AMINO ACID CONCENTRATION BY A FREE CELL NEOPLASM. STRUCTURAL INFLUENCES*

BY THOMAS R. RIGGS, BARBARA A. COYNE, AND HALVOR N. CHRISTENSEN

(From the Deparhzent of Biochemistry and Nutrition, Tufts College Medical School, Boston, Massachusetts)

(Received for publication, January 4, 1954)

The process by which cells transfer amino acids into their interiors against concentration gradients is by no means limited to the naturally occurring amino acids; in fact the activity is considerably greater for some substances not known to occur in proteins (2, 3). In the series of homolo- gous straight chain diamino acids a striking maximum in accumulation by the Ehrlich mouse ascites carcinoma cell has been observed with the 2,4- diamino acid; the 2,3 acid does not fall far behind. For ornithine and lysine the activity is very much weaker. Two explanations have been considered for the strong concentration of the 2,3- and 2,4diamino acids. (1) That the distance between the amino groups is optimal for the forma- tion of stable five- and six-membered rings with an atom of the “carrier” which presumably is involved in active amino acid transfer. (2) That the important effect of the distal amino group is to lower the pK’ of the a-amino group, to favor its combination with the carrier. If it is the un- charged a-amino group by which the amino acid combines with the carrier, the lower the pK of this group the greater would be the stability anticipated for the derivative. This should apply, for example, to pyridoxylidene de- rivatives (4).

In the present study, the importance of the distance between the (Y- amino group and another nitrogenous group has been verified by further examples. Two amino acids showing strong uptake, presumably because of the presence of a second nitrogenous group, namely cr,y-diaminobutyric acid and tryptophan, have been studied in detail and their accumulation shown to resemble in almost all respects that of glycine. Advantage has also been taken of the favorable effect of a second nitrogenous group to introduce structures which absorb light in the ultraviolet or the visible range with the hope that the concentration process might be microscopi- cally visualized.

*This investigation was supported in part by a grant (No. C-1268) from the National Cancer Institute, National Institutes of Health, United States Public Health Service, and by a grant from the Abbott Laboratories. A preliminary report of part of the work has been published (1).

395

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396 FREE CELL NEOPLASM AND AMINO ACIDS

The ring formation suggested in the active transport of diamino acids conceivably could occur by coordination with a metal. Participation of metals is also suggested by the finding that pyridoxal takes part in amino acid transfer (4), together with the indications obtained by Metzler and Snell (5) that iron and aluminum serve to stabilize the pyridoxylidene de- rivatives of the amino acids. Finally there is the association between deranged copper metabolism (6, 7) and disturbed amino acid distribution in Wilson’s disease. Accordingly evidence has been sought, particularly by the study of chelating agents, for the participation of a metallic ion in the transfer process. No indications of this nature were obtained.

Finally, the second possible explanation of the strong accumulation of certain diamino acids has been rather inadequately tested by the synthesis of p-chloro-L-alanine. The pK’ of the ammo group was lowered to 8.2 by introduction of the chlorine atom. Not better, but poorer, accumula- tion was obtained for this substance, but the result was probably associated with its degradation rather than due to the shift of the pK.

EXPERIMENTAL

Procedures for collecting, incubating, and extracting the tumor cells and for analyzing for water, glycine, arginine, sodium, potassium, and chloride have been described elsewhere (8, 9, 2). A brief acid hydrolysis was used to convert acetylglycine and glycinamide to glycine for analysis. Tryp- tophan and other indolyl compounds were determined by the method of Bates (10) applied to water extracts deproteinized by holding at 100” at pH 5 for 10 minutes (referred to here as hot water extracts). a! ,r-Diamino- butyric acid and other aliphatic diamino acids were estimated either as described before (2) or by applying the calorimetric procedure of Moore and Stein (11) to the precipitate by phosphotungstic acid, and correcting for the relatively small amount of color given by parallel control prepara- tions. p-Aminobenzoic acid, anthranilic acid, and m-aminophenylglycine were determined on hot water extracts by the Bratton and Marshall method (12). The pyridyl amino acids, the derivatives of phenylglycine, and other aromatic substances were determined by observing the light absorption of hot water extracts at favorable wave-lengths. &Chloro- alanine was determined by measuring the organic chlorine present in 0.75 N nitric acid extracts; that is, the chlorine released upon heating the ex- tract in a sealed tube with nitric acid and silver nitrate at 130” for 3 hours.

