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Fate of glucose in Ascaris lumbricoides
EXPERIMENTAL
PARASITOLOGY
Fate
471-479
of Glucose Nathan
Department
8,
in Ascaris
Entner
of Preventive
(Submitted
(1959)
lumbricoides~
and Celia
Medicine,
New York
New
N.
York,
for publication,
Gonzalez University
Medical School,
Y.
28 June 1958)
It has been shown (Entner, 1957) that enzymes of the pentose-phosphate pathway occur in muscle of Ascaris lumbricoides. Since the enzymes of the glycolytic scheme have also been demonstrated to be present in significant amounts in Ascaris (Bueding and Most, 1953; Rathbone and Reese, 1954), it seemed desirable to determine the relative contribution of each pathway for glucose metabolism in the intact worm. In the present paper, the fate of radioactive glucose, labeled in various positions and supplied to live Ascaris during a 24-hour period, has been followed in an attempt to evaluate the contribution of each of the two pathways. The use of labeled glucose has found wide application in the study of pathways of glucose metabolism by microorganisms, plants and animals (Horecker and Mehler, 1955; Gunsalus et aE., 1955; Racker, 1954; Wood, 1955). In the last cited review, Wood has presented an excellent critical evaluation of the use and interpretation of results obtained with glucose labeled in different positions. The rationale for using glucose labeled in specific carbon atoms is based upon the fact that the fate of the carbon atoms of the hexose molecule is different in the two pathways. In the glycolytic pathway, hexose is split into two equivalent halves; Cl is equivalent to C6, C2 to C5, and C3 to C4, the latter two carbon atoms being the predominant source of carbon dioxide. While in the pentosephosphate pathway, the first three carbon atoms are dissimilar to the last three and Cl is the largest source of COZ . 1 This investigation was carried out under the sponsorship of the Commission on Parasitic Diseases of the Armed Forces Epidemiological Board and was supported by the Office of the Surgeon General, Department of the Army. 471
472
ENTNER MATERIALS
AND GONZALEZ AND METHODS
Pig Ascaris was obtained from the local slaughterhouse. All worms used in experiments were thoroughly washed and kept at 37°C for 24 hours in a salt solution (Cavier and Savel, 1952) maintained sterile by addition of penicillin, streptomycin and gantrisine (3,4-dimethyl-5sulfanilamido-isosazole; also sulfasoxazole). At the end of this period the worms, only sterile ones being used, were placed in fresh salt-antibiotic media and were allowed to incubate an additional 24 hours in the presence of radioactive glucose, either uniformly labeled (Gl-U-C14), or labeled in the 1, 2 or 6 positions (Gl-l-Ci4, Gl-2-Cl4 or Gl-6-C”). After the second day the worms were removed, washed and either fractionated or frozen immediately for subsequent fractionation. The incubation medium, after removal of a small aliquot for carbohydrate and total acid determinations, was concentrated under a stream of air in a warm water bath, acidified and the fermentation acids were then extracted into ether, Volatile fatty acids were further separated on buffered celite columns (Bueding and Yale, 1951), and the fractions titrated with .Ol N NaOH. Degradation of acetic acid was accomplished by the method described by Abraham and Hassid (1957). It was determined with known samples of uniformly labeled acetate and methyl labeled acetate that no cross contamination of the methyl and carboxyl groups occurred. The method resulted in yields of 90 % or above of the carboxyl group and approximately 80 % of the methyl group. All of the methods used for fractionation of the helminths are described in Methods of Enzymology, Vol. III (Colowick and Kaplan, 1957). The worms were minced and homogenized with 3 volumes of .04 M Tris buffer, pH 8.9 in an ice bath. An aliquot was removed for determination of glycogen. The sample was hydrolyzed in a boiling water bath for 30 min with 2 volumes of 30% KOH. The glycogen was precipitated with 60% (final volume) ethanol, washed several times with 60% ethanol, dissolved and determined calorimetrically with anthrone in sulfuric acid. Alternatively, a separate batch of worms was used for glycogen, when it was found that the amount of incorporation of labeled sugar into glycogen was a constant function of the amount of sugar used. The remainder of the homogenate was treated with 3 volumes of cold 10% trichloracetic acid (TCA). The precipitate that formed contained protein, lipid, nucleic acid and some glycogen. A total of 10 washings of the precipitate with cold 10% TCA (8X) and 5 % TCA (2X) removed
FATE OF GLUCOSE IN ASCARIS LUMBRICOIDES
473
