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Effect of carcinolipin on protein synthesis in cell-free systems
BIOCHIMICA ET BIOPHYSICA ACTA
I49
E F F E C T OF C A R C I N O L I P I N ON P R O T E I N S Y N T H E S I S IN C E L L - F R E E SYSTEMS JAN HRADEC*
Chester Beatty Research Institute, Institute o[ Cancer Research, Royal Cancer Hospital, London (Great Britain) (Received July 6th, 196o)
SUMMARY
Carcinolipin, the carcinogenic lipid substance from egg-yolks, markedly stimulates the incorporation of labeled valine, phenylalanine and lysine into the proteins of isolated microsomes in the presence of cell sap. Also the carboxyl-activation of amino acids is greatly enhanced in the presence of this substance, whereas the incorporation of amino acids into soluble ribonucleic acid as well as transfer of amino acids from pH 5 enzymes into microsomal proteins is stimulated to a smaller extent.
INTRODUCTION
A protein synthesis-affecting substance was recently isolated from egg-yolks ~ as well as from animal tissues 2. For its lipid nature and carcinogenic activity the designation Carcinolipin was proposed for this factor. Carcinolipin markedly affects the incorporation of labeled amino acids into proteins of Ehrlich ascites cells and tissue homogenates in vitro. Some doses of this substance stimulate this process while others on the contrary have inhibitory activity 2. It is believed that various enzymic systems present in whole cells and tissue homogenates might be affected by Carcinolipin and that this could be the reason for these diverse results. A need was therefore felt for a more suitable system which would permit a more thorough study into the mechanism of this action. It was shown that a cell free system can be prepared from rat liver which incorporates amino acids into proteins in vitro 4. Evidence is accumulating that reactions occurring within this system may be concerned in protein synthesis in vivo, too. Furthermore, it is possible to define certain steps during this process. Thus carboxylactivation of amino acids seems to be the first reaction necessary for protein synthesis ~. The aminoacyl adenylate formed in this way is then incorporated into the soluble ribonucleic acid of the cell sap 6. Transfer of amino acids from the soluble RNA to microsomes, where the actual protein synthesis takes place, is assumed to be the Abbreviations: ATP, adenosine triphosphate; Tris, trisIhydroxymethyl) aminomethane; RNA, ribonucleic acid. * Present address: Department of Biochemistry, Oncological Institute, Prague 8 (Czechoslovakia).
Biochim. Biophys.:t~ta, 47 (1901) x49 .t57
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ultimate step in this processL Suitable experimental techniques were devised making possible the study of these various stages of protein biosynthesis separatelyS,< s. Evidence is accumulating that this schema must by no means be taken as the only possible pathway of protein synthesis. Further alternate reactions are without doubt involved in this whole process 9 and on the other hand, some of the steps described above do not seem to be absolutely necessary for protein synthesis in cell-free systems 1°. In any event, the pathway of protein synthesis in the cell free system of rat liver as described by ZAMECNIK'Sgroup represents at the present time the only system of protein synthesis studied in greater detail. Furthermore, the system used by these authors is relatively simple and might be free of enzymes which were probably the reason of our diverse results in experiments in which the effect of Carcinolipin was tested on protein synthesis in Ehrlich ascites cells 2. For these reasons this experimental system was chosen as a most suitable one for further elucidation of the effect of Carcinolipin on protein synthesis. MATERIALS AND METHODS
Animals Wistar albino rats of both sexes, weighing 12o-15o g, bred at this institute were kept on a mixed diet.
Radioactive compounds Generally labeled L-I14Qvaline, L-I14CJphenylalanine, L-I14C~lysine, and L-~4C laspartic acid, with an average specific activity approx, i mC/mmole, were obtained from the Radiochemical Centre, Amersham, Bucks., England. LabeledEa~Plsodium pyrophosphate (5-3 me/mmole) was obtained from the same source.
Preparation of subcellularfractions Rats were killed b y breaking their necks, livers quickly removed, plunged into ice-cold 0.25 M sucrose, and chopped with scissors. Pooled liver tissue from 2-3 rats was usually used for one experiment. To each gram of liver 3 ml of medium 11 (0.025 M KC1, o . o o 4 M MgCI~. 6 H~O, 0.035 M KHCOs, o . o I 6 M K~HPO4 and 0.0026 M KH2PO4 in a 0.25 M sucrose solution) was added and the whole placed in an icecold tube of a Potter-Elvehjem homogenizer fitted with a polyethylene pestle. 5-6 upand-down strokes were carried out to prepare a homogeneous suspension. This was transferred to the tubes (11. 5 ml each) of a Spinco centrifuge (Model L, No. 40 head) and spun at 18.ooo x g for Io min. 7.5 ml of the supcrnatant was transferred to another tube and 3.5 ml of the above medium added. The resulting suspension was centrifuged at lO5,OOO × g for 45 min. The supernatant cell sap fraction was withdrawn and used without further preparation in some experiments. Microsomal sediment after wiping the inside of the tube with filter paper was gently homogenized in 0.5 ml of sucrose medium. All these procedures were carried out at 0-5 ° and the subcellular fractions maintained at the same temperature throughout. For the preparation of p H 5 enzymes for some experiments, the supernatant fraction was brought to p H 4.8 by addition of i M acetic acid. After IO rain the resulting precipitate was spun at 18,ooo x g for 15 min, washed twice by homogenizing Biochim. Biophys. Acta, 47 (1901) 149 q 5 7
CARCINOLIPIN AND PROTEIN SYNTHESIS
I5t
it in water with subsequent centrifugation, and finally homogenized in medium A of HOAGLAND6 to obtain approx. IO mg of protein/ml. These procedures were again performed at 0-5 ° throughout.