Resu1t.s

Is Transport of Diaminobutyric Acid and Tryptophan into Cells Active? -The extreme avidity of the cells for the cationic diaminobutyric acid conceivably might have an origin quite different from the accumulation of

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T. R. RIGGS, B. A. COYNE, AND H. N. CHRISTENSEN 397

the neutral amino acids. The activity, however, proved to be highly tem- perature-sensitive, as was that for tryptophan (Fig. 1). Furthermore the accumulation of these amino acids was readily inhibited by 2,4-dinitro- phenol, by cyanide, by Versene, and by the presence of other amino acids (Table I). Exposing the cells first to clupeine for 20 minutes practically abolished the uptake of diaminobutyric acid (Table I). Clupeine itself came to be associated with the cells, either upon their surface or interior, as shown by the intense color given by cellular extracts with the Sakaguchi reaction. a-Amino acid determinations (13) after acid hydrolysis of cel- lular extracts confirmed this conclusion (see Table IV) .l The cell boundary

0 L -‘%,y-DIAMINOBUTYRATE

x L - TRYPTOPHAN

TEMPERATURE, ‘G

FIG. 1. Temperature sensitivity of the concentration of or,r-diaminobutyric acid and tryptophan. The distribution ratio at 37.5” has been taken as 100 per cent, and ratios at other temperatures are given a relative basis.

as observed under phase-contrast microscopy appeared to become greatly sharpened upon the addition of clupeine. The accumulation of glycine was also strongly inhibited by low levels of clupeine (Table II).

In the rat, cy,r-diaminobutyric acid had a strong neurotoxic action. A dose of 7 mM per kilo subcutaneously resulted in preconvulsive and con- vulsive behavior and death. Injection of 2 mM per kilo for 7 days led to little change in behavior, but upon section of the central nervous system the following changes were reported by Dr. G. F. Meissner of the Depart- ment of Pathology of this School: “The ganglion cells of the cerebellum and cerebral cortex showed focal degenerative changes. These cells appeared shrunken and pyknotic. This change was particularly conspicuous in the Purkinje cells of the cerebellum.”

1 Dr. Herbert Fischer, University of Frankfurt, who was associated with this experiment, is continuing the study of the possible entrance of protamines into cells.

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398 FREE CELL NEOPLASM AND AMINO ACIDS

TABLE I Inhibition of Uptake of La,y-Diaminobutyric Acid and Tryptophan

The extracellular phase was Krebs’ Ringer-bicarbonate.

Experi-

mNeoqt

118 157 176 161 137 160 160 137 137 137 133 139 139 139

Amino acid

Diaminobutyric “ “ ‘I “

Tryptophan “ ‘I ‘I “ “ “ “ ‘I

Initial mine aci

lW.4

nargni 15 21.8 27.1 21.8 25 10 10 5

25 50 10 7.3 7.3

36.4

id Inhibitor

I

Clupeine, 0.5st 2,4-Dinitrophenol, 1 mM

“ 1 “ NaCN, 5 mM Tryptophan, 25 mM NaCN, 5 mM 2,4-Dinitrophenol, 1 mM L-Diaminobutyrate, 25 mM (5 n)S

“ 25 “ (1 “) “ 25 “ (0.5 n)

Glycine, 50 mM (5 n) “ 36.4 nm (5 n) I‘ 7.3 rnM (1 n) “ 36.4 mM (1 n)

-

:

--

-

klative istribu- tion

ratio*

3 23 17 14 64 35 37 65 93

116 19 23 55 81

* Cellular concentration versus extracellular concentration; value in absence of the inhibitor taken as 100.

t Cells pretreated 20 minutes with clupeine; solution then removed. $ n = the initial extracellular concentration of tryptophan.

TABLE II Inhibition of Glycine Accumulation by Clupeine

Extracellular fluid, Krebs’ Ringer-bicarbonate solution (KRB) containing ini- tially 15 mM per liter Of glycine. -

Experiment No Clupeine treatment

119

118

121

119

119 --

Pretreated 20 min. with clupeine, O.l%, in glycine-free KRB

Pretreated 20 min. with clupeine, 0.5yo, in glycine-free KRB

Pretreated 14 min. with clupeine, O.l%, in glycine-free KRB

Pretreated la min. with clupeine, 0.5yo, in glycine-free KRB

0.1% clupeine present during incubation with glycine

--

Relative dis- tribution ratio

for glycine’

48

22

43

51

44

* Value = 100 in the absence of the inhibitor.

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T. R. RIGGS, B. A. COYNE, AND H. N. CHRISTENSEN 399

The D isomer2 of diaminobutyric acid was more weakly accumulated than the L form; for example, in one instance after 2 hours of incubation with the D form the cellular level was only 52 mu and the extracellular 26 m&r (3).