all traces of glycogen. The combined TCA extracts represented “acid solubles,,’ after removal of glycogen with ethanol precipitation. The TCA precipitated material was then washed once with a small amount of ethanol, and lipid was removed first by refluxing with 200 ml of ethanol:ether (3: 1) for 2 hours, and then with chloroform:methano1 (1: 1) for one hour. The combined supernatant solvents were concentrated, and lipid was determined gravimetrically. Nucleic acid was extracted from the remaining precipitate with hot 10 % NaCl, three extractions each of one hour duration being conducted in a boiling water bath. The nucleic acid was precipitated with 2 volumes of 95 % ethanol, washed and redissolved. RNA was determined with orcinol, and purified yeast RNA served as a standard. Most of the nucleic acid was RNA, and tests with diphenylamine showed that DNA was present in relatively insignificant amounts. The precipitate remaining after the hot NaCl extraction was taken to represent protein. It was washed with 95 % ethanol, then ether, dried in vacua and finally weighed. Respiratory carbon dioxide was collected in separate flasks containing a test tube in which COzfree KOH had been added. At the end of each experiment the medium was acidified by insertion of a separate tube containing acid, and the flask was sealed and shaken for 15 min. The COz was converted to barium carbonate, and the latter was washed free of excess alkali and barium ions by several washings with Cot-free water. Radioactivity of samples, plated and dried on stainless steel planchets, was determined with a thin-window, gas-flow Geiger tube. Acids were plated as the sodium salts and COz as barium carbonate. Special care was taken to determine the weight per unit area plated in the case of glycogen, BaC03 and protein, so that corrections for self-absorption could be made. RESULTS
Early experiments indicated that when Ascuris is supplied with labeled glucose, radioactive carbon enters into every major fraction of the helminth, and the highest amount of incorporation is in glycogen and the fermentation acids. In most carbohydrate fermentations, the fermentation products represent reduced carbon originating from the carbohydrate. Since the acids produced by Ascaris appeared to be suitable material for comparing differences in distribution of labeled carbon atoms, an experiment was first designed to demonstrate whether glucose can
474
ENTNER
AND
GONZALEZ
TABLE I Substrate (am% worm)
1. Labeled glucose (8.7 g)
Total activity of glycogen cts/min
Total activity of acids cts/min (am’t of acid)
1st 24 hrs 31,400
11,300 (.83 mM)
2nd 24 hrs 2. Labeled glucose (6.9 g) 3. Unlabeled glucose (7.5 d 4. Unlabeled glucose (7.4 d 5. No glucose (6.1 d
57,400
11,348 (.82 mM) 1,731 (.73 mM) 2,178 (.91 mM) 5,444 (.59 mM)
30,300 20,400 6,800
Each flask containing 4 wormswassuppliedwith 400 mg glucose(in 100ml) except in Expt. %5, 2nd 24 hours. The total radioactivity in the addedlabeled sugarwas83,000cts/min.
serve as a direct source of fatty acids, and that it does not at first enter into some kind of metabolic pool. The experiments in Table I show that a large part of the acids does arise directly from glucose. In these experiments, the worms were first
allowed to become radioactive by incubation with Gl-U-Cl4 for a period of 24 hours. They were then carefully washed and re-incubated for an additional 24 hours with radioactive, unlabeled or no glucose. It is clearly shown that a significant part of the acids come directly from glucose and only a small amount from radioactive carbon in the worm unless glucose is omitted (Expt. 5). The experiments further indicate that glucose markedly inhibits the utilization of already synthesized glycogen, and that glycogen synthesis continues at an almost constant rate.
A more complete investigation
of the distribution
patterns of carbon
atoms from glucose labeled in various positions is shoa
III
and IV. The worms and incubation
in Tables II,
media were fractionated
and
fractions measured according to procedures described under “Methods”.
It soon became apparent that the relative weights of the worms could not serve as a comparison for activity. Slender, non-egglaying, female worms were used almost exclusively. Smaller worms as a rule have rela-
FATE
OF
GLUCOSE
IN
ASCARIS
475
LUMBRICOIDES
tively more protein, less body fluid and considerably less reproductive tract, and on a unit weight basis they are more active metabolically than large worms. We felt that a more comparable reflection of distribution patterns of different carbon atoms from glucose could be expressed on the basis of the amount of consumed glucose. This is supported to some extent by the interesting finding that in every case, very nearly half of the consumed glucose is incorporated into glycogen, independently of the weight of the worms. The results in Table II are, therefore, expressed in terms of amount of radioactive glucose used. Table III represents the further fractionation of the volatile fatty acids. Apparently, some variation in the amounts of fatty acid occurs, particularly in the C-2 and C-5 acids. On the whole, there appears to be a distinct difference in the patterns between the products from Gl-l-Cl4 and those from Gl-6-CY4. TABLE
II
Fractionation of ASCARIS After Feeding on Radioactive Glucose GOT24 HOUTS The amount of radioactivity (cts/min) appearing in each fraction is based on 100,000 cts/min of glucose consumed. Labeled sugar added.. Number and (wt) of worms in experiment.. Amount and (cts/min) of glucose consumed. .