Incubation procedures In experiments in which the incorporation was followed of labeled amino acids into microsomal proteins, the incubation mixture contained 0.2 ml of microsomal suspension, o.I ml of cell sap fraction, 0.05 ml ATP (I/*mole), 0.05 ml MgCl~ (5 /*moles), o.17 ml phosphoenolpyruvate (IO/*moles, kindly prepared by Dr. P. COHN after the method of OHLMEYER12), o.13 ml of medium, and o.I ml of Carcinolipin suspension (or o.i ml of medium in control mixtures). Carcinolipin suspension was prepared by homogenizing this substance finely in the incubation medium using a Potter-Elvehjem homogenizer. The stock suspension (IO mg/ml) was kept at 20 ° and diluted appropriately with the medimn when necessary. The tubes containing the incubation mixture were placed in a constant temperature b a t h and shaken at 37 °. After equilibration (1-2 rain), o.i ml (I/*mole) of the labeled amino acid solution in water was added to each tube and incubation continued under Na-Co2(95:5). The reaction was stopped by adding 0.4 M solution of perchloric acid. When the incorporation of labeled amino acids into p H 5 enzymes was studied, the incubation mixture contained 2 ml of pH 5 enzymes suspension (20 mg of protein), o.I ml of ATP solution (20/,moles), and o.I ml of Carcinolipin suspension (or o.I ml of medium A in control mixtures). After equilibration the same quantity of amino acid solution was added and mixtures incubated in air. The reaction was again stopped by addition of perchloric acid. In experiments on transfer of labeled amino acids from p H 5 enzymes to microsomal protein the method of HOAGLAND et al. 6 was used. The incubation mixtures contained IO mg of prelabeled pH 5 enzymes. Labeling of pH 5 enzymes was carried out by incubating IO ml of pH 5 enzymes suspension with 15o/*moles of ATP and 15/*moles of labeled amino acid in air at 37 ° for IO min. Reaction was stopped by immersing the tube in ice-cold water. 2o ml of ice-cold water was then added to the incubation mixture and p H brought to 5.o by I M acetic acid. The resulting precipitate was spun down at 18,ooo x g. It was then twice again reprecipitated by acetic acid, three times washed with water and ultimately finely homogenized in medium A to give a suspension containing io mg of protein/ml. The method of HOAGLAND et alfi was used for studying the amino acid catalyzed phosphorus exchange between pyrophosphate and ATP. I/*mole of radioactive pyrophosphate was added to each incubation mixture containing pH 5 enzymes and ATP and amino acids in Tris buffer. In each experiment experimental mixtures containing different quantities of Carcinolipin were incubated simultaneously with two control samples containing o.i ml incubation medium instead of the active substance.
Preparation of samples for radioactivity assay Proteins precipitated at the end of the incubation were washed three times with 0.4 M perehloric acid and twice with an ethanol-ether-chloroform mixture (2:2:1) leaving them in this mixture at least 4 h each time. The precipitate was then dissolved
Biockim. ]3iophys. ~-~cta,47 (190I) 149 157
152
J. HRADEC
in 5 ml of I M sodium hydroxide and proteins precipitated again by 3 ml of 6 M hydrochloric acid. After this 5 ml of 0.4 M perchloric acid were added and the mixture incubated for 20 min at 9 o°. After centrifugation the precipitate was washed with cold 0.4 M perchloric acid and twice with acetone. The precipitate was then dried at 60 °. When the incorporation of labeled amino acids was stttdied into the soluble RNA, the precipitated proteins of each tube were divided into two equal samples. They were washed 4 times with 0.2 M perchloric acid, once with ethanol-o.2 M perchloric acid (5 : I), once with ethanol, and incubated in ethanol-ether (3 : I) at 5 °0 for IO rain. After this, the first of both duplicate mixtures was washed twice with acetone and the precipitate dried at 60 °. From the other precipitate, RNA was extracted with io ml of IO % NaC1 leaving the tubes for 30 min in a boiling water bath. This was followed by two washings with acetone and drying of the precipitate at 60 ° .
Radioactivity assay This was carried out on infinitely thick samples of protein precipitates plated on polyethylene discs. Samples of less than infinite thickness were corrected to infinite thickness using an experimentally determined curve obtained with proteins of known specific activity. An EHM 2 S end-window Geiger-Miiller tube in conjunction with an N 529 scaling unit (Ekco Electronics Ltd., Southend-on-Sea, England) was used for counting. 32p samples were counted in io-ml samples in an annular liquid counter type M 5 M (2oth Century Electronics Ltd., New Addington, Surrey). RESULTS
Effect of various doses of Carcinolipin on the incorporation of labeled amino acids into the proteins of microsomes Fig. I shows the effect of various doses of Carcinolipin on the incorporation of labeled valine, phenylalanine, lysine, and aspartic acid into the microsomal protein in the presence of the corresponding soluble cell fraction. Incorporation of valine is markedly enhanced by this substance, the most effective dose being 0.0I #g. A similar stimulation is also found in the case of phenylalanine, the optimal dose being 0.i-I/~g, however. Large doses of Carcinolipin inhibit the incorporation of labeled lysine to microsomal protein to some extent, lower doses having a similar enhancing effect as in the case of valine and phenylalanine. A closely corresponding optimal dose, i.e. 0.i/~g, was found when using lysine, too. Widely different results, however, were obtained, when the effect of Carcinolipin was studied on the incorporation of labeled aspartic acid into microsomal protein. As given in Fig. I, incorporation of this amino acid is greatly reduced in the presence of Carcinolipin. Not only high doses of this substance have an inhibitory activity as in the case of lysine. Also the usual stimulating doses show the most pronounced inhibitory effect when using aspartic acid. When trying to explain this apparently anomalous behavior of aspartic acid it was thought that it might be caused by a more rapid labeling of protein in the presence of Carcinolipin. This swifter incorporation followed by a more rapid loss of label might well result in a lower radioactivity in experimental protein samples after the relatively long incubation period which was used in these experiments. Biochim. Biophys..d cta, 47 (190~) 149 -I 57
CARCINOLIPIN ANT) PROTEIN SYNTHESIS
153
To examine this possibility, further experiments were performed using shorter incubation periods. The results of these have shown (see Fig. 2) that the incorporation of labeled lysine into microsomal protein proceeds more swiftly under the influence of Carcinolipin, the highest radioactivity being in the protein after 20 rain of incubation. After this, some loss of label occurs in the experimental samples when compared with the controls where highest labeling was found not until 25 min. Similar enhancement of incorporation is caused by Carcinolipin in the case of aspartic acid. However, the highest radioactivity is found in experimental protein samples after IO rain of incubation. After this, a very rapid loss of labeling occurs resulting in a lower specific activity of experimental samples when compared with the normal ones after 15 min of incubation. This lower radioactivity of experimental samples remains until the end of the experiment after 25 min, although at this time the specific activity of experimental samples is somewhat higher than it was after 15 rain. i
i
/
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t~
)
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/
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i
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140"
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/ 60. 40-
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.04. .02"
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Fig. I. Effect of v a r i o u s d o s e s of Carcinolipin on t h e i n c o r p o r a t i o n of labeled a m i n o acids into p r o t e i n s of m i c r o s o m e s . E a c h c u r v e is b a s e d on v a l u e s o b t a i n e d in four i n d i v i d u a l e x p e r i m e n t s w i t h different b a t c h e s of liver tissue. O - - - ~ , L-valine; O----O , L-phenyla l a n i n e ; © © , L-lysine; O - - O , L-aspartic acid.