In all of the preceding experiments the behavior of a,y-diaminobutyric acid, and that of tryptophan as well, was very like that of the commonly occurring amino acids, except for the extremely active uptake with di- aminobutyric acid.

Further Observations on Tryptophan Accumulation-In comparison with other amino acids of similar molecular size (2), tryptophan is strongly ac-

.

20 30 40 50 60

EXTRACELLULAR k TRYPTOPHAN

m M/KILO WATER

FIG. 2. Relationship between the extracellular and cellular concentrations of tryptophan at an approximately steady state. Krebs’ Ringer-bicarbonate medium in which tryptophan replaced part of the sodium chloride isosmotically. Tempera- ture 37”; time 2 to 4 hours.

cumulated, gradients of 30 to 40 mM between the cell interior and exterior being observed. Fig. 2 represents a study of the relation between the extracellular and cellular levels after 2 to 4 hours of incubation. This is very similar to the curve recorded earlier for glycine (9). We have hoped to see whether crystallization within the cell might be produced if a cell concentrated an amino acid beyond the limit of its solubility in the cell fluid. Such a result would bear upon the state of the amino acid within the cells and possibly also upon the etiology of cystinosis. The solubility of tryptophan in a salt solution simulating the cell fluid was about 65 to 75 mM at 37”. Levels distinctly higher than this were not found for the cells, nor were crystals observed under the microscope.

nn-Tryptophan was more weakly accumulated than the L form (Table

* Gift of Dr. Jesse Greenstein, National Institutes of Health.

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400 FREE CELL NEOPLASM AND AMINO ACIDS

III). The absence of the primary amino group (as exemplified by indole- propionic acid and two homologues) largely prevented entrance into the cells; the absence of the carboxyl group, as illustrated by tryptamine, caused a more moderate decrease in the accumulation. The presence of a methyl group on the indole nitrogen was also unfavorable. Similar indications of the necessity of the amino and carboxyl groups were indicated by the absent or slight concentration of glycinamide and N-acetylglycine (Table IV). A number of diamines, aminobenzoates, and pyridine carboxylates, also quinoline, failed to show distributional asymmetries approaching those of the amino acids (Table IV). From these results a combination of an

TABLE III

Uptake of Substances Related to Tryptophan

Compound Distribution ratio Relative

distribution ratio

143 L-Tryptophan 143 nL-Tryptophan

143 N-Methylindolyl-nL-alanine 143 Tryptamine HCl 148 “ “ 155 “ I‘ 143 &(Indole-3)-propionate 136 Indole-3-acetate 145 r-(Indole-3)-butyrate

144 N-Acetyl-nL-tryptophan

* Compared to L-tryptophan. t Compared to DL-tryptophan.

10.9/1.72 = 6.34 100* 9.03/1.76 = 5.13 81*

7.69/2.30 = 3.34 W 9.86/2.10 = 4.69 74* 65/30 = 2.2 109/5-i = 2.0 2.05/3.40 = 0.60 9* 3.00/10.4 = 0.29 1.87/4.79 = 0.39

1.57/6.47 = 0.24 gt

aromatic amino group and a carboxyl group in 1,2 or 1,3 relation is seen not to meet the requirements for concentrative activity; the failure here may be a consequence of the low pK of the amino group, or may be steric in origin. On the other hand the second nitrogenous group is stimulating, even if it is on, or in, an aromatic ring (see below). The necessity of a carboxyl group adjoining the first amino group for characteristic accumula- tion is also emphasized. In taurine a sulfonic acid group appears to re- place the carboxyl group satisfactorily (14) ; accumulation of p-alanine and taurine indicates that the two groups need not be on the same carbon (14). The amino acid, m-kynurenine, was concentrated about as strongly as m-tryptophan (Table IV).

Study of Other Amino Acids Having Second Nitrogenous Group Near a-Amino Group-The series p-(2-pyridyl)alanine,3 P-(3-pyridyl)alanine,s

* Gift of Dr. Carl Niemann, California Institute of Technology.

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T. R. RIGGS, B. A. COYNE, AND H. N. CHRISTENSEN 401

and p-(3-pyridyl)-cr-aminobutyric acid4 showed clearly the advantage of having 3 carbons rather than 4 carbons between the nitrogen atoms (Table V) . The activity for 3-pyridylalanine and for 2-pyridyl+-aminobutyric acid was almost exactly the same, indicating that the spacing rather than

TABLE IV

Concentration of Various Substances by Carcinoma Cells

Suspensions of 1 part of cells in 5 to 7 parts of Krebs’ Ringer-bicarbonate, incu- bated 1 to 3 hours. The glycine derivatives were split to the following extent during incubation: Experiment 151, 11 per cent; Experiment 117, 7 per cent; Experiment 150, 17 per cent; Experiment 149, 7 per cent; Experiment 144,43 per cent.