Glycogen............... .
Protein. Lipid. Nucleic
acid..
Acid solubles. co2 ..
Gl-1-W
Gi-204
4 (7.5 g)
4 (5.0 d
3 (4.92 g)
3 (6.25 g)
1.67 mil4 (60,000)
1.33 m.M (63,100)
0.95 mA4 (34,800)
1.83 m&f (87,500)
Radioactivity
Fraction
Acids
Gl-U-W
52,200 (389 w) 16,900 (0.83 mM) 4,410 (895 w) 714 (33 mid 5,310 (24.2 mg) 3,920 8,100
in cts/min (Total of each fraction)
49,100
(272md 18,300 (0.73 mill) 10,900 (518 md 2,120 (32md 206 (4.8 md 4,660 2,540
52,000 (440 mg) 24,800 (0.63 mill) 15,700 (678 me) (20 rng: 947 (3.6 w) 5,150 1,170
Gl&C’4
amount
49,200
(342w) 20,700 (0.79 m&f) 5,700 (475 mg) 147 (44 md 1,056
(8.0 md 4,460 1,290
476
ENTNER
AND
GONZALEZ
TABLE III Fatty Acid Fractionation appearing in each fraction total fatty acids.
Amount
of radioactivity
Fraction
GI-U-C”
Gl-1-C”
for each 1,000 cts of
Gl-6-C’”
ci-2-c"
Expt.
C6 c5 c4 c3 c2
I
II
I
II
I
II
I
II
205 405 87 205 102
258 384 43 203 118
198 420 42 187 153
191 350 49 232 177
304 242 75 127 249
307 408 72 138 73
270 288 65 104 272
328 410 77 109 76
TABLE Per Cent Radioactivity
Gl-U-C” Gl-1-C” Gl-2-C” Gl-6-C”
IV in Acetate Carbon* COOH
CHa
50 27 65 31.5
50 67.5 36 65.5
* Each value represents the average of two determinations.
The high labeling of the CO2 from Gl-U-C14, in comparison to CO2 from other labeled carbons, presumably arises from the 3 and 4 carbon atoms of glucose and indicates a predominant functioning of glycolysis. The amount of labeling in the methyl group of acetate from Gl-l-Cl4 and Gl-6-Cl4 (Table IV) would be a further indication of the extent of activity of the glycolytic pathway. While the 1 and 6 carbon atoms of glucose gives rise to the major portion of the methyl group of acetate, there also seemsto be considerable randomnization. DISCUSSION
In someorganisms, the distribution of carbon from radioactive glucose gives a clear picture of the pathway of glucose metabolism involved. For example, in the lactobacilli, each particular carbon atom of glucose appears almost exclusively in one position of a product. Thus, it has been
FATE
OF
GLUCOSE
IN
ASCARIS
LUMBRICOIDES
477
established that the homofermentative lactobacilli use the glycolytic scheme exclusively (Gibbs et al., 1950), while in heterofermentative lactobacilli only the pentose-phosphate pathway functions (Gunsalus and Gibbs, 1952). In other organisms, such as the propionic acid bacteria, in which there is considerable mixing of labeled carbon, and in animals in which there are no definite fermentation products, the interpretation of results is attended with considerable uncertainty (Wood, 1955; Wood et al., 1955). In Ascaris fermentations the labeling in acetate and COZ is somewhat similar to what has been found in the propionic bacteria. The predominance of the methyl group from l- and 6-labeled glucose and carboxyl from 2-labeled glucose is in accordance with the glycolytic pathway, while the presence of labeling in the alternate carbon can be due to one of several possibilities. There may be some functioning of the pentosephosphate pathway2 or there may be some kind of symmetrical intermediate. In the propionic acid bacteria, in which both acetate and propionate carbons are randomnized, it has been shown that propionate is in equilibrium with symmetrical succinic acid by virtue of the reversible addition of an active form of CO2 . Another possibility is a rapid COz fixation mechanism such as has been shown to occur in Ascuris (Saz and Hubbard, 1957). It has also been pointed out by Wood (1955) that the two trioses from glycolysis may not be in equilibrium if limiting amounts of triose-phosphate isomerase are present. That the pentose-phosphate functions in live Ascuris for the synthesis of nucleic acid pentose is indicated by the relatively low activity of the nucleic acid from Gl-l-C14. Another important role which the pentosephosphate pathway no doubt plays in Ascaris is the formation of reduced TPN (Hiatt et al., 1958), which among other things is an absolute requirement for lipid synthesis. One of the more constant and interesting findings is the amount of incorporation of radioactive glucose into glycogen. The results have been obtained several times, and without exception very nearly half of the 2 In the previous paper already cited (Entner, 1957), we were unable to report the demonstration of transaldolase, an enzyme which imparts to the pentose-phosphate a cyclic process which can in turn account for mixing of labeled carbon. We have since been able to demonstrate transaldolase by employing the reverse reaction, namely, by observing the formation of sedoheptulose-phosphate when fructose-phosphate and erythrose-phosphate are incubated with muscle extracts of Ascaris. The authors are indebted to Dr. David B. Sprinson for a sample of erythrosel-phosphate.