Ib
~
~
25
rime (min)
Fig. z. Effect of Carcinolipin on t h e incorporation of labeled lysine ~ , o.t tig of Carcinolipin, a n d aspartic acid ~ , 5 Fg of Carcinolipin, into m i c r o s o m a l protein. Control m i x t u r e s are g i v e n in full lines, e x p e r i m e n t a l in d a s h e d line. Values o b t a i n e d in one p a r t i c u l a r experim e n t are g i v e n (two o t h e r s similar e x p e r i m e n t s were p e r f o r m e d in each i n s t a n c e yielding c o r r e s p o n d i n g results).
It is obvious from these results, that this anomalous labeling of microsomal protein under the influence of Carcinolipin may really be the reason of the inhibitory effect of this substance on the incorporation of aspartic acid. It is not yet clear, however, why this rapid loss of label after io min of incubation appears only in the case of aspartie acid. When the effect of Carcinolipin was tested on the incorporation of labeled valine and phenylalanine in the same way, curves were obtained closely similar to those with lysine (Fig. 2), the specific activity of protein samples being only somewhat B i o c h i m . B i o p h y s . ,4cta, 47 (196I} 140-t57
154
J. HRADEC
higher. It will need further elucidation why such a rapid loss of labeling occurs just in the case of aspartic acid while only moderate losses of activity are found when using other amino acids.
E~ect of Carcinolipin on the carboxyl-activation of amino acids When a suspension of o.I/xg of Carcinolipin was added to the incubation mixture, a greater amino acid-catalyzed pyrophosphate-ATP exchange occurred when compared with the control samples without this substance. As seen in Table I, this enhancing effect varies with different amino acids. A striking effect is seen when lysine is used whereas only slight stimulation is shown in the case of phenylalanine. It is clear from these results, however, that Carcinolipin stimulates the carboxyl-activation of amino acids to a much greater extent than the incorporation of amino acids into microsomal protein. TABLE I EFFECT
OF
CARCINOLIPIN
ON
AMINO
ACID
ACTIVATION
All v a l u e s g i v e n in this table are a v e r a g e s f r o m four e x p e r i m e n t s . M i x t u r e A c o n t a i n e d p h e n y l alanine, lysine, leucine a n d valine; m i x t u r e B, glycine, threonine, arginine a n d t r y p t o p h a n ; m i x t u r e C, alanine, serine, isoleucine a n d histidine. Per cent c~a°'P e.rcham, e A rain6 acid
Valine Phenylalanine Lysine Mixture A Mixture B Mixture C
Cvntrol
Experime~!lal
4.9 3.5 o. 3 2.6 6.2 0.9
12.6 4.4 5.7 4.2 10.2 9..5
Effect of Carcinolipin on the incorporation of labeled amino acids into soluble RNA It was found in these experiments that essentially all radioactivity (approx. 97 %) is removed from precipitated pH 5 enzymes by extraction with hot NaC1. This suggested that practically all activity is present in the soluble RNA of pH 5 enzymes. As seen in Fig. 3, addition of o.i ~g of Carcinolipin into a reaction mixture containing pH 5 enzymes and ATP results in a more rapid labeling of pH 5 enzymes with lysine than in the control mixture. Closely similar curves were obtained when labeled valine or phenylalanine were used instead of lysine. It is apparent that the highest radioactivity is present in experimental samples after 6 min of incubation and after this a rather rapid decline of specific activity occurs. On the other hand, labeling of pH 5 enzymes in the corresponding control samples proceeds for the whole incubation period. It is also obvious that the highest specific radioactivity of the control samples is roughly the same as in the experimental ones, although this highest value is obtained sooner in this latter case. This is in good correlation with the concept that a limited number of combining sites is present in the soluble RNA 13 which of course cannot be affected by the action of Carcinolipin. E~ect o/Carcinolipin on the trans[er o/amino acids/rom pH 5 enzymes to microsomal protein In this study of the terminal stage of protein biosynthesis in a cell-free system, o.i/~g of Carcinolipin was added to a reaction mixture containing prelabeled pH 5 Biochim. Biophys. Acta, 47 (I961) I 4 9 - i 5 7
CARCINOLIPIN AND PROTEIN SYNTHESIS
155
enzymes, mierosomes, ATP, phosphoenolpyruvate and guanosin triphosphate. As shown in Table II, Carcinolipin proved to possess a definite stimulating activity at this stage also. Whereas maximal incorporation of labeled valine into microsomal proteins of control samples was reached in 25 min of incubation, the highest specific
.12-
o.to~
"~
//d j
.08-
.06.04-f .02-
Time (rain) Fig. 3. Effect of o.i # g of Carcinolipin on t h e i n c o r p o r a t i o n of labeled lysine into p H 5 e n z y m e s . Full line d e n o t e s t h e control m i x t u r e , d a s h e d t h e e x p e r i m e n t a l sample. Values g i v e n here are a v e r a g e s of t h r e e similar e x p e r i m e n t s . T A B L E II EFFECT
OF CARCINOLIPIN pH
ON
THE
TRANSFER
5 ENZYMES
TO
OF VARIOUS
MICROSOMAL
LABELED
AMINO
ACIDS
FROM
PROTEINS
All values are specific activities of m i c r o s o m a l p r o t e i n precipitates (/,C/g of protein) in i n d i v i d u a l e x p e r i m e n t s .