Clupeine sulfate* 149t/64.5t = 2.3 cu-Methyl-nL-glutamate1 0.5/12.0 = 0.04 DLa,r-Diaminoglutarate 8.0/5.6 = 1.4 Glycinamide ss/50 = 1.8

“ 21.4/31.9 = 0.67 “ 11.2/8.0 = 1.4

Acetylglycine 6.9/12.4 = 0.56 “ 3.7/2.88 = 1.28

oL-Kynurenine 28.4/7.2 = 3.94 “ 18.1/2.72 = 6.65

Sodium kynurenate 1.23/5.04 = 0.24 Quinoline 8.11/3.88 = 2.1 Sodium p-aminobenzoate 5.88/13.2 = 0.45

‘I anthranilate 2.91/7.44 = 0.39 I‘ nicotinate 8.78/17.6 = 0.50 “ picolinate 10.1/21.0 = 0.48

@-Chloro-L-alanine 5.5/5.3 = 1.04 I‘ 21/20 = 1.05

* Gift of Dr. Herbert Fischer, University of Frankfurt. t After acid hydrolysis, as millimoles of carbon dioxide released by ninhydrin. $ Gift of Dr. Karl Pfister, Merck and Company, Inc.

118 127 132 151 117 150 149 144 141 142 145 147 179

218

rnY pn 2.

(2%) 10 5

60 30 10 10

5 10

5 5 5

10 10 10 10 10 27.7

Substance Added

to wspension

Distribution ratio

the position of the side chain on the pyridine ring was the critical feature. These substances were all used in the DL form. Comparison with the phen- ylalanine shows that the nitrogen in the ring greatly increased the concen- tration obtained.

A similar effect was obtained by introducing an amino group on the ben- zene ring of phenylglycine. m-Aminophenylglycine, obtained by direct nitration of nn-cY-phenylglycine, followed by reduction (15), was concen-

4 Gift of Professor J. H. Burckhalter, University of Kansas.

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402 FREE CELL NEOPLASM AND AMINO ACIDS

trated much more strongly than was phenylalanine or phenylglycine. p-Aminophenylglycine was concentrated to almost the same degree (Table V). These two isomers resemble ornithine and lysine as to the separation of the two amino groups. o-Aminophenylglycine, on the other hand, cor- responds to (~,y-diaminobutyric acid, and probably would show much stronger accumulation, especially in the L form. p-Aminophenylglycine was prepared from phenacylamine by way of phenylthioglyoxaline, phenyl-

TABLE V

Concentrative Uptake of Aromatic Amino Acids

The ratio given is for the cellular concentration divided by the extracellular, both in millimoles per kilo of water.

Experi- ment No.

148 178

231

174

190 151 264

190

200

Substance

2-Pyridyl-DL-alanine “

3-Pyridyf-nL-alanine 2-Pyridyl-nL-alanine 3-Pyridyl-nL-alanine r-(2-Pyridyl)-I%-a-aminobutyrate m-Nitro-nn-phenylglycine

“

3’-Amino-L-tyrosine m-Amino-Dr.-phenylglycine p-Amino-on-phenylglycine m-Amino-nn-phenylglycine r-(2,4-Dinitroanilino)-L-alanine ~-(Aminonitroanilino)-~a-aminobu-

tyric (A) -y-(Aminonitroanilino)-~-a-aminobu-

tyric (B)

Final distribution ratio

my per 1.

10 10 10 10 10 10 10

5 10 40 10 10

Saturated ‘I

37.4/6.56 = 5.70 31.3/9.88 = 3.17 25.5/13.1 = 1.95 39.7/5.78 = 6.87 30.8/9.04 = 3.41 33.3/9.36 = 3.56 22.3/8.82 = 2.53 10.4/4.0 = 2.60 34.8/3.96 = 8.79 74.9/37.4 = 2.00 31.5/10.2 = 3.09 29.0/10.5 = 2.76 4.24/0.84 = 5.05 4.06/0.65 = 6.25

7.2 9.3/5.2 = 1.8

glyoxalme, 4-p-nitrophenylglyoxaline, 5-nitro(4-p-nitrophenyl)glyoxaline, and the corresponding diamino compound (16). 4-o-Nitrophenylglyoxaline (16) was obtained in the same sequence and nitrated to 5-nitro(4-o-nitro- phenyl)glyoxaline, which was recrystallized from hot water. With slow heating this melted poorly, beginning at HO”, uncorrected. Complete and instantaneous melting occurred with very rapid heating at about 184”. The reduction product with chlorostannous acid at room temperature crys- tallized as the chlorostannate; the product on removal of tin crystallized readily from absolute ethanol. In contrast to the 5-amino(4-p-aminophen- yl)glyoxaline, this product failed to yield an a-amino acid upon acid hy- drolysis at 170”. A ring closure of the reduction product, analogous to the

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T. R. RIGGS, B. A. COYNE, AND H. N. CHRISTENSEN 403

Fischer indole synthesis, seems likely. Other courses toward the prepara- tion of o-aminophenylglycine are under examination.

