film for 1 week or longer in order to discern the areas of paper that contained radioactivity. The radioactive spots were cut from the chromatograms and quantitatively eluted with 5 ml of 0.4 N HCl followed by 5 ml of 95 per cent ethyl alcohol. The eluates were neutralized, plated, and counted. The cell layer was fractionated by a modification of the method of Schmidt and Thannhauser [IT]. The acid-soluble fraction was extracted in three washings of 10 per cent trichloroacetic acid, followed by the removal of the lipid components in 80 per cent ethyl alcohol, 100 per cent ethyl alcohol, 25 per cent chloroform in ethyl alcohol, and dry ether, in that order. To the dry pellet of protein and nucleic acidcontaining residues was added 0.5 ml of 0.3 A’ KOH. After incubating overnight the solution was neutralized with 1 N HCI and acidified with 0.15 ml of 50 per cent trichloroacetic acid and the deoxyribonucleotides were removed by centrifugation. They were washed twice with hot 5 per cent trichloroacetic acid. The protein residue was “1 - 621801
Experimental
Cell Research
27
P. A. Kites,
510
I$. Sinclair
und C. I\‘trymorrth
estimated hg the tnethotl of Lowry [ 101. Glucose was detern~inetl b>- tttc glucose osidase-perosidase tnethod using glucostat reagent (\Vorthington Riochentiwl C:orp., Freehold, N.J.) and lactic acid by tltc method of Barker and Sunnnet-son 1I I.
EXPERIMENTAL
Combustion of’ glutaruinc by growing ce/l.sPSuhline D ‘it)5 L cells were for 3 days in synthetic medium MD iO5/1 containing uniformly labeled W-glutamine. During this interval the growing cells converted some of the radioactive amino acid to carbon dioxide, to cell constituents, and to soluble substances that were released into the medium. The carbon dioxide accounted for no more than 15 per cent of the total amount of glutamine that was administered to the fresh medium. incubated
II.
TABLE
Conversion of unifobrmly labeled WI-glutamine carbon dioxide by growing L cells.
Experitnent
Glutamine
added initially
Glutatnine
110.
pmoles
cptu % 10-j
pmoles
cprn y 10-j
1 ‘2
19.2 19.2
32.5 30.2
2.54 2.81
4.29 1.42
r
Total volume of medium: 5.0 ml. Incubation:
in MD 705/I
to
converted to carbon dioxide “b conversion 13.2 l-l.6
72 hr at 37°C.
To begin with, 19.2 ,~moles of glutamine was present in each vessel. Within 3 days much but not all of this amino acid was converted to metabolic products. The amount of glutamine that was recovered as respiratory carbon dioxide has been recorded in Table II. The fresh medium contained 7.2 pmoles of glutamic acid which may be oxidized by the growing cells along with the glutamine. Therefore the total amount of oxidation of these two amino acids is probably greater than that determined by the recovery of WO, from glutamine. Since about 2.5 +noles of glutamine was recovered as carbon dioxide and i.2 /[moles of glutamic acid was present initially the total consumption of these t\vo amino acids by combustion could have been no more than 9.7 Llmoles, and probabl! was a good deal less than this. Did this much osidation supply a major part of the energy needed for cell function? The principal source of energy for the L-cells is probably glucose, an excess of which was included in the formula (Table I). During growth the Experimental
Cell
Research
27
Glutamine
metabolism
311
culture converted a portion of the consumed glucose to lactic acid (Table III). Experience has shown that about 20 per cent of the used glucose accumulates as lactic acid. TAHLE III. Fermentation
of glucose in JlD 705/l Glucose
initial &moles) final @moles) used @moles) yield from glucose used (per cent) Total
volume
of growth
medium:
7.0 ml.
by growing Lactic
acid
-
195 146 49
19 -
Incubation:
L cells.
19 72 hr at 37%.