obtained
Incubation perio, l (rain) Labelled amino acid
L-valine L-phenylalanine L-aspartic acid
Control Experimental Control E x p e r i m e n t al Control Experimental
5
11~
15
25
o.i 47 o. 165 o.162 0.21 o 0.030 0.048
o.293 o.298 0.349 o. 427 0.o6o 0.095
o.327 o.374 0.387 o. 47 o 0.o86 o.I42
o.362 o.353 o.36S 0.33 ~ o.o02 0.088
activity was found after 15 min in experimental samples, some loss of label occurring after this time. The highest radioactivity of experimental samples was only slightly higher than that of the controls. Corresponding results were also obtained when phenylalanine was used in the same way. In this case, however, maximal label was found to be present after 15 min of incubation in both experimental as well as control samples. After this a very slight decline of specific activity was found in control samples, whereas a heavy loss of label was observed in experimental mixtures. The highest specific activity of control samples was distinctly lower than that of experimental ones. The same type of curve was obtained when lysine was used in some experiments instead of phenylalanine. An excellent correlation was found when comparing the extent of transfer of labeled amino acids from p H 5 enzymes to microsomal protein obtained in present
Biochim. Biophys. Acta, 47
(196I) 149 t57
156
j. HRADEC
experiments with results given by HOAGLAND et al. ~. Whereas these authors describe an average transfer of 20 to 30 % of amino acids in this type of experiment, an average transfer of 24 % of valine, 23 % of phenylalanine, and 18 % of lysine was found in the present study. DISCUSSION
Only stimulatory effects on the protein synthesis were caused by Carcinolipin in the present study. The inhibition of the incorporation of labeled lysine and aspartie acid into microsomal protein in the presence of the highest doses of this substance may probably be regarded as a mere non-specific toxic effect of excessive amounts of the active substance. No inhibition was found at lower doses as in our experiments where Ehrlich ascites cells or tissue homogenates were used 2. It is probable that an effect of Carcinolipin on other enzymes, absent from the cell-free system used in the present experiments, might be the reason for a partial inhibition of protein synthesis in whole cells or homogenates under the influence of certain doses of this substance. It is obvious that Carcinolipin is capable of stimulating protein biosynthesis at each step, although distinct quantitative differences exist in its action at various stages of protein synthesis. A striking effect is exhibited by this substance on the amino acid activation whereas only slight stinlulatory effect could be demonstrated on the transfer of amino acids from pH 5 enzymes into microsomes and in particular on the incorporation of labeled amino acids into soluble RNA. It is not clear, however, from the present experiments if Carcinolipin exhibits a direct effect on protein synthesis. Since ATP is necessary for each step of protein synthesis studied here, it cannot be excluded that Carcinolipin acts upon the metabolism of this compound affecting in this way protein synthesis only indirectly 11. However, this does not seem very probable, although Carcinolipin seems to stimulate the adenosinetriphosphatase activity in homogenates to some extent 14. It was shown above that great differences do exist in the stimulating effect of Carcinolipin on amino acid activation varying with the amino acid used. Similarly, varying stimulatory effects were exhibited by this substance at other stages of protein synthesis, depending on the amino acid used. Furthermore, it was shown that Carcinolipin stimulates net-synthesis of serum albumin in liver slices in vitro 14. From this it seems very probable that Carcinolipin is capable of specific stimulation of protein synthesis. It has been shown recently that lipids may play an important role in protein synthesis, although it is not quite clear at this moment if phospholipids 15 or other types of lipids 1~ are involved. The idea that Carcinolipin bears some relation to these substances seems very attractive. The ribose moiety of Carcinolipin li might well take a part in amino acid transport. This possibility is being investigated further. ACKNOWLEDGEMENTS
I wish to express my deepest gratitude to Professor A. HADDOW,who enabled me to obtain a Visiting Scholarship of the Chester Beatty Research Institute, for his encouragement and interest in this work. Also I give my best thanks to Professor J. A. V. BUTLER, who enabled me to do these experiments in his laboratories. I am very grateful to Dr. P. COliN for his many valuable suggestions, discussions and friendly help. Also the valuable advice of Dr. G. D. HUNTER and all other members of the Biochim. Biophys. Acta, 47 (I961) 149-157
CARCINOLIPIN AND PROTEIN SYNTHESIS
157
staff of Pollard's Wood Research Station (of the Institute of Cancer Research, Royal Cancer Hospital) is gratefully acknowledged. This investigation has been supported by grants to the Chester Beatty Research Institute (Institute of Cancer Research, Royal Cancer Hospital) from the British Empire Cancer Campaign, the Jane Coffin Childs Memorial Fund for Medical Research, the Anna Fuller Fund and the National Cancer Institute of the National Institutes of Health, U.S. Public Health Service. The author is a visiting scholar of the Chester Beatty Institute, Institute of Cancer Research, Royal Cancer Hospital. REFERENCES 1 2 3 4 5 6
j . HRADEC, Nature, 182 (1958) 52. j . HRADEC AND A. ~TROUFOV.~ Biochim. Biophys. Acta, 4 ° (196o) 32. j . I4RADEC AND J. I'{RUML, Nature, 185 (196o) 551~. C. ZAMECNIK AND E . B, KELLER, J. Biol. Chem., 209 (I954) 337M. g . HOAGLAND, E . g . KELLER AND P. C.. ZAMECNIK, J . Biol. Chem., 218 (1956) 345. M. B. HOAGLAND, M. L. STEPHENSON, J. F. SCOTT, L. I. HECHT AND P. C. ZAMECNIK, J. Biol.
Chem., 23I (1958) 241. 7 R. B. LOFTFIELI), Progr. in Biophys. and Biophys. Chem., 8 (1957) 347. 8 p . C. ZAMECNIK AND E . B. KELLER, J. Biol. Chem., 221 (1956) 45. 9 R. W. HENDLER, Science, 128 (1958) 143. 10 p. COHN, Biochim. Biophys. Acta, 33 (I959) 28411 p. Cohn, personal communication. 12 p. OHLMEYER, J. Biol. Chem., 19o (1951) 21. 13 1D N. CAMPBELL, personal communication. 14 j . HRADEC, manuscript in preparation.
15 R. W . HENDLER, J. Biol. Chem., 234 (1959) 1466. 1~ j . L. HAINING, T. FUKUI AND 13. AXELROI), J. Biol. Chem., 235 (196o) i6o.