478
ENTNER
AND GONZALEZ
glucose consumed is incorporated into glycogen. It would appear that with the use of radioactive sugar, Ascaris could almost serve as a standard for the study of glycogen synthesis. There appears to be a relatively high incorporation of labeled carbon from glucose into the protein fraction. It was not determined whether the radioactive carbon was in amino acids or was the non-nitrogenous part of some conjugated proteins. SUMMARY
1. Radioactive glucose has been given to Ascaris lumbricoides in sterile salt solution, and the fate of the labeled carbons have been followed. 2. Cl4 from glucose enters into every major fraction of Ascaris and is highest in glycogen and acids. Half of the sugar consumed is incorporated into glycogen, and a significant part of the acids come directly from glucose. 3. Distribution patterns were observed with Gl-U-CY4, Gl-l-C4, Gl2-P and Gl-6-C14. The labeling of the CO1 and acetate indicates the major pathway in Ascaris to be the glycolytic pathway. However, the difference in patterns from Gl-l-Cl4 and Cl-6-CY4, and a considerable amount of randomnization in acetate carbons, indicate a small contribution by the pentose-phosphate pathway and possibly the functioning of other mechanisms. ACKNOWLEDGMENT The authors would like to express their thanks to Dr. S. Abraham for his generous help with suggestions and loan of equipment for acetate degradations. REFERENCES S., AND HASSID, W. Z. 1957. The synthesis and degradation of isotopitally labeled carbohydrates and carbohydrate intermediates. Methods in Enzymology. (Ed. by Colowick, S. and Kaplan, N.) Vol. IV, pp. 533-535. BUEDING, E., AND MOST, H. 1953. Helminths: metabolism, nutrition, and chemotherapy. Ann. Rev. Microbial. 7, 295-326. BUEDING, E., AND YALE, H. W. 1951. Production of d-methyl-butyric acid by bacteria-free Ascaris lumbricoides. J. Biol. Chem. 193,411-423. CAMER, R., AND SAVEL, J. 1952. Etude des conditions de vie de 1’ Ascaris du Port, Ascaris lumbricoides Linne 1958. Compt. rend. X%,1216-1218. COLOWICK, S. P., AND KAPLAN, N. 0. (Eds.). 1957. Methods in Enzymology. Vol. III. Academic Press Inc., New York. ENTNER, N. 1957. The occurrence of the pentose-phosphate pathway in Ascaris lumbricoides. Arch. Biochem. Biophys. 71,52-61. ABRAHAM,
FATE
OF
GLUCOSE
IN
ASCARIS
LUMBRICOIDES
479
M., DUMROSE, R., BENNETT, F. A., AND BUBECH, M. R. 1950. On the mechanism of bacterial fermentation of glucose to lactic acid studied with C14-glucose. J. Biol. Chem. l&545-549. GUNSALUS, I. C., AND GIBBS, M. 1952. The heterolactic fermentation. II. Position of Cl4 in the products of glucose dissimilation by Leuconostoc mesenteroides J. Biol. Chem. 194, 871-875. GUNSALUS, I. C., HORECKER, B. L., AND WOOD, W. A. 1955. Pathways of carbohydrate metabolism in microorganisms. Bact. Revs. 19, 79-129. HIATT, H. H., GOLDSTEIN, M., LAREAU, J., AND HORECKER, B. L. 1958. The pathway of hexose synthesis from pyruvate in muscle. J. Biol. Chem. 231,303-307. HORECKER, B. L., AND MEHLER, A. H. 1955. Carbohydrate metabolism. Ann. Rev. GIBBS,
Biochem. 24, 207-274. RACKER, E. 1954. Alternate pathways of glucose and fructose metabolism. Advances in Enzymol. l&141-182. RATHBONE, L., AND REES, K. R. 1954. Glycolysis in Ascaris lumbricoides from the pig. Biochem. et Biophys. Acta l&126-133. SAZ, H. J., AND HUBBARD, J. A. 1957. The oxidative decarboxylation of malate by Ascaris lumbricoides. J. Biol. Chem. 226, 921-933. WOOD, H. G. 1955. Significance of alternate pathways in the metabolism of glucose. Physiol. Revs. 36, 841-359. WOOD, H. G., STJERNHOLM, R., AND LEAVER, F. W. 1955. The metabolism of labeled glucose by the propionic acid bacteria. J. Bact. 70, 510-520.