These aromatic compounds have interested us because of their high ab- sorption in the ultraviolet region, leading to the hope that their movement into the cell could be visualized microscopically. This calls for a combina- tion of strong accumulation and a high extinction coefficient at a wave- length at which the cells themselves show little absorption, preferably above 310 rnp. Fig. 3 represents the absorption of a tumor cell suspension

.800-

= .600-

iii 2 .500- w 0

.400 -

d * .300-

F $ zoo-

I I I I I I I I I I I ’ 240 260 280 300 320 340 360 380 400 420 440

WAVELENGTH IN MILLIMICRONS

FIG. 3. Optical density of carcinoma cell suspension at various wave-lengths. Beckman spectrophotometer; an approximately 1 per cent suspension; preparation freed of erythrocytes by brief osmotic shock.

(freed of erythrocytes by brief osmotic shock) as observed by the Beckman spectrophotometer.

Absorption maxima of the more promising substances considered so far were about as follows: Z-pyridylalanine, 261 rnp; m-aminophenylglycine, 288 rnp; and tryptophan, 279 rnp. In tumor cells the absorption was too high and too variable in the region of these values to permit observation of the uptake of these amino acids by the Land model II color-translating microscope (17, 18) .I The following compounds were prepared to examine further the possibility of visualizing the concentration process: (1) the prod- ucts of the reaction of diazotized 3-aminopyridine and diazotized p-nitro- aniline with m-aminophenylglycine.‘ These orange-red products were not characterized, being obviously too insoluble for use. (2) b-(2,4-Dinitro-

6 For access to this instrument we are indebted to the Polaroid Corporation and a scientific committee which has made recommendations concerning its use.

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404 FREE CELL NEOPLASM AND AMINO ACIDS

aniline)-L-alanine and y-(2 ,4-dinitroanilino)-L-CY-aminobutyric acid, by the action of 2,4-dinitrofluorobenzene on the copper salts of a,@-diamino- propionic acid and a!, y-diaminobutyric acid, respectively, as described by Sanger (19) for the corresponding derivatives of ornithine. Interestingly, the copper salts of these two amino acids reacted as if the distal amino group were free, although the results of Albert have indicated that their copper salts probably involved principally the two amino groups, rather than the a-amino and carboxyl groups (20). (3) The reduction products of these substances by ammonium sulfide. In each case two isomeric O-(aminonitroanilino)-a-amino acids were isolated by fractional crystalli- zation: A, the less soluble and more abundant, forming brown crystals and

250 300 350 400 450 500 550 600

WAVELENGTH IN MILLIMICRONS

FIG. 4. Absorption of positional isomers of r-(aminonitroanilino)-L-cx-aminobuty- ric acid at various wave-lengths. The letters A and B correspond to the terminology of the text.

yellow solutions; and B, the more soluble, forming deep red crystals and intensely red solutions. The two isomeric derivatives of butyric acid were readily separated also by chromatography on paper with the solvent sys- tem 1:l lutidine and collidine, both water-saturated. The retardation factor for the A isomer was 0.54, for the B isomer, 0.39. In view of the poor value of melting points in characterizing amino acids, these dinitro and mononitro derivatives were characterized by their absorption spectra (Fig. 4). Which nitro group had been reduced in each case was not deter- mined. The red (B) amino acid derivative of butyric acid upon titration with the glass electrode showed pK’ values of 2.2 (approximate), 4.1, and 9.1. The middle value undoubtedly was due to the aromatic amino group.