From the information provided in Table III the extent of consumption of glucose that occurred esclusirely by glycolysis can be calculated. By difference the amount of glucose that was metabolized by pathways not leading to the accumulation of lactic acid can he estimated. Among the latter possibilities are retention as storage polysaccharide and combustion to carbon dioxide and water by conventional osidative metabolism. These must account for up to 40 pmoles of glucose. Only a small fraction of the used glucose that did not accumulate as lactic acid could supply as much energy for cell function as the estimated maximum amount of osidized glutamine and glutamic acid. Therefore, the utilization of glutamine as fuel is probably not of primary importance to the L cells growing in the defined medium ND 705/l. Conversion of glutamine to other amino acids.--lifter the culture was exposed to WI:-glutamine and the used medium separated from the cells the amino acids of this solution were converted to their dinitrophenyl derivatives which were resolved by two-dimensional paper chromatography. The chromatograms were exposed to S-ray film to identify the regions that contained radioactivity. Even after 3 days incubation the culture still contained much of the glutamine that was added to the medium. The only labeled amino acids that appeared in the medium as a result of exposure of the growing L cells to 14C-glutamine were glutamic acid and proline, the former in relatively large amounts (Fig. 1). Aspartic acid and alanine acquired no detectible radioactivity. The radioactive chromatographic spots were eluted quantitatively from the paper and counted and the proportion of radioactivit! Experimental
Cell Research
27
I’. ;I. Kilos,
312
K. Sinclair
cm1 C. \I’ctyttzoiifl~
in each spot u-as cakiihtrt~. ;\pprosirnatc~y 40 JN!L’ cent 01’ the aclministrtytl glutamintk still remained at the cnci of the csperimrnt. ;\bout 20 per c*cnt ot the original glutamine had accuniulated as glutaniic acid and 3 per c*ctnt ah proline. -At that time the medium still containctl t\vo-thirds of the original radioactivity. The sum of the amounts of radioactivity in the glutamine, glutamic acid, and proline of the medium approximates the total recover! in the liquid. It seems reasonahlc to conclude that proclucts, other than l
origin toluene:
chloroethonol:
pyridine+lO:
6~3
Fig. l.-‘&dimensional chromatogram of the dinitrophenyl amino acids of MD 705/l after 3 days exposure to growing L cells. Initially the medium contained uniformly labeled %-glutamine. The striped spots locate radioactive areas corresponding to the indicated amino acids. Xo spots other than those shown exhibited any radioactivity detectible by intimate exposure to S-ray film for 3 weeks.
glutamic medium
acid and proline, are quantitatively
of glutamine small.
metabolism
that accumulate
in the
Incorporation of glutcimine and its products into cell materid-Some of the glutamine of RID 703/l was incorporated into the cells. Analysis of the cellular matter revealed that carbon-14 n-as present in small but significant amounts in the protein, DNA, RNA, and lipid fractions. An acid soluble extract of the whole cells contained little radioactivity compared to the other fractions that were studied. This means that a large pool of free glutatnine, glutamic acid and proline was not present within the cells at the time the experiment was terminated. The total amount of cell protein was measured only after the removal of the acid soluble, lipid, DNA, and RN.1 fractions. 769 and 904 ilg of protein \vere present in experiments 1 and 2 respectively. The estent of incorporation into the cell fractions has been recorded in Table I\‘. These data indicate that a relatively large proportion of the cellular radioactivity resides in the protein and lipid fractions. t’tilizcrtion of glutamine in synthetic and semi-synthetic mrdia.--Esperiments were carried out to cotnpare the utilization of glutamine by L cells growing in a synthetic medium to that of cells growing in a medium suppleExperimental
Cell
Reseurch
27
Glutamine
metabolism
313
The semi-synthetic medium mented with a complex biological derivative. ML 838/l (Table I) which was derived from PIIL 192/2 [20] by including double concentrations of amino acids and excluding albumin differs from MD 705/l in several respects, foremost amongst which are: 1IL 838/l (1) contains 0.3 per cent peptone; (2) has only l/7 as much glutamine (in these TABLE
I\‘.
Incorporation of I’-W-glutnmine of growing L cells. cpni/pg
Cell constituent Protein Lipid DNA RNA
Exp.
cell protein
1
Exp.
99.5 49.6 10.3 17.1
TABLE
T’. Irtilizntion
Medium
Glutamine added pmoles
Per cent 2
19.2 pmoles,
Total
of growth
1
1100 cpm/,ug.
glutamine Exp.
2
2.3 0.94 0.6-L 0.67
Total
volume
of growth
medium:
of glutamine of MD 705/l nnd ML 838/I by L cells for combustion and protein synthesis.
Carbon
2.1 19.2 volume
of total
2.-l 1.2 0.25 0.11
Glutamine
ML 838/l RID 705/l
Exp.
85.5 35.4 21.0 25.0
Radioactive glutamine administered: 5.0 ml. Incubation: 72 hr at 37°C.
into constituents
medium:
(pmoles)
converted
dioxide
to Protein
Total
Per mg cell protein
Total
Per mg cell protein
0.47 2.51
0.70 3.30
0.18 0.45
0.27 0.33
7.0 ml.