PARASITOLOGY
Fate
471-479
of Glucose Nathan
Department
8,
in Ascaris
Entner
of Preventive
(Submitted
(1959)
lumbricoides~
and Celia
Medicine,
New York
New
N.
York,
for publication,
Gonzalez University
Medical School,
Y.
28 June 1958)
It has been shown (Entner, 1957) that enzymes of the pentose-phosphate pathway occur in muscle of Ascaris lumbricoides. Since the enzymes of the glycolytic scheme have also been demonstrated to be present in significant amounts in Ascaris (Bueding and Most, 1953; Rathbone and Reese, 1954), it seemed desirable to determine the relative contribution of each pathway for glucose metabolism in the intact worm. In the present paper, the fate of radioactive glucose, labeled in various positions and supplied to live Ascaris during a 24-hour period, has been followed in an attempt to evaluate the contribution of each of the two pathways. The use of labeled glucose has found wide application in the study of pathways of glucose metabolism by microorganisms, plants and animals (Horecker and Mehler, 1955; Gunsalus et aE., 1955; Racker, 1954; Wood, 1955). In the last cited review, Wood has presented an excellent critical evaluation of the use and interpretation of results obtained with glucose labeled in different positions. The rationale for using glucose labeled in specific carbon atoms is based upon the fact that the fate of the carbon atoms of the hexose molecule is different in the two pathways. In the glycolytic pathway, hexose is split into two equivalent halves; Cl is equivalent to C6, C2 to C5, and C3 to C4, the latter two carbon atoms being the predominant source of carbon dioxide. While in the pentosephosphate pathway, the first three carbon atoms are dissimilar to the last three and Cl is the largest source of COZ . 1 This investigation was carried out under the sponsorship of the Commission on Parasitic Diseases of the Armed Forces Epidemiological Board and was supported by the Office of the Surgeon General, Department of the Army. 471
472
ENTNER MATERIALS
AND GONZALEZ AND METHODS
Pig Ascaris was obtained from the local slaughterhouse. All worms used in experiments were thoroughly washed and kept at 37°C for 24 hours in a salt solution (Cavier and Savel, 1952) maintained sterile by addition of penicillin, streptomycin and gantrisine (3,4-dimethyl-5sulfanilamido-isosazole; also sulfasoxazole). At the end of this period the worms, only sterile ones being used, were placed in fresh salt-antibiotic media and were allowed to incubate an additional 24 hours in the presence of radioactive glucose, either uniformly labeled (Gl-U-C14), or labeled in the 1, 2 or 6 positions (Gl-l-Ci4, Gl-2-Cl4 or Gl-6-C”). After the second day the worms were removed, washed and either fractionated or frozen immediately for subsequent fractionation. The incubation medium, after removal of a small aliquot for carbohydrate and total acid determinations, was concentrated under a stream of air in a warm water bath, acidified and the fermentation acids were then extracted into ether, Volatile fatty acids were further separated on buffered celite columns (Bueding and Yale, 1951), and the fractions titrated with .Ol N NaOH. Degradation of acetic acid was accomplished by the method described by Abraham and Hassid (1957). It was determined with known samples of uniformly labeled acetate and methyl labeled acetate that no cross contamination of the methyl and carboxyl groups occurred. The method resulted in yields of 90 % or above of the carboxyl group and approximately 80 % of the methyl group. All of the methods used for fractionation of the helminths are described in Methods of Enzymology, Vol. III (Colowick and Kaplan, 1957). The worms were minced and homogenized with 3 volumes of .04 M Tris buffer, pH 8.9 in an ice bath. An aliquot was removed for determination of glycogen. The sample was hydrolyzed in a boiling water bath for 30 min with 2 volumes of 30% KOH. The glycogen was precipitated with 60% (final volume) ethanol, washed several times with 60% ethanol, dissolved and determined calorimetrically with anthrone in sulfuric acid. Alternatively, a separate batch of worms was used for glycogen, when it was found that the amount of incorporation of labeled sugar into glycogen was a constant function of the amount of sugar used. The remainder of the homogenate was treated with 3 volumes of cold 10% trichloracetic acid (TCA). The precipitate that formed contained protein, lipid, nucleic acid and some glycogen. A total of 10 washings of the precipitate with cold 10% TCA (8X) and 5 % TCA (2X) removed
FATE OF GLUCOSE IN ASCARIS LUMBRICOIDES
473