The solubilities of the dinitrophenyl derivatives of the diamino acids and of their brown aminonitrophenyl derivatives were rather low for the in- tended purpose. Nevertheless the r(-dinitroanilino)+aminobutyric acid

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T. R. RIGGS, B. A. COYNE, AND FL N. CHRISTENSEN 405

was tested; the neoplastic cells were a distinct, uniform yellow color at a magnification of 970 times after 2 hours of incubation with a saturated solution of this amino acid in Krebs’ bicarbonate medium. During this experiment an excess of the amino acid was present in the medium in the form of microscopic crystals. This result is not considered very satisfac- tory because of uncertainty as to the state of the colored amino acid inside the cell, considering that the depth of color corresponded to concentrations far above the limit of solubility. The solubility of the red y-(aminonitro- aniline)-cr-aminobutyric acid (B) was adequate; saturated aqueous solu- tions appeared to be opaque, not unlike a sample of blood in redness. The tumor cells accumulated this substance enough to appear deeper in color than ery-throcytes present in the same field, and here again the coloration was uniform and not localized in any special part of the cell. Direct analysis in a separate experiment showed that the levels were disappoint- ingly less than twice as great inside the cells as outside. Study of this and other nitrophenylamino acids indicated a shortcoming of the nitro group as an auxochrome, namely, the danger of inhibition of the concentration process in its presence; the strong inhibition by 2,4-dinitrophenol is re- called (9).

Possible Participation of Metal in Amino Acid Transfer-Such chelating agents as ELhydroxyquinoline (1 mM) or (Y ,a’-dipyridyl (2 mM) were without inhibitory effect on the concentration of glycine from a 2 mM solution. Versene (the disodium salt of ethylenediaminetetraacetic acid) was dis- tinctly inhibitory to the concentration of glycine (Fig. 5), of a,~-diamino- butyric acid, and of tryptophan. For example, 5 mM Versene decreased the distribution ratio reached starting with a 25 mM solution of diamino- butyric acid to 58 per cent of its uninhibited value; 10 mM Versene, to 51 per cent. 10 mM Versene decreased the concentration of tryptophan (10 mM) to 70 per cent of its control value.

The inhibitory effect of Versene on glycine accumulation was completely released by calcium, magnesium, strontium, copper, or nickel, 1.1 mM of which were added per millimole of Versene (Table VI). Zinc produced a partial release of inhibition, but cobalt and barium were ineffective in this respect. Cobalt or strontium alone at about 1 mM was without significant effect on the transfer process. Copper by itself was inhibitory in the range 0.005 to 1 mM. Cobalt or nickel at 9 mM largely eliminated the concentra- tion of QI ,y-diaminobutyric acid. The addition of either ferric, aluminum, manganous, cupric, or cobalt salts at 0.1 mM levels along with 1 mM pyri- doxal failed to give stronger concentration of glycine than with pyridoxal alone.

Versene is itself an amino acid analogue, and its inhibitory effect may be due to a competition for a non-metallic acceptor quite as well as for a metal.

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40G FREE CELL NEOPLASM AND AMINO ACIDS

The disappearance of the inhibition when a metal is added does not imply direct participation by the metal in the amino acid transfer process, but only that the metal has changed Versene to a less inhibitory form. The

GLYClNE,mM/L

FIG. 5. Decreasing inhibition of the concentration of glycine by 10 mM Versene as the glycine level is raised.

TABLE VI

Reversal of Versene Znhibition by Metallic Ions

Glycine was at 2 mM, Versene at 1 mM, and the metal at, 1.1 mM per liter.

Experiment No.

124

126

129

-

--

-

Metal

None

2; co++ None Sr++ None Ba++ ZIP cu++ Ni++

_-

-

Relative distribution ratio

48 106

91 54 71 92 64 68 79 98

102

inhibitory effect of a given level of Versene was rapidly decreased by in- creasing the level of glycine; the degree of inhibition depended upon the ratio of the Versene to the glycine level (Fig. 5). This provides no evi- dence for the nature of the group for which the two may be competing. A significant fact, however, is that the affiity of the two for the acceptor is of a similar order. Considering the inferior binding of metals by an amino acid, this could argue against a metallic nature. Furthermore, if the

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T. R. RIGGS, B. A. COYNE, AND H. N. CHRISTENSEN 407

superior accumulation of cr, y-diaminobutyric acid were due to the greater stability of a metal-chelation product formed by it, then the accumulation of this amino acid should be more resistant to inhibition by Versene. The results cited above in the text show that the diamino acid, on the contrary, was more sensitive to inhibition by Versene.

Further evidence against the involvement of a metal in the “carrier” for amino acid transport was obtained by study of two derivative@ of ethylenediamine related to the tetraacetic acid derivative, namely N, N’- dimethylethylenediaminediacetic acid and hydroxyethylaminediacetic acid. In these compounds the glycine structure can be traced in two rather than four ways, but the compounds retain considerable metal-binding proper- ties. At 10 mM they were without significant effect upon the concentration process for glycine at 2 mM.

Attempts to study the action of iminodiacetice acid and sarcosine were unsuccessful because of the extensive production of formaldehyde by their reaction with ninhydrin in the glycine determination.