Incubation:
72 hr at 37’C.
experiments only one-eighth as much). Thus glutamine is used by the L cells in both culture conditions (Table 1’) but there is a quantitatke difference. By the end of the incubation period most of the glutamine in ML 838/l was used up. However only 2.4 pmoles was added to begin with compared to 19.2 pmoles in MD 705/l. In ML 838/l only about l/6 as much glutamine was converted to respiratory carbon dioxide per unit of cell protein as in PtlD 705/l. But although there is much less conversion of glutamine to carbon dioxide in blL 838/l the amount of incorporation into cell protein is relatively constant. Erperimentnl
Cell
Research
27
The specific activity of tlir c*ell protcin synthcsizetl iu thts ~JL’CSC’II(~(~ 01’ ;I ratlioacti\~r amino acid tlcpentls on the specific activity or that anlit :14.*itl. If 1Gglutaniine is incorporated intcj protein y1 .w the amount of inc*c~rlJor;ltion should be intlepentlent of the amount of glutamic acitl that is prescbnt. On the other hand, if glutamine is incorporated onl,~ after convc~rsion to glutamic acitl or to some other amino acitl by \vay of glutaniic acid, then the amount of incc)rporalion will be influencecl by the amount of diluting gliitanniv acid. Table \’ shows that the incorporation of glutamine into protein, per unit of cell protein, differs onlv slightlv between the t\vo cultural contli tions. On the one hand hlD 705/l contains about three times as much glutaminc as glutamic acid while on the other hand 111, XMjl contains ahout three times as much glutamic acid as glutamine. Therefore there is a 9 fold ditference betlveen the ratio of glutamine to glutamic acid in these t\vcJ cultural circumstances. This difference nould be clen greater if the amount of glutamic acid in peptone \vas considered. However, since the results indicate that theamount of incorporation of glutamine into cell protein is not allected by dilution the glutamine used for cell synthesis must be incorporated without change or at least in a manner by which glutamic acid is not an intermediate. Similar results by Sansom and Barr! I16 j hare shown that glutamine and glutamir acid are incorporated without interconversion into Plasma proteins anal casein in the lactating goat.
DISCUSSION
Pasielta et crl. [ 15 j sholvetl that accumulation or depletion of certain amino acids in synthetic medium hl 150 by L cells depends on glutamine. \Vhen it was left out of the medium the cells consumed these amino acids and accumulation \vas prevented. Thus, glutamic acid, proline, threonine, tyrosine, methionine, lysine, arginine, saline, and isoleucine disappeared from the deficient medium. Holyever, when both glutamic acid and glutamine were excluded from the culture medium glutamic acid and tyrosine accumulated in the liquitl and the seven other amino acids underlvent no major change in concentration. Eagle and his co-workers stipulate the necessity for at least 1.3 amino acids in the growth medium of all serially propagated cultures [2 j. These essentials include glutamine and all the amino acids (escept glutamic acid and proline) mentioned above that are influenced by glutamine. Thus glutamine is required by the cells to maintain adequate quantities of other essential amino acids. Glutamine may provide structural elements, such as carbon or nitrogen, for synthesis or maintenance of the essential nutrilite Erperimentnl
Cell
Rt~~nrch
27
Glutamine
315
metabolism
or it may be incorporated into an enzyme that participates in the synthesis or preservation of the nutrilite. In the experiments reported here uniformly-labeled IGglutamine of the medium n-as converted to WI-glutamic acid and proline. This indicates a direct conversion of glutamine to these two amino acids. Since this metabolic conversion takes place the amount of glutamic. acid and proline produced by the cells should depend on the amount of available glutamine. The fact that radioactivity was not detected in any other amino acids of the medium precludes the conversion of the carbon structure of glutamine to that of the seven other cited amino acids. Therefore these seven amino acids are influenced by glutamine indirectly. Since even with added glutamine they must be supplied in the medium the cell does not synthesize them fast enough to support growth. Glutamic acid cannot replace glutamine in sparing the seven amino acids [3, 151. Alaninc was not supplied by medium MD 703/l and it has to be manufactured by the cell. However during 3 days of growth none of the radioactivity of glutamine was incorporated into alanine of the medium. Aspartic acid was supplied in MD 705/l and it too did not become labeled. This is unespetted since the extensive recovery of the carbon of glutamine as carbon dioxide suggests that the tricarbosylic acid cycle disposes of much of the glutamine. hspartic acid could be formed from the osaloacetic acid [9] of the cycle but in this system it does not happen.
SUMMARY
1. During growth on synthetic medium hlD i&5/1 mouse L cells consume uniformly labeled X-glutamine. The cells convert this amino acid to carbon dioxide, cell substances and soluble estracellular products. 2. In 3 days carbon dioxide accounts for approsimately 13 per cent of the administered glutamine. This amount is not sufficient to be considered as a principal source of energy for the cells. 3. The only amino acids of the medium that are fnrmed from the carbon structure of glutamine are glutamic acid and proline. 4. The carbon of glutamine is incorporated in small but significant amounts into protein. lipid, DNA\, and RN.1 of the cell. 5. The incorporation of glutamine into protein is constant and unaffected by dilution with glutamic acid.