all traces of glycogen. The combined TCA extracts represented “acid solubles,,’ after removal of glycogen with ethanol precipitation. The TCA precipitated material was then washed once with a small amount of ethanol, and lipid was removed first by refluxing with 200 ml of ethanol:ether (3: 1) for 2 hours, and then with chloroform:methano1 (1: 1) for one hour. The combined supernatant solvents were concentrated, and lipid was determined gravimetrically. Nucleic acid was extracted from the remaining precipitate with hot 10 % NaCl, three extractions each of one hour duration being conducted in a boiling water bath. The nucleic acid was precipitated with 2 volumes of 95 % ethanol, washed and redissolved. RNA was determined with orcinol, and purified yeast RNA served as a standard. Most of the nucleic acid was RNA, and tests with diphenylamine showed that DNA was present in relatively insignificant amounts. The precipitate remaining after the hot NaCl extraction was taken to represent protein. It was washed with 95 % ethanol, then ether, dried in vacua and finally weighed. Respiratory carbon dioxide was collected in separate flasks containing a test tube in which COzfree KOH had been added. At the end of each experiment the medium was acidified by insertion of a separate tube containing acid, and the flask was sealed and shaken for 15 min. The COz was converted to barium carbonate, and the latter was washed free of excess alkali and barium ions by several washings with Cot-free water. Radioactivity of samples, plated and dried on stainless steel planchets, was determined with a thin-window, gas-flow Geiger tube. Acids were plated as the sodium salts and COz as barium carbonate. Special care was taken to determine the weight per unit area plated in the case of glycogen, BaC03 and protein, so that corrections for self-absorption could be made. RESULTS
Early experiments indicated that when Ascuris is supplied with labeled glucose, radioactive carbon enters into every major fraction of the helminth, and the highest amount of incorporation is in glycogen and the fermentation acids. In most carbohydrate fermentations, the fermentation products represent reduced carbon originating from the carbohydrate. Since the acids produced by Ascaris appeared to be suitable material for comparing differences in distribution of labeled carbon atoms, an experiment was first designed to demonstrate whether glucose can
474
ENTNER
AND
GONZALEZ
TABLE I Substrate (am% worm)
1. Labeled glucose (8.7 g)
Total activity of glycogen cts/min
Total activity of acids cts/min (am’t of acid)
1st 24 hrs 31,400
11,300 (.83 mM)
2nd 24 hrs 2. Labeled glucose (6.9 g) 3. Unlabeled glucose (7.5 d 4. Unlabeled glucose (7.4 d 5. No glucose (6.1 d
57,400
11,348 (.82 mM) 1,731 (.73 mM) 2,178 (.91 mM) 5,444 (.59 mM)
30,300 20,400 6,800
Each flask containing 4 wormswassuppliedwith 400 mg glucose(in 100ml) except in Expt. %5, 2nd 24 hours. The total radioactivity in the addedlabeled sugarwas83,000cts/min.
serve as a direct source of fatty acids, and that it does not at first enter into some kind of metabolic pool. The experiments in Table I show that a large part of the acids does arise directly from glucose. In these experiments, the worms were first
allowed to become radioactive by incubation with Gl-U-Cl4 for a period of 24 hours. They were then carefully washed and re-incubated for an additional 24 hours with radioactive, unlabeled or no glucose. It is clearly shown that a significant part of the acids come directly from glucose and only a small amount from radioactive carbon in the worm unless glucose is omitted (Expt. 5). The experiments further indicate that glucose markedly inhibits the utilization of already synthesized glycogen, and that glycogen synthesis continues at an almost constant rate.
A more complete investigation
of the distribution
patterns of carbon
atoms from glucose labeled in various positions is shoa
III
and IV. The worms and incubation
in Tables II,
media were fractionated
and
fractions measured according to procedures described under “Methods”.
It soon became apparent that the relative weights of the worms could not serve as a comparison for activity. Slender, non-egglaying, female worms were used almost exclusively. Smaller worms as a rule have rela-
FATE
OF
GLUCOSE
IN
ASCARIS
475
LUMBRICOIDES
tively more protein, less body fluid and considerably less reproductive tract, and on a unit weight basis they are more active metabolically than large worms. We felt that a more comparable reflection of distribution patterns of different carbon atoms from glucose could be expressed on the basis of the amount of consumed glucose. This is supported to some extent by the interesting finding that in every case, very nearly half of the consumed glucose is incorporated into glycogen, independently of the weight of the worms. The results in Table II are, therefore, expressed in terms of amount of radioactive glucose used. Table III represents the further fractionation of the volatile fatty acids. Apparently, some variation in the amounts of fatty acid occurs, particularly in the C-2 and C-5 acids. On the whole, there appears to be a distinct difference in the patterns between the products from Gl-l-Cl4 and those from Gl-6-CY4. TABLE
II
Fractionation of ASCARIS After Feeding on Radioactive Glucose GOT24 HOUTS The amount of radioactivity (cts/min) appearing in each fraction is based on 100,000 cts/min of glucose consumed. Labeled sugar added.. Number and (wt) of worms in experiment.. Amount and (cts/min) of glucose consumed. .