LowerinS of pK’ of a-Amino Group-Albert has reported that the pK’ of the a-amino group of a,~-diaminobutyric acid is 8.24 and that of cr,p- diaminopropionic is 6.69 (20). The possibility that pyridoxylidene deriva- tives were formed during accumulation (4) led to the idea that a lower pK should result in more stable derivatives of this character; the pK of diaminobutyric acid might represent an optimum for formation and dis- sociation of the amino acid-carrier complex. Introduction of an electron- attracting group which has no hydrogen ion dissociation of its own might in this case increase the concentration of an amino acid. Accordingly, &chloro-n-alanine was prepared from L-serine (2 1). Titration by the glass electrode gave pK’ values of 1.8 (carboxyl group) and 8.2 (amino group) at 25” and ionic strength 0.05 and 0.1 respectively. Upon incubation with this amino acid, the tumor cells swelled strongly, leading us to anticipate strong accumulation. Instead organic chlorine was only slightly higher inside the cells than outside; the amino acid was scarcely concentrated at all (Table IV). Two observations indicate that this result was not due simply to the lowered pK’ of the amino group; first, 37 per cent of the chlorine of the amino acid was released as inorganic chloride in 2 hours of incubation with the tumor cells; second, p-chloroalanine was a much stronger inhibitor of the concentration of glycine (Table VII) than was ala- nine or other similar amino acids (2). Accordingly the poor accumulation of /%chloroalanine is attributed to its partial degradation by the cell sus- pension rather than to its physical properties. This amino acid is under further study.

6 We are indebted to Dr. Albert E. Frost and Dr. John J. Singer of the Bersworth Chemical Company, Framingham, Massachusetts, forversene and the several related compounds.

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408 FREE CELL NEOPLASM AND AMINO ACIDS

Fluoroacetic acid was tested for inhibiting action on glycine accumula- tion in connection with chloroalanine, because some results of Awapara (22) suggested to us that this substance might be inhibitory to the process. The results were negative (Table VII). The sulfonic acid analogue of glycine was also without effect upon glycine accumulation.

Shifts of Potassium and Sodium during Uptake of Amino Acids-Hereto- fore two types of electrolyte shift have been seen during the concentration of amino acids. The first type serves to maintain electroneutrality; e.g., the uptake of potassium and sodium accompanying that of glutamate (9, 23, 24) or the uptake of chloride and the displacement of potassium

TABLE VII

Inhibition of Glycine Accumulation by Various Substances

1 part of tumor cells in 5 to 7 parts of Krebs’ Ringer-bicarbonate medium. Gly- tine added to an initial level of 2 mM per liter of suspension. Distribution ratio taken as 100 in absence of the inhibitor.

Experiment No inhibitor level

222

219

p-Chloro-L-alanine “ “ “ “

Sodium fluoroacetate ‘I “ “ aminomethylene sulfonate

-

-

WSY

10 5.5 2 0.5

10 4.8

13.8 10

-

-

R&tiW distribution

ratio

26 38 31 78 16

116 110

98

and sodium accompanying the accumulation of diaminobutyrate (3). In the second type of ion shift, preservation of electroneutrality does not suffice as an explanation. For example, the uptake of large amounts of glycine is accompanied by a considerable exchange of the cellular potassium for sodium (9, 2). This loss of potassium is smaller than the simultaneous gain of glycine. This interesting change now has been obtained also with L-tryptophan, m-amino-nn-phenylglycine, and 2-pyridyl-nn-alanine (Table VIII). With the uptake of tryptamine both types of shift were evident; that is, part of the potassium leaving the cell was replaced by the organic cation and part by sodium (Table VIII). In every case so far en- countered the strong uptake of an uncharged amino acid has been accom- panied by a shift of sodium for potassium.

Displacement of One Amino Acid from Cells by Another-As a corollary to the competition which has been observed among amino acids for con-

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T. R. RIGGS, B. A. COYNE, AND H. N. CHRISTENSEN 409

centration (9,Z) one would expect that the addition of a new amino acid to the cell suspension would cause the displacement from the cells of one

TABLE VIII Shifts of Potassium and Sodium during Uptake of Amino Acids and of Tryptamine

The amino acid gradients are expressed in millimoles per kilo of water, and A[K+] and A[Na+] in milliequivalents per kilo of cell water.

Experi- ment No.