Experimenlul
Cell Research
27
REFERENCES 1. BAHKEH, .J. B. and Sunrn~~nst)x, \V. H.. ./. Bird. (:/wm. 138, 535 (18.11). 2. EAGLE. H., kkEEK%S, A. E. and LF.\.Y. \I., J. Exf’ff. ,Iletf. 107, 643 (195X). :<. EAGLE, H., Oyaara. V. I., LEVY. \I.. HOHT~N. (:. I.. and FLEISCHKAN. R.. ./. ljisd. (,‘hrm. 218, 607 (1956). -1. EAGI.E, H., Oya~.\,'\'. I.. I.EVU, 31. and FREEM.\X. A. E.. ./. Hiol. (Xem. 226, 191 (1957). 5. GOLDTHWAIT, D. A., J. Bid. Chem. 221. 555 (1956). 6. HARTKAK, S. C., LEYENBERO, B. and BucH,
Experimenful
Cell Research
27
Cell Reseurch
GLUTAMINE
27, 307-316
METABOLISM BY ANIMAL CELLS GROWING A SYNTHETIC MEDIUM1 P. A. KITOS,
Department
307
(196:‘)
R. SINCLAIR*
IN
and C. WAYMOUTH
of Biochemistry, Unioersity of Kansas, Lawvence, Kansas, and Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine, U.S.rl. Received
September
25, 1961
A
NUTRIENT medium for the continuous propagation of animal cells in vitro must supply the raw materials needed for maintenance and replication. Several recipes for synthetic media that satisfy the minimum requirements for survival and growth have been designed [A, 11, 13, 21,221. In almost every instance, regardless of cell line, glutamine is included as a principal constituent. \Vhether it is essential for growth remains to be established [2, 3, 211. In synthetic medium MD 705/l the growth of NCTC clone 929 (strain L) mouse cells is stimulated by a concentration of glutamine greater than that used in diet hlL 838/l [T] (Table I). The latter medium contains a supplement of peptone. The extent to which the extra glutamine is used for energy and structural purposes has not been demonstrated. However certain well demonstrated functions such as the donation of its gamma-amide nitrogen to appropriate acceptors in the biosynthesis of aminosugars, purines, and some amino acids [5,6, 8, 12, 11, 191implicate it as an important unit in biosynthetic processes. The carbon skeleton of glutamine is susceptible to metabolic dissimilation by a pathway similar to that for glutamic acid. This paper describes the fate of the carbon skeleton of glutamine during the growth of L cells in synthetic and semi-synthetic media.
MATERIALS
AND
METHODS
Subline D 705 mouse L cells were cultivated through 50 or 60 serial passagesin synthetic medium MD 705/l. This medium differs from MB 752/l reported by Waymouth [21] in that it contains the trace minerals iron, copper, manganese,zinc, molybdenum, and cobalt and that it contains only half as much magnesium sulfate. Thus
the principal
difference
between
the two media
is that
MD 705/l
has added
1 This investigation was supported in part by ACS institutional grant to the University of Kansas and by .4CS grants P-87A and IN-19 and PHS grant CRT-5013 to the Roscoe B. Jackson Memorial Laboratory. 2 Present address:
Department
of Zoology,
The
University
of Edinburgh, Experimental
Scotland. Cell Research
27
308
P.
;I.
1Cito.s.
K.
Sindtrir
cu7tl
C.