Glycogen............... .
Protein. Lipid. Nucleic
acid..
Acid solubles. co2 ..
Gl-1-W
Gi-204
4 (7.5 g)
4 (5.0 d
3 (4.92 g)
3 (6.25 g)
1.67 mil4 (60,000)
1.33 m.M (63,100)
0.95 mA4 (34,800)
1.83 m&f (87,500)
Radioactivity
Fraction
Acids
Gl-U-W
52,200 (389 w) 16,900 (0.83 mM) 4,410 (895 w) 714 (33 mid 5,310 (24.2 mg) 3,920 8,100
in cts/min (Total of each fraction)
49,100
(272md 18,300 (0.73 mill) 10,900 (518 md 2,120 (32md 206 (4.8 md 4,660 2,540
52,000 (440 mg) 24,800 (0.63 mill) 15,700 (678 me) (20 rng: 947 (3.6 w) 5,150 1,170
Gl&C’4
amount
49,200
(342w) 20,700 (0.79 m&f) 5,700 (475 mg) 147 (44 md 1,056
(8.0 md 4,460 1,290
476
ENTNER
AND
GONZALEZ
TABLE III Fatty Acid Fractionation appearing in each fraction total fatty acids.
Amount
of radioactivity
Fraction
GI-U-C”
Gl-1-C”
for each 1,000 cts of
Gl-6-C’”
ci-2-c"
Expt.
C6 c5 c4 c3 c2
I
II
I
II
I
II
I
II
205 405 87 205 102
258 384 43 203 118
198 420 42 187 153
191 350 49 232 177
304 242 75 127 249
307 408 72 138 73
270 288 65 104 272
328 410 77 109 76
TABLE Per Cent Radioactivity
Gl-U-C” Gl-1-C” Gl-2-C” Gl-6-C”
IV in Acetate Carbon* COOH
CHa
50 27 65 31.5
50 67.5 36 65.5
* Each value represents the average of two determinations.
The high labeling of the CO2 from Gl-U-C14, in comparison to CO2 from other labeled carbons, presumably arises from the 3 and 4 carbon atoms of glucose and indicates a predominant functioning of glycolysis. The amount of labeling in the methyl group of acetate from Gl-l-Cl4 and Gl-6-Cl4 (Table IV) would be a further indication of the extent of activity of the glycolytic pathway. While the 1 and 6 carbon atoms of glucose gives rise to the major portion of the methyl group of acetate, there also seemsto be considerable randomnization. DISCUSSION
In someorganisms, the distribution of carbon from radioactive glucose gives a clear picture of the pathway of glucose metabolism involved. For example, in the lactobacilli, each particular carbon atom of glucose appears almost exclusively in one position of a product. Thus, it has been
FATE
OF
GLUCOSE
IN
ASCARIS
LUMBRICOIDES
477
established that the homofermentative lactobacilli use the glycolytic scheme exclusively (Gibbs et al., 1950), while in heterofermentative lactobacilli only the pentose-phosphate pathway functions (Gunsalus and Gibbs, 1952). In other organisms, such as the propionic acid bacteria, in which there is considerable mixing of labeled carbon, and in animals in which there are no definite fermentation products, the interpretation of results is attended with considerable uncertainty (Wood, 1955; Wood et al., 1955). In Ascaris fermentations the labeling in acetate and COZ is somewhat similar to what has been found in the propionic bacteria. The predominance of the methyl group from l- and 6-labeled glucose and carboxyl from 2-labeled glucose is in accordance with the glycolytic pathway, while the presence of labeling in the alternate carbon can be due to one of several possibilities. There may be some functioning of the pentosephosphate pathway2 or there may be some kind of symmetrical intermediate. In the propionic acid bacteria, in which both acetate and propionate carbons are randomnized, it has been shown that propionate is in equilibrium with symmetrical succinic acid by virtue of the reversible addition of an active form of CO2 . Another possibility is a rapid COz fixation mechanism such as has been shown to occur in Ascuris (Saz and Hubbard, 1957). It has also been pointed out by Wood (1955) that the two trioses from glycolysis may not be in equilibrium if limiting amounts of triose-phosphate isomerase are present. That the pentose-phosphate functions in live Ascuris for the synthesis of nucleic acid pentose is indicated by the relatively low activity of the nucleic acid from Gl-l-C14. Another important role which the pentosephosphate pathway no doubt plays in Ascaris is the formation of reduced TPN (Hiatt et al., 1958), which among other things is an absolute requirement for lipid synthesis. One of the more constant and interesting findings is the amount of incorporation of radioactive glucose into glycogen. The results have been obtained several times, and without exception very nearly half of the 2 In the previous paper already cited (Entner, 1957), we were unable to report the demonstration of transaldolase, an enzyme which imparts to the pentose-phosphate a cyclic process which can in turn account for mixing of labeled carbon. We have since been able to demonstrate transaldolase by employing the reverse reaction, namely, by observing the formation of sedoheptulose-phosphate when fructose-phosphate and erythrose-phosphate are incubated with muscle extracts of Ascaris. The authors are indebted to Dr. David B. Sprinson for a sample of erythrosel-phosphate.