145

151

155

Substance studied

L-Tryptophan, 20 mu ‘I 40 “ “ 40 “ ‘I 40 “ ‘I 60 “

m-Amino-rm-phenylglycine, 40 mM 2-Pyridyl-nL-alanine, 40 mhr Tryptamine HCl, 64.3 mM

‘I “ 64.3 “ m-Amino-nn-phenylglycine, 60.3 IIW

AIUiIlO Time acid

gradient -__

hrs.

6 31 2 31 4 38 6 22 6 16 4 38 4 77 1 55 4 45 4 13

-46 +28 -3 -10

-27 +12 -52 +47 -58 +57 -34 +46 -49 +25 -85 +33 -85 +28 -69 +90

TABLE IX

Displacement of One Amino Acid by Another or by Potassium Ion

The amino acid gradients are expressed in millimoles per kilo of water. L-DiAB = L-cu, y-diaminobutyric acid.

152A B

159A B C D

175A B C

1st amino acid 2nd amino acid

L-Tryptophan Glycine L-DiAB

‘I

Glycine ‘I

L-Tryptophan “ “

_-

Gradient for 1st amino acid

- I Gradient for

2nd amino acid (final)

Half-way Final .-

Glycine 48 23 52 L-Tryptophan 50 55 19 Glycine 114 53 23 L-Tryptophan 114 114 11 L-DiAB 51 37 50 L-Tryptophan 51 34 17 Glycine 37 8.3 27 L-DiAB 37 8.8 10 K+ 37 12 58 (K+)

already accumulated. This effect has been seen in the intact animal (25) and probably is a major factor in the deleterious nutritional effect of amino acid imbalance. Table IX illustrates this effect for the ascites tumor cell. In these experiments the suspension was incubated 2 or 3 hours with the

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410 FREE CELL NEOPLASM AND AMINO ACIDS

first amino acid at a 30 to 60 mM level; then after separation of a portion for analysis a second amino acid was added to the suspension at a 25 to 60 mM level and the incubation continued for 2 or 3 hours. Glycine displaced tryptophan rather better than tryptophan displaced glycine (Experiments 152B, 159D). Elevation of potassium to a 30 mM level also caused an exodus of the accumulated amino acid (Experiment 175C).

SUMMARY

1. In the study of the concentration of amino acids by the Ehrlich mouse ascites carcinoma cell, the origin of the particularly strong con- centration of certain dinitrogenous acids has been sought.

2. In the sensitivity of the process to temperature and to inhibitors and in stereochemical specificity, ar,rdiaminobutyric acid and tryptophan are very similar to other amino acids that have been studied.

3. A neurotoxic action of Q! ,y-diaminobutyric acid in the rat has been described.

4. Further evidence was obtained for the importance of the amino group, of the carboxyl group, and of a second nitrogenous group at an appropriate distance from the a-amino group. The importance of the spacing is illus- trated particularly well with a series of pyridine-substituted amino acids.

5. Several colored amino acids have been synthesized. These were studied, along with some ultraviolet-absorbing amino acids, in an effort to visualize the concentration process microscopically. One colored amino acid, although not particularly strongly concentrated, produced an approxi- mately uniform coloration of the cells.

6. The possibility was considered that chelation of a metal might be in- volved in the superior accumulation of certain dinitrogenous amino acids. Among several metal-chelating agents only ethylenediaminetetraacetic acid was significantly inhibitory to the concentration of amino acids. The nature of this inhibition was not such as to support the participation of a metal in the process.

7. The possibility was considered that a second nitrogenous group might improve concentration of an amino acid merely by lowering the pK of the amino group. Introduction of a chlorine atom on the p-carbon of alanine lowered the pK of the amino group to 8.2. The resulting compound was not well concentrated, but this behavior was probably associated with its partial degradation.

8. Tryptophan, m-aminophenylglycine, and 2-pyridylalanine, like gly- tine, cause the loss of potassium and the gain of sodium by cells upon their uptake.

9. One strongly concentrated amino acid tended to displace from the cells another which had already been accumulated.

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T. R. RIGGS, B. A. COYNE, AND H. N. CHRISTENSEN 411

BIBLIOGRAPHY

1. Riggs, T. R., Coyne, B. A., and Christensen, H. N., Federation Proc., 12, 258 (1953).

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3. Christensen, H. N., Riggs, T. R., Fischer, H., and Palatine, I. M., J. Biol. Chem., 198, 17 (1952).

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(1949). 24. Davies, R., Folkes, J. P., Gale, E. F., and Bigger, L. C., Biochem. J., 64, 430

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Halvor N. ChristensenThomas R. Riggs, Barbara A. Coyne and

INFLUENCESSTRUCTURALFREE CELL NEOPLASM.

AMINO ACID CONCENTRATION BY A

1954, 209:395-411.J. Biol. Chem. 

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