I\‘clymouth
trace elements. The inoculum for each esperimcnt was grown in a single (:arrcl LX<..‘, flask (approximate area of growth surface: 10 cm’). The cells were harvested X days after planting during which time there was a single change of nutrient. The cells were
mg/ I (JO ml common to IUD X/l and ntL 83X/l
Ingredient
600.0 15.0 30.0 8.0 224.0 500.0 1.75 2.5 1.5 15.0 -I .3 m,.a 6.5 6.0
NaCl KC1
Na,HPO, KH,PO, NaHCO, Glucose Xscorbic acid Hypoxanthine Glutathione L-Glutamic acid L-Threonine L-Arginine * HCI L-Valine L-Aspartic acid Glycine L-Proline L-Leucine L-hlethionine L-Phenylalanine L-Tyrosine L-Tryptophan L-Isoleucine L-Cystine
.i.O
-5.0 5.0 5.0 C5.0 4.0 4.0 2.5 1.5
mg/IIlo Ingredient CaC1, . 2H,O CatNO,), * 4H,O iugc1,. 6~~0 MgSO, . SH,O FeSO, CuSO, * 5H,O JInSO, . H,O ZnSO, * 7H,O (NH,),hlo,O,, . 4H,O COCI, * 6H,O Choline . HCl Thiamine * HCI Calcium pantothenate Riboflavin Pyridoxine . HCI Folic acid Biotiu m-Inositol nicotinamide vitamin B,, Peptone Glutamine Cysteine * HCl L-l.ysine * HCI L-Histidine * HCl
I\ID
705/l 12.U (J 24.0 10.0 0.026 0.025 0.008 0.015 0.012 0.011 25.0 1.0 0.1 (1.1 0.1 0.05 0.002 0.1 0.1 0.02 0 35.0 9.0 24.0 15.0
ml 111. 838/l 0 ‘0 (I
20 I) 0 0 0 0 0 0 0.5 0.05 0.05 0.05 0.0 I 0.0 I I) 0 (1 500.1 j.ll i.5 16.0 r.:,
removed from the glass surface by gentle scraping with a capillary pipette and clumps of cells were broken up by repeated squirting through the tip. The cells were suspended in 8 ml of MD 705/l and were transferred to a T 60 flask (approximate area of growth surface: 60 cm*). The vessel was stoppered and incubated at 37°C for 4 days. The cells attached normally to the glass and by the fourth day developed as an uniform layer over the bottom of the flask. By means of a capillary pipette the used culture medium was removed and discarded. To the cell layer was added 7 ml of fresh MD 705/l which contained 2.8 mg Experimentul
Cell
Reseurch
27
Glutamine
metabolism
309
of uniformly labeled %-glutamine (Schwarz Laboratories, Inc.) of specific activity 1.96 pc/mg. The resulting concentration of glutamine in the medium was S/7 that stipulated in Table I for MD 705/l. The vessel was stoppered with a tightly fitting rubber vial seal and returned to the incubator for 72 hr. At the end of the incubation period the flask was inverted without opening it and slanted to permit drainage and complete separation of the medium from the cell layer. To the medium was added by hypodermic needle 2 ml of IO per cent perchloric acid. The gaseous system was flushed for 2 hr by passing-in carbon dioxide free air and bubbling the exhaust gases through 1 N NaOH in a scrubbing tower. The carbon dioxide was trapped in the alkali as carbonate and was recovered quantitatively as the barium salt by precipitation with barium chloride. Then the reaction vessel was opened and the acidic medium removed and retained for analysis. The cell layer was washed with two 2 ml portions of fresh non-radioactive MD 705/l and retained for analysis. Samples were prepared for radioactive counting as follows: The barium carbonate precipitate was washed twice with 50 ml of water and was resuspended in 50 ml of 70 per cent ethyl alcohol. Infinitely thin barium carbonate plates were prepared directly on 1.5 inch aluminum planchets. The radioactivity was determined using a Nuclear thin-window gas-flow Geiger counter. When practicable, large enough counts were collected to assure less than 5 per cent standard error of counting. Replicate samples were counted routinely. All other liquid samples were prepared for counting by direct plating. Acidic and alkaline samples were neutralized before being applied to the planchet. Free amino acids in the medium were converted to their dinitrophenyl (DNP) derivatives and resolved by paper chromatography [IS]. -4 1.0 ml sample of the acidified medium was brought to pH 8.0 by the addition of 0.5 N NaOH. Unchanged fluorodinitrobenzene was extracted with ether and the pH of the aqueous layer brought to zero by the addition of 1 N HCl. The DNP-amino acids were extracted in five washings of ether and, after evaporating the solvent, taken up in acetone and spotted near one corner of a sheet of chromatography paper (Whatman No. 1, 184 x 22)“). The first dimension separation was made with a mixture of toluene, chloroethanol, and pyridine (10: 6: 3), both the resolving solution and the paper being equilibrated with 0.8 N ammonia. The paper was dried and 1.5 AI phosphate buffer (1.0 Af NaH,PO,, 0.5 Al Na,HPO,) was used for resolution in the second dimension. The developed chromatograms were exposed to no-screen X-ray film for 1 week or longer in order to discern the areas of paper that contained radioactivity. The radioactive spots were cut from the chromatograms and quantitatively eluted with 5 ml of 0.4 N HCl followed by 5 ml of 95 per cent ethyl alcohol. The eluates were neutralized, plated, and counted. The cell layer was fractionated by a modification of the method of Schmidt and Thannhauser [IT]. The acid-soluble fraction was extracted in three washings of 10 per cent trichloroacetic acid, followed by the removal of the lipid components in 80 per cent ethyl alcohol, 100 per cent ethyl alcohol, 25 per cent chloroform in ethyl alcohol, and dry ether, in that order. To the dry pellet of protein and nucleic acidcontaining residues was added 0.5 ml of 0.3 A’ KOH. After incubating overnight the solution was neutralized with 1 N HCI and acidified with 0.15 ml of 50 per cent trichloroacetic acid and the deoxyribonucleotides were removed by centrifugation. They were washed twice with hot 5 per cent trichloroacetic acid. The protein residue was “1 - 621801
Experimental
Cell Research
27
P. A. Kites,
510
I$. Sinclair
und C. I\‘trymorrth
estimated hg the tnethotl of Lowry [ 101. Glucose was detern~inetl b>- tttc glucose osidase-perosidase tnethod using glucostat reagent (\Vorthington Riochentiwl C:orp., Freehold, N.J.) and lactic acid by tltc method of Barker and Sunnnet-son 1I I.