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glucose consumed is incorporated into glycogen. It would appear that with the use of radioactive sugar, Ascaris could almost serve as a standard for the study of glycogen synthesis. There appears to be a relatively high incorporation of labeled carbon from glucose into the protein fraction. It was not determined whether the radioactive carbon was in amino acids or was the non-nitrogenous part of some conjugated proteins. SUMMARY
1. Radioactive glucose has been given to Ascaris lumbricoides in sterile salt solution, and the fate of the labeled carbons have been followed. 2. Cl4 from glucose enters into every major fraction of Ascaris and is highest in glycogen and acids. Half of the sugar consumed is incorporated into glycogen, and a significant part of the acids come directly from glucose. 3. Distribution patterns were observed with Gl-U-CY4, Gl-l-C4, Gl2-P and Gl-6-C14. The labeling of the CO1 and acetate indicates the major pathway in Ascaris to be the glycolytic pathway. However, the difference in patterns from Gl-l-Cl4 and Cl-6-CY4, and a considerable amount of randomnization in acetate carbons, indicate a small contribution by the pentose-phosphate pathway and possibly the functioning of other mechanisms. ACKNOWLEDGMENT The authors would like to express their thanks to Dr. S. Abraham for his generous help with suggestions and loan of equipment for acetate degradations. REFERENCES S., AND HASSID, W. Z. 1957. The synthesis and degradation of isotopitally labeled carbohydrates and carbohydrate intermediates. Methods in Enzymology. (Ed. by Colowick, S. and Kaplan, N.) Vol. IV, pp. 533-535. BUEDING, E., AND MOST, H. 1953. Helminths: metabolism, nutrition, and chemotherapy. Ann. Rev. Microbial. 7, 295-326. BUEDING, E., AND YALE, H. W. 1951. Production of d-methyl-butyric acid by bacteria-free Ascaris lumbricoides. J. Biol. Chem. 193,411-423. CAMER, R., AND SAVEL, J. 1952. Etude des conditions de vie de 1’ Ascaris du Port, Ascaris lumbricoides Linne 1958. Compt. rend. X%,1216-1218. COLOWICK, S. P., AND KAPLAN, N. 0. (Eds.). 1957. Methods in Enzymology. Vol. III. Academic Press Inc., New York. ENTNER, N. 1957. The occurrence of the pentose-phosphate pathway in Ascaris lumbricoides. Arch. Biochem. Biophys. 71,52-61. ABRAHAM,
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M., DUMROSE, R., BENNETT, F. A., AND BUBECH, M. R. 1950. On the mechanism of bacterial fermentation of glucose to lactic acid studied with C14-glucose. J. Biol. Chem. l&545-549. GUNSALUS, I. C., AND GIBBS, M. 1952. The heterolactic fermentation. II. Position of Cl4 in the products of glucose dissimilation by Leuconostoc mesenteroides J. Biol. Chem. 194, 871-875. GUNSALUS, I. C., HORECKER, B. L., AND WOOD, W. A. 1955. Pathways of carbohydrate metabolism in microorganisms. Bact. Revs. 19, 79-129. HIATT, H. H., GOLDSTEIN, M., LAREAU, J., AND HORECKER, B. L. 1958. The pathway of hexose synthesis from pyruvate in muscle. J. Biol. Chem. 231,303-307. HORECKER, B. L., AND MEHLER, A. H. 1955. Carbohydrate metabolism. Ann. Rev. GIBBS,
Biochem. 24, 207-274. RACKER, E. 1954. Alternate pathways of glucose and fructose metabolism. Advances in Enzymol. l&141-182. RATHBONE, L., AND REES, K. R. 1954. Glycolysis in Ascaris lumbricoides from the pig. Biochem. et Biophys. Acta l&126-133. SAZ, H. J., AND HUBBARD, J. A. 1957. The oxidative decarboxylation of malate by Ascaris lumbricoides. J. Biol. Chem. 226, 921-933. WOOD, H. G. 1955. Significance of alternate pathways in the metabolism of glucose. Physiol. Revs. 36, 841-359. WOOD, H. G., STJERNHOLM, R., AND LEAVER, F. W. 1955. The metabolism of labeled glucose by the propionic acid bacteria. J. Bact. 70, 510-520.