Biochim. Biophys. Acta, 47 (i 961) 149-157
I49
E F F E C T OF C A R C I N O L I P I N ON P R O T E I N S Y N T H E S I S IN C E L L - F R E E SYSTEMS JAN HRADEC*
Chester Beatty Research Institute, Institute o[ Cancer Research, Royal Cancer Hospital, London (Great Britain) (Received July 6th, 196o)
SUMMARY
Carcinolipin, the carcinogenic lipid substance from egg-yolks, markedly stimulates the incorporation of labeled valine, phenylalanine and lysine into the proteins of isolated microsomes in the presence of cell sap. Also the carboxyl-activation of amino acids is greatly enhanced in the presence of this substance, whereas the incorporation of amino acids into soluble ribonucleic acid as well as transfer of amino acids from pH 5 enzymes into microsomal proteins is stimulated to a smaller extent.
INTRODUCTION
A protein synthesis-affecting substance was recently isolated from egg-yolks ~ as well as from animal tissues 2. For its lipid nature and carcinogenic activity the designation Carcinolipin was proposed for this factor. Carcinolipin markedly affects the incorporation of labeled amino acids into proteins of Ehrlich ascites cells and tissue homogenates in vitro. Some doses of this substance stimulate this process while others on the contrary have inhibitory activity 2. It is believed that various enzymic systems present in whole cells and tissue homogenates might be affected by Carcinolipin and that this could be the reason for these diverse results. A need was therefore felt for a more suitable system which would permit a more thorough study into the mechanism of this action. It was shown that a cell free system can be prepared from rat liver which incorporates amino acids into proteins in vitro 4. Evidence is accumulating that reactions occurring within this system may be concerned in protein synthesis in vivo, too. Furthermore, it is possible to define certain steps during this process. Thus carboxylactivation of amino acids seems to be the first reaction necessary for protein synthesis ~. The aminoacyl adenylate formed in this way is then incorporated into the soluble ribonucleic acid of the cell sap 6. Transfer of amino acids from the soluble RNA to microsomes, where the actual protein synthesis takes place, is assumed to be the Abbreviations: ATP, adenosine triphosphate; Tris, trisIhydroxymethyl) aminomethane; RNA, ribonucleic acid. * Present address: Department of Biochemistry, Oncological Institute, Prague 8 (Czechoslovakia).
Biochim. Biophys.:t~ta, 47 (1901) x49 .t57
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J. HRADEC
ultimate step in this processL Suitable experimental techniques were devised making possible the study of these various stages of protein biosynthesis separatelyS,< s. Evidence is accumulating that this schema must by no means be taken as the only possible pathway of protein synthesis. Further alternate reactions are without doubt involved in this whole process 9 and on the other hand, some of the steps described above do not seem to be absolutely necessary for protein synthesis in cell-free systems 1°. In any event, the pathway of protein synthesis in the cell free system of rat liver as described by ZAMECNIK'Sgroup represents at the present time the only system of protein synthesis studied in greater detail. Furthermore, the system used by these authors is relatively simple and might be free of enzymes which were probably the reason of our diverse results in experiments in which the effect of Carcinolipin was tested on protein synthesis in Ehrlich ascites cells 2. For these reasons this experimental system was chosen as a most suitable one for further elucidation of the effect of Carcinolipin on protein synthesis. MATERIALS AND METHODS
Animals Wistar albino rats of both sexes, weighing 12o-15o g, bred at this institute were kept on a mixed diet.
Radioactive compounds Generally labeled L-I14Qvaline, L-I14CJphenylalanine, L-I14C~lysine, and L-~4C laspartic acid, with an average specific activity approx, i mC/mmole, were obtained from the Radiochemical Centre, Amersham, Bucks., England. LabeledEa~Plsodium pyrophosphate (5-3 me/mmole) was obtained from the same source.
Preparation of subcellularfractions Rats were killed b y breaking their necks, livers quickly removed, plunged into ice-cold 0.25 M sucrose, and chopped with scissors. Pooled liver tissue from 2-3 rats was usually used for one experiment. To each gram of liver 3 ml of medium 11 (0.025 M KC1, o . o o 4 M MgCI~. 6 H~O, 0.035 M KHCOs, o . o I 6 M K~HPO4 and 0.0026 M KH2PO4 in a 0.25 M sucrose solution) was added and the whole placed in an icecold tube of a Potter-Elvehjem homogenizer fitted with a polyethylene pestle. 5-6 upand-down strokes were carried out to prepare a homogeneous suspension. This was transferred to the tubes (11. 5 ml each) of a Spinco centrifuge (Model L, No. 40 head) and spun at 18.ooo x g for Io min. 7.5 ml of the supcrnatant was transferred to another tube and 3.5 ml of the above medium added. The resulting suspension was centrifuged at lO5,OOO × g for 45 min. The supernatant cell sap fraction was withdrawn and used without further preparation in some experiments. Microsomal sediment after wiping the inside of the tube with filter paper was gently homogenized in 0.5 ml of sucrose medium. All these procedures were carried out at 0-5 ° and the subcellular fractions maintained at the same temperature throughout. For the preparation of p H 5 enzymes for some experiments, the supernatant fraction was brought to p H 4.8 by addition of i M acetic acid. After IO rain the resulting precipitate was spun at 18,ooo x g for 15 min, washed twice by homogenizing Biochim. Biophys. Acta, 47 (1901) 149 q 5 7
CARCINOLIPIN AND PROTEIN SYNTHESIS
I5t
it in water with subsequent centrifugation, and finally homogenized in medium A of HOAGLAND6 to obtain approx. IO mg of protein/ml. These procedures were again performed at 0-5 ° throughout.