EXPERIMENTAL
Combustion of’ glutaruinc by growing ce/l.sPSuhline D ‘it)5 L cells were for 3 days in synthetic medium MD iO5/1 containing uniformly labeled W-glutamine. During this interval the growing cells converted some of the radioactive amino acid to carbon dioxide, to cell constituents, and to soluble substances that were released into the medium. The carbon dioxide accounted for no more than 15 per cent of the total amount of glutamine that was administered to the fresh medium. incubated
II.
TABLE
Conversion of unifobrmly labeled WI-glutamine carbon dioxide by growing L cells.
Experitnent
Glutamine
added initially
Glutatnine
110.
pmoles
cptu % 10-j
pmoles
cprn y 10-j
1 ‘2
19.2 19.2
32.5 30.2
2.54 2.81
4.29 1.42
r
Total volume of medium: 5.0 ml. Incubation:
in MD 705/I
to
converted to carbon dioxide “b conversion 13.2 l-l.6
72 hr at 37°C.
To begin with, 19.2 ,~moles of glutamine was present in each vessel. Within 3 days much but not all of this amino acid was converted to metabolic products. The amount of glutamine that was recovered as respiratory carbon dioxide has been recorded in Table II. The fresh medium contained 7.2 pmoles of glutamic acid which may be oxidized by the growing cells along with the glutamine. Therefore the total amount of oxidation of these two amino acids is probably greater than that determined by the recovery of WO, from glutamine. Since about 2.5 +noles of glutamine was recovered as carbon dioxide and i.2 /[moles of glutamic acid was present initially the total consumption of these t\vo amino acids by combustion could have been no more than 9.7 Llmoles, and probabl! was a good deal less than this. Did this much osidation supply a major part of the energy needed for cell function? The principal source of energy for the L-cells is probably glucose, an excess of which was included in the formula (Table I). During growth the Experimental
Cell
Research
27
Glutamine
metabolism
311
culture converted a portion of the consumed glucose to lactic acid (Table III). Experience has shown that about 20 per cent of the used glucose accumulates as lactic acid. TAHLE III. Fermentation
of glucose in JlD 705/l Glucose
initial &moles) final @moles) used @moles) yield from glucose used (per cent) Total
volume
of growth
medium:
7.0 ml.
by growing Lactic
acid
-
195 146 49
19 -
Incubation:
L cells.
19 72 hr at 37%.
From the information provided in Table III the extent of consumption of glucose that occurred esclusirely by glycolysis can be calculated. By difference the amount of glucose that was metabolized by pathways not leading to the accumulation of lactic acid can he estimated. Among the latter possibilities are retention as storage polysaccharide and combustion to carbon dioxide and water by conventional osidative metabolism. These must account for up to 40 pmoles of glucose. Only a small fraction of the used glucose that did not accumulate as lactic acid could supply as much energy for cell function as the estimated maximum amount of osidized glutamine and glutamic acid. Therefore, the utilization of glutamine as fuel is probably not of primary importance to the L cells growing in the defined medium ND 705/l. Conversion of glutamine to other amino acids.--lifter the culture was exposed to WI:-glutamine and the used medium separated from the cells the amino acids of this solution were converted to their dinitrophenyl derivatives which were resolved by two-dimensional paper chromatography. The chromatograms were exposed to S-ray film to identify the regions that contained radioactivity. Even after 3 days incubation the culture still contained much of the glutamine that was added to the medium. The only labeled amino acids that appeared in the medium as a result of exposure of the growing L cells to 14C-glutamine were glutamic acid and proline, the former in relatively large amounts (Fig. 1). Aspartic acid and alanine acquired no detectible radioactivity. The radioactive chromatographic spots were eluted quantitatively from the paper and counted and the proportion of radioactivit! Experimental
Cell Research
27
I’. ;I. Kilos,
312
K. Sinclair
cm1 C. \I’ctyttzoiifl~
in each spot u-as cakiihtrt~. ;\pprosirnatc~y 40 JN!L’ cent 01’ the aclministrtytl glutamintk still remained at the cnci of the csperimrnt. ;\bout 20 per c*cnt ot the original glutamine had accuniulated as glutaniic acid and 3 per c*ctnt ah proline. -At that time the medium still containctl t\vo-thirds of the original radioactivity. The sum of the amounts of radioactivity in the glutamine, glutamic acid, and proline of the medium approximates the total recover! in the liquid. It seems reasonahlc to conclude that proclucts, other than l
origin toluene:
chloroethonol:
pyridine+lO:
6~3
Fig. l.-‘&dimensional chromatogram of the dinitrophenyl amino acids of MD 705/l after 3 days exposure to growing L cells. Initially the medium contained uniformly labeled %-glutamine. The striped spots locate radioactive areas corresponding to the indicated amino acids. Xo spots other than those shown exhibited any radioactivity detectible by intimate exposure to S-ray film for 3 weeks.