Incubation procedures In experiments in which the incorporation was followed of labeled amino acids into microsomal proteins, the incubation mixture contained 0.2 ml of microsomal suspension, o.I ml of cell sap fraction, 0.05 ml ATP (I/*mole), 0.05 ml MgCl~ (5 /*moles), o.17 ml phosphoenolpyruvate (IO/*moles, kindly prepared by Dr. P. COHN after the method of OHLMEYER12), o.13 ml of medium, and o.I ml of Carcinolipin suspension (or o.i ml of medium in control mixtures). Carcinolipin suspension was prepared by homogenizing this substance finely in the incubation medium using a Potter-Elvehjem homogenizer. The stock suspension (IO mg/ml) was kept at 20 ° and diluted appropriately with the medimn when necessary. The tubes containing the incubation mixture were placed in a constant temperature b a t h and shaken at 37 °. After equilibration (1-2 rain), o.i ml (I/*mole) of the labeled amino acid solution in water was added to each tube and incubation continued under Na-Co2(95:5). The reaction was stopped by adding 0.4 M solution of perchloric acid. When the incorporation of labeled amino acids into p H 5 enzymes was studied, the incubation mixture contained 2 ml of pH 5 enzymes suspension (20 mg of protein), o.I ml of ATP solution (20/,moles), and o.I ml of Carcinolipin suspension (or o.I ml of medium A in control mixtures). After equilibration the same quantity of amino acid solution was added and mixtures incubated in air. The reaction was again stopped by addition of perchloric acid. In experiments on transfer of labeled amino acids from p H 5 enzymes to microsomal protein the method of HOAGLAND et al. 6 was used. The incubation mixtures contained IO mg of prelabeled pH 5 enzymes. Labeling of pH 5 enzymes was carried out by incubating IO ml of pH 5 enzymes suspension with 15o/*moles of ATP and 15/*moles of labeled amino acid in air at 37 ° for IO min. Reaction was stopped by immersing the tube in ice-cold water. 2o ml of ice-cold water was then added to the incubation mixture and p H brought to 5.o by I M acetic acid. The resulting precipitate was spun down at 18,ooo x g. It was then twice again reprecipitated by acetic acid, three times washed with water and ultimately finely homogenized in medium A to give a suspension containing io mg of protein/ml. The method of HOAGLAND et alfi was used for studying the amino acid catalyzed phosphorus exchange between pyrophosphate and ATP. I/*mole of radioactive pyrophosphate was added to each incubation mixture containing pH 5 enzymes and ATP and amino acids in Tris buffer. In each experiment experimental mixtures containing different quantities of Carcinolipin were incubated simultaneously with two control samples containing o.i ml incubation medium instead of the active substance.
Preparation of samples for radioactivity assay Proteins precipitated at the end of the incubation were washed three times with 0.4 M perehloric acid and twice with an ethanol-ether-chloroform mixture (2:2:1) leaving them in this mixture at least 4 h each time. The precipitate was then dissolved
Biockim. ]3iophys. ~-~cta,47 (190I) 149 157
152
J. HRADEC
in 5 ml of I M sodium hydroxide and proteins precipitated again by 3 ml of 6 M hydrochloric acid. After this 5 ml of 0.4 M perchloric acid were added and the mixture incubated for 20 min at 9 o°. After centrifugation the precipitate was washed with cold 0.4 M perchloric acid and twice with acetone. The precipitate was then dried at 60 °. When the incorporation of labeled amino acids was stttdied into the soluble RNA, the precipitated proteins of each tube were divided into two equal samples. They were washed 4 times with 0.2 M perchloric acid, once with ethanol-o.2 M perchloric acid (5 : I), once with ethanol, and incubated in ethanol-ether (3 : I) at 5 °0 for IO rain. After this, the first of both duplicate mixtures was washed twice with acetone and the precipitate dried at 60 °. From the other precipitate, RNA was extracted with io ml of IO % NaC1 leaving the tubes for 30 min in a boiling water bath. This was followed by two washings with acetone and drying of the precipitate at 60 ° .
Radioactivity assay This was carried out on infinitely thick samples of protein precipitates plated on polyethylene discs. Samples of less than infinite thickness were corrected to infinite thickness using an experimentally determined curve obtained with proteins of known specific activity. An EHM 2 S end-window Geiger-Miiller tube in conjunction with an N 529 scaling unit (Ekco Electronics Ltd., Southend-on-Sea, England) was used for counting. 32p samples were counted in io-ml samples in an annular liquid counter type M 5 M (2oth Century Electronics Ltd., New Addington, Surrey). RESULTS
Effect of various doses of Carcinolipin on the incorporation of labeled amino acids into the proteins of microsomes Fig. I shows the effect of various doses of Carcinolipin on the incorporation of labeled valine, phenylalanine, lysine, and aspartic acid into the microsomal protein in the presence of the corresponding soluble cell fraction. Incorporation of valine is markedly enhanced by this substance, the most effective dose being 0.0I #g. A similar stimulation is also found in the case of phenylalanine, the optimal dose being 0.i-I/~g, however. Large doses of Carcinolipin inhibit the incorporation of labeled lysine to microsomal protein to some extent, lower doses having a similar enhancing effect as in the case of valine and phenylalanine. A closely corresponding optimal dose, i.e. 0.i/~g, was found when using lysine, too. Widely different results, however, were obtained, when the effect of Carcinolipin was studied on the incorporation of labeled aspartic acid into microsomal protein. As given in Fig. I, incorporation of this amino acid is greatly reduced in the presence of Carcinolipin. Not only high doses of this substance have an inhibitory activity as in the case of lysine. Also the usual stimulating doses show the most pronounced inhibitory effect when using aspartic acid. When trying to explain this apparently anomalous behavior of aspartic acid it was thought that it might be caused by a more rapid labeling of protein in the presence of Carcinolipin. This swifter incorporation followed by a more rapid loss of label might well result in a lower radioactivity in experimental protein samples after the relatively long incubation period which was used in these experiments. Biochim. Biophys..d cta, 47 (190~) 149 -I 57
CARCINOLIPIN ANT) PROTEIN SYNTHESIS
153
To examine this possibility, further experiments were performed using shorter incubation periods. The results of these have shown (see Fig. 2) that the incorporation of labeled lysine into microsomal protein proceeds more swiftly under the influence of Carcinolipin, the highest radioactivity being in the protein after 20 rain of incubation. After this, some loss of label occurs in the experimental samples when compared with the controls where highest labeling was found not until 25 min. Similar enhancement of incorporation is caused by Carcinolipin in the case of aspartic acid. However, the highest radioactivity is found in experimental protein samples after IO rain of incubation. After this, a very rapid loss of labeling occurs resulting in a lower specific activity of experimental samples when compared with the normal ones after 15 min of incubation. This lower radioactivity of experimental samples remains until the end of the experiment after 25 min, although at this time the specific activity of experimental samples is somewhat higher than it was after 15 rain. i
i
/
16o-
t~
)
/
/
/
i
/
140"
,,/~ "~ J2-
120.
u~
tO0, 80-
.'lO-
o....'/~,
/I o ..'~.1o
/ 60. 40-
¥
0/ • i
"~ "\
i. j,.,~
"\
¶ • ,,
.~
/
i .08.06.
e/p"
.04. .02"
20-
~ 10 - n m g
Fig. I. Effect of v a r i o u s d o s e s of Carcinolipin on t h e i n c o r p o r a t i o n of labeled a m i n o acids into p r o t e i n s of m i c r o s o m e s . E a c h c u r v e is b a s e d on v a l u e s o b t a i n e d in four i n d i v i d u a l e x p e r i m e n t s w i t h different b a t c h e s of liver tissue. O - - - ~ , L-valine; O----O , L-phenyla l a n i n e ; © © , L-lysine; O - - O , L-aspartic acid.