glutamic medium
acid and proline, are quantitatively
of glutamine small.
metabolism
that accumulate
in the
Incorporation of glutcimine and its products into cell materid-Some of the glutamine of RID 703/l was incorporated into the cells. Analysis of the cellular matter revealed that carbon-14 n-as present in small but significant amounts in the protein, DNA, RNA, and lipid fractions. An acid soluble extract of the whole cells contained little radioactivity compared to the other fractions that were studied. This means that a large pool of free glutatnine, glutamic acid and proline was not present within the cells at the time the experiment was terminated. The total amount of cell protein was measured only after the removal of the acid soluble, lipid, DNA, and RN.1 fractions. 769 and 904 ilg of protein \vere present in experiments 1 and 2 respectively. The estent of incorporation into the cell fractions has been recorded in Table I\‘. These data indicate that a relatively large proportion of the cellular radioactivity resides in the protein and lipid fractions. t’tilizcrtion of glutamine in synthetic and semi-synthetic mrdia.--Esperiments were carried out to cotnpare the utilization of glutamine by L cells growing in a synthetic medium to that of cells growing in a medium suppleExperimental
Cell
Reseurch
27
Glutamine
metabolism
313
The semi-synthetic medium mented with a complex biological derivative. ML 838/l (Table I) which was derived from PIIL 192/2 [20] by including double concentrations of amino acids and excluding albumin differs from MD 705/l in several respects, foremost amongst which are: 1IL 838/l (1) contains 0.3 per cent peptone; (2) has only l/7 as much glutamine (in these TABLE
I\‘.
Incorporation of I’-W-glutnmine of growing L cells. cpni/pg
Cell constituent Protein Lipid DNA RNA
Exp.
cell protein
1
Exp.
99.5 49.6 10.3 17.1
TABLE
T’. Irtilizntion
Medium
Glutamine added pmoles
Per cent 2
19.2 pmoles,
Total
of growth
1
1100 cpm/,ug.
glutamine Exp.
2
2.3 0.94 0.6-L 0.67
Total
volume
of growth
medium:
of glutamine of MD 705/l nnd ML 838/I by L cells for combustion and protein synthesis.
Carbon
2.1 19.2 volume
of total
2.-l 1.2 0.25 0.11
Glutamine
ML 838/l RID 705/l
Exp.
85.5 35.4 21.0 25.0
Radioactive glutamine administered: 5.0 ml. Incubation: 72 hr at 37°C.
into constituents
medium:
(pmoles)
converted
dioxide
to Protein
Total
Per mg cell protein
Total
Per mg cell protein
0.47 2.51
0.70 3.30
0.18 0.45
0.27 0.33
7.0 ml.
Incubation:
72 hr at 37’C.
experiments only one-eighth as much). Thus glutamine is used by the L cells in both culture conditions (Table 1’) but there is a quantitatke difference. By the end of the incubation period most of the glutamine in ML 838/l was used up. However only 2.4 pmoles was added to begin with compared to 19.2 pmoles in MD 705/l. In ML 838/l only about l/6 as much glutamine was converted to respiratory carbon dioxide per unit of cell protein as in PtlD 705/l. But although there is much less conversion of glutamine to carbon dioxide in blL 838/l the amount of incorporation into cell protein is relatively constant. Erperimentnl
Cell
Research
27
The specific activity of tlir c*ell protcin synthcsizetl iu thts ~JL’CSC’II(~(~ 01’ ;I ratlioacti\~r amino acid tlcpentls on the specific activity or that anlit :14.*itl. If 1Gglutaniine is incorporated intcj protein y1 .w the amount of inc*c~rlJor;ltion should be intlepentlent of the amount of glutamic acitl that is prescbnt. On the other hand, if glutamine is incorporated onl,~ after convc~rsion to glutamic acitl or to some other amino acitl by \vay of glutaniic acid, then the amount of incc)rporalion will be influencecl by the amount of diluting gliitanniv acid. Table \’ shows that the incorporation of glutamine into protein, per unit of cell protein, differs onlv slightlv between the t\vo cultural contli tions. On the one hand hlD 705/l contains about three times as much glutaminc as glutamic acid while on the other hand 111, XMjl contains ahout three times as much glutamic acid as glutamine. Therefore there is a 9 fold ditference betlveen the ratio of glutamine to glutamic acid in these t\vcJ cultural circumstances. This difference nould be clen greater if the amount of glutamic acid in peptone \vas considered. However, since the results indicate that theamount of incorporation of glutamine into cell protein is not allected by dilution the glutamine used for cell synthesis must be incorporated without change or at least in a manner by which glutamic acid is not an intermediate. Similar results by Sansom and Barr! I16 j hare shown that glutamine and glutamir acid are incorporated without interconversion into Plasma proteins anal casein in the lactating goat.