Ib
~
~
25
rime (min)
Fig. z. Effect of Carcinolipin on t h e incorporation of labeled lysine ~ , o.t tig of Carcinolipin, a n d aspartic acid ~ , 5 Fg of Carcinolipin, into m i c r o s o m a l protein. Control m i x t u r e s are g i v e n in full lines, e x p e r i m e n t a l in d a s h e d line. Values o b t a i n e d in one p a r t i c u l a r experim e n t are g i v e n (two o t h e r s similar e x p e r i m e n t s were p e r f o r m e d in each i n s t a n c e yielding c o r r e s p o n d i n g results).
It is obvious from these results, that this anomalous labeling of microsomal protein under the influence of Carcinolipin may really be the reason of the inhibitory effect of this substance on the incorporation of aspartic acid. It is not yet clear, however, why this rapid loss of label after io min of incubation appears only in the case of aspartie acid. When the effect of Carcinolipin was tested on the incorporation of labeled valine and phenylalanine in the same way, curves were obtained closely similar to those with lysine (Fig. 2), the specific activity of protein samples being only somewhat B i o c h i m . B i o p h y s . ,4cta, 47 (196I} 140-t57
154
J. HRADEC
higher. It will need further elucidation why such a rapid loss of labeling occurs just in the case of aspartic acid while only moderate losses of activity are found when using other amino acids.
E~ect of Carcinolipin on the carboxyl-activation of amino acids When a suspension of o.I/xg of Carcinolipin was added to the incubation mixture, a greater amino acid-catalyzed pyrophosphate-ATP exchange occurred when compared with the control samples without this substance. As seen in Table I, this enhancing effect varies with different amino acids. A striking effect is seen when lysine is used whereas only slight stimulation is shown in the case of phenylalanine. It is clear from these results, however, that Carcinolipin stimulates the carboxyl-activation of amino acids to a much greater extent than the incorporation of amino acids into microsomal protein. TABLE I EFFECT
OF
CARCINOLIPIN
ON
AMINO
ACID
ACTIVATION
All v a l u e s g i v e n in this table are a v e r a g e s f r o m four e x p e r i m e n t s . M i x t u r e A c o n t a i n e d p h e n y l alanine, lysine, leucine a n d valine; m i x t u r e B, glycine, threonine, arginine a n d t r y p t o p h a n ; m i x t u r e C, alanine, serine, isoleucine a n d histidine. Per cent c~a°'P e.rcham, e A rain6 acid
Valine Phenylalanine Lysine Mixture A Mixture B Mixture C
Cvntrol
Experime~!lal
4.9 3.5 o. 3 2.6 6.2 0.9
12.6 4.4 5.7 4.2 10.2 9..5
Effect of Carcinolipin on the incorporation of labeled amino acids into soluble RNA It was found in these experiments that essentially all radioactivity (approx. 97 %) is removed from precipitated pH 5 enzymes by extraction with hot NaC1. This suggested that practically all activity is present in the soluble RNA of pH 5 enzymes. As seen in Fig. 3, addition of o.i ~g of Carcinolipin into a reaction mixture containing pH 5 enzymes and ATP results in a more rapid labeling of pH 5 enzymes with lysine than in the control mixture. Closely similar curves were obtained when labeled valine or phenylalanine were used instead of lysine. It is apparent that the highest radioactivity is present in experimental samples after 6 min of incubation and after this a rather rapid decline of specific activity occurs. On the other hand, labeling of pH 5 enzymes in the corresponding control samples proceeds for the whole incubation period. It is also obvious that the highest specific radioactivity of the control samples is roughly the same as in the experimental ones, although this highest value is obtained sooner in this latter case. This is in good correlation with the concept that a limited number of combining sites is present in the soluble RNA 13 which of course cannot be affected by the action of Carcinolipin. E~ect o/Carcinolipin on the trans[er o/amino acids/rom pH 5 enzymes to microsomal protein In this study of the terminal stage of protein biosynthesis in a cell-free system, o.i/~g of Carcinolipin was added to a reaction mixture containing prelabeled pH 5 Biochim. Biophys. Acta, 47 (I961) I 4 9 - i 5 7
CARCINOLIPIN AND PROTEIN SYNTHESIS
155
enzymes, mierosomes, ATP, phosphoenolpyruvate and guanosin triphosphate. As shown in Table II, Carcinolipin proved to possess a definite stimulating activity at this stage also. Whereas maximal incorporation of labeled valine into microsomal proteins of control samples was reached in 25 min of incubation, the highest specific
.12-
o.to~
"~
//d j
.08-
.06.04-f .02-
Time (rain) Fig. 3. Effect of o.i # g of Carcinolipin on t h e i n c o r p o r a t i o n of labeled lysine into p H 5 e n z y m e s . Full line d e n o t e s t h e control m i x t u r e , d a s h e d t h e e x p e r i m e n t a l sample. Values g i v e n here are a v e r a g e s of t h r e e similar e x p e r i m e n t s . T A B L E II EFFECT
OF CARCINOLIPIN pH
ON
THE
TRANSFER
5 ENZYMES
TO
OF VARIOUS
MICROSOMAL
LABELED
AMINO
ACIDS
FROM
PROTEINS
All values are specific activities of m i c r o s o m a l p r o t e i n precipitates (/,C/g of protein) in i n d i v i d u a l e x p e r i m e n t s .