DISCUSSION
Pasielta et crl. [ 15 j sholvetl that accumulation or depletion of certain amino acids in synthetic medium hl 150 by L cells depends on glutamine. \Vhen it was left out of the medium the cells consumed these amino acids and accumulation \vas prevented. Thus, glutamic acid, proline, threonine, tyrosine, methionine, lysine, arginine, saline, and isoleucine disappeared from the deficient medium. Holyever, when both glutamic acid and glutamine were excluded from the culture medium glutamic acid and tyrosine accumulated in the liquitl and the seven other amino acids underlvent no major change in concentration. Eagle and his co-workers stipulate the necessity for at least 1.3 amino acids in the growth medium of all serially propagated cultures [2 j. These essentials include glutamine and all the amino acids (escept glutamic acid and proline) mentioned above that are influenced by glutamine. Thus glutamine is required by the cells to maintain adequate quantities of other essential amino acids. Glutamine may provide structural elements, such as carbon or nitrogen, for synthesis or maintenance of the essential nutrilite Erperimentnl
Cell
Rt~~nrch
27
Glutamine
315
metabolism
or it may be incorporated into an enzyme that participates in the synthesis or preservation of the nutrilite. In the experiments reported here uniformly-labeled IGglutamine of the medium n-as converted to WI-glutamic acid and proline. This indicates a direct conversion of glutamine to these two amino acids. Since this metabolic conversion takes place the amount of glutamic. acid and proline produced by the cells should depend on the amount of available glutamine. The fact that radioactivity was not detected in any other amino acids of the medium precludes the conversion of the carbon structure of glutamine to that of the seven other cited amino acids. Therefore these seven amino acids are influenced by glutamine indirectly. Since even with added glutamine they must be supplied in the medium the cell does not synthesize them fast enough to support growth. Glutamic acid cannot replace glutamine in sparing the seven amino acids [3, 151. Alaninc was not supplied by medium MD 703/l and it has to be manufactured by the cell. However during 3 days of growth none of the radioactivity of glutamine was incorporated into alanine of the medium. Aspartic acid was supplied in MD 705/l and it too did not become labeled. This is unespetted since the extensive recovery of the carbon of glutamine as carbon dioxide suggests that the tricarbosylic acid cycle disposes of much of the glutamine. hspartic acid could be formed from the osaloacetic acid [9] of the cycle but in this system it does not happen.
SUMMARY
1. During growth on synthetic medium hlD i&5/1 mouse L cells consume uniformly labeled X-glutamine. The cells convert this amino acid to carbon dioxide, cell substances and soluble estracellular products. 2. In 3 days carbon dioxide accounts for approsimately 13 per cent of the administered glutamine. This amount is not sufficient to be considered as a principal source of energy for the cells. 3. The only amino acids of the medium that are fnrmed from the carbon structure of glutamine are glutamic acid and proline. 4. The carbon of glutamine is incorporated in small but significant amounts into protein. lipid, DNA\, and RN.1 of the cell. 5. The incorporation of glutamine into protein is constant and unaffected by dilution with glutamic acid.
Experimenlul
Cell Research
27
REFERENCES 1. BAHKEH, .J. B. and Sunrn~~nst)x, \V. H.. ./. Bird. (:/wm. 138, 535 (18.11). 2. EAGLE. H., kkEEK%S, A. E. and LF.\.Y. \I., J. Exf’ff. ,Iletf. 107, 643 (195X). :<. EAGLE, H., Oyaara. V. I., LEVY. \I.. HOHT~N. (:. I.. and FLEISCHKAN. R.. ./. ljisd. (,‘hrm. 218, 607 (1956). -1. EAGI.E, H., Oya~.\,'\'. I.. I.EVU, 31. and FREEM.\X. A. E.. ./. Hiol. (Xem. 226, 191 (1957). 5. GOLDTHWAIT, D. A., J. Bid. Chem. 221. 555 (1956). 6. HARTKAK, S. C., LEYENBERO, B. and BucH,
Experimenful
Cell Research
27