obtained
Incubation perio, l (rain) Labelled amino acid
L-valine L-phenylalanine L-aspartic acid
Control Experimental Control E x p e r i m e n t al Control Experimental
5
11~
15
25
o.i 47 o. 165 o.162 0.21 o 0.030 0.048
o.293 o.298 0.349 o. 427 0.o6o 0.095
o.327 o.374 0.387 o. 47 o 0.o86 o.I42
o.362 o.353 o.36S 0.33 ~ o.o02 0.088
activity was found after 15 min in experimental samples, some loss of label occurring after this time. The highest radioactivity of experimental samples was only slightly higher than that of the controls. Corresponding results were also obtained when phenylalanine was used in the same way. In this case, however, maximal label was found to be present after 15 min of incubation in both experimental as well as control samples. After this a very slight decline of specific activity was found in control samples, whereas a heavy loss of label was observed in experimental mixtures. The highest specific activity of control samples was distinctly lower than that of experimental ones. The same type of curve was obtained when lysine was used in some experiments instead of phenylalanine. An excellent correlation was found when comparing the extent of transfer of labeled amino acids from p H 5 enzymes to microsomal protein obtained in present
Biochim. Biophys. Acta, 47
(196I) 149 t57
156
j. HRADEC
experiments with results given by HOAGLAND et al. ~. Whereas these authors describe an average transfer of 20 to 30 % of amino acids in this type of experiment, an average transfer of 24 % of valine, 23 % of phenylalanine, and 18 % of lysine was found in the present study. DISCUSSION
Only stimulatory effects on the protein synthesis were caused by Carcinolipin in the present study. The inhibition of the incorporation of labeled lysine and aspartie acid into microsomal protein in the presence of the highest doses of this substance may probably be regarded as a mere non-specific toxic effect of excessive amounts of the active substance. No inhibition was found at lower doses as in our experiments where Ehrlich ascites cells or tissue homogenates were used 2. It is probable that an effect of Carcinolipin on other enzymes, absent from the cell-free system used in the present experiments, might be the reason for a partial inhibition of protein synthesis in whole cells or homogenates under the influence of certain doses of this substance. It is obvious that Carcinolipin is capable of stimulating protein biosynthesis at each step, although distinct quantitative differences exist in its action at various stages of protein synthesis. A striking effect is exhibited by this substance on the amino acid activation whereas only slight stinlulatory effect could be demonstrated on the transfer of amino acids from pH 5 enzymes into microsomes and in particular on the incorporation of labeled amino acids into soluble RNA. It is not clear, however, from the present experiments if Carcinolipin exhibits a direct effect on protein synthesis. Since ATP is necessary for each step of protein synthesis studied here, it cannot be excluded that Carcinolipin acts upon the metabolism of this compound affecting in this way protein synthesis only indirectly 11. However, this does not seem very probable, although Carcinolipin seems to stimulate the adenosinetriphosphatase activity in homogenates to some extent 14. It was shown above that great differences do exist in the stimulating effect of Carcinolipin on amino acid activation varying with the amino acid used. Similarly, varying stimulatory effects were exhibited by this substance at other stages of protein synthesis, depending on the amino acid used. Furthermore, it was shown that Carcinolipin stimulates net-synthesis of serum albumin in liver slices in vitro 14. From this it seems very probable that Carcinolipin is capable of specific stimulation of protein synthesis. It has been shown recently that lipids may play an important role in protein synthesis, although it is not quite clear at this moment if phospholipids 15 or other types of lipids 1~ are involved. The idea that Carcinolipin bears some relation to these substances seems very attractive. The ribose moiety of Carcinolipin li might well take a part in amino acid transport. This possibility is being investigated further. ACKNOWLEDGEMENTS
I wish to express my deepest gratitude to Professor A. HADDOW,who enabled me to obtain a Visiting Scholarship of the Chester Beatty Research Institute, for his encouragement and interest in this work. Also I give my best thanks to Professor J. A. V. BUTLER, who enabled me to do these experiments in his laboratories. I am very grateful to Dr. P. COliN for his many valuable suggestions, discussions and friendly help. Also the valuable advice of Dr. G. D. HUNTER and all other members of the Biochim. Biophys. Acta, 47 (I961) 149-157
CARCINOLIPIN AND PROTEIN SYNTHESIS
157
staff of Pollard's Wood Research Station (of the Institute of Cancer Research, Royal Cancer Hospital) is gratefully acknowledged. This investigation has been supported by grants to the Chester Beatty Research Institute (Institute of Cancer Research, Royal Cancer Hospital) from the British Empire Cancer Campaign, the Jane Coffin Childs Memorial Fund for Medical Research, the Anna Fuller Fund and the National Cancer Institute of the National Institutes of Health, U.S. Public Health Service. The author is a visiting scholar of the Chester Beatty Institute, Institute of Cancer Research, Royal Cancer Hospital. REFERENCES 1 2 3 4 5 6
j . HRADEC, Nature, 182 (1958) 52. j . HRADEC AND A. ~TROUFOV.~ Biochim. Biophys. Acta, 4 ° (196o) 32. j . I4RADEC AND J. I'{RUML, Nature, 185 (196o) 551~. C. ZAMECNIK AND E . B, KELLER, J. Biol. Chem., 209 (I954) 337M. g . HOAGLAND, E . g . KELLER AND P. C.. ZAMECNIK, J . Biol. Chem., 218 (1956) 345. M. B. HOAGLAND, M. L. STEPHENSON, J. F. SCOTT, L. I. HECHT AND P. C. ZAMECNIK, J. Biol.
Chem., 23I (1958) 241. 7 R. B. LOFTFIELI), Progr. in Biophys. and Biophys. Chem., 8 (1957) 347. 8 p . C. ZAMECNIK AND E . B. KELLER, J. Biol. Chem., 221 (1956) 45. 9 R. W. HENDLER, Science, 128 (1958) 143. 10 p. COHN, Biochim. Biophys. Acta, 33 (I959) 28411 p. Cohn, personal communication. 12 p. OHLMEYER, J. Biol. Chem., 19o (1951) 21. 13 1D N. CAMPBELL, personal communication. 14 j . HRADEC, manuscript in preparation.
15 R. W . HENDLER, J. Biol. Chem., 234 (1959) 1466. 1~ j . L. HAINING, T. FUKUI AND 13. AXELROI), J. Biol. Chem., 235 (196o) i6o.
Biochim. Biophys. Acta, 47 (i 961) 149-157