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Studies on the intermediary metabolism of the aquatic snail, Australorbis glabratus
Studies on the Intermediary Metabolism of the Aquatic Snail, Australorbis glabratus’ Eugene C. Weinbach’ From
the Federal Security Agency, Service, National Institutes Microbiological Institute,3 Received
United States Public of Health, National Bethesda, Maryland
October
Health
7. 1952
INTRODUCTION
Fundamental studies relating to comparative cellular metabolism among a wide variety of organisms are of intrinsic interest for biochemistry and underlie the approach to many practical problems. Thus the importance of a rational approach to the problem of eradicating tremat’ode diseases by a systematic study of the physiology of their intermediate hosts has been pointed out by von Brand, Nolan, and Mann (1). Such an approach includes studies of metabolic pathways which, theoretically at least, may be found vulnerable to attack by chemical agents. Previous studies, employing intact, living snails to investigate the mode of action of certain molluscacides, indicated the need for investigation on the cellular level (2). The present paper reports the results of such an investigation. It is concerned mainly with aerobic metabolic activities of minced tissues of Australorbis glabratus, the intermediate snail host of the human blood fluke SchistosomamansoG, in the West Indies and South America. METHODS ~1~t.sfralorbi.s glabrafus, the only species of snail used for this study, u-as lab orat’org-reared from Venezuelan specimens and was kept in balanced aquaria in :t room with a minimum temperature of 21°C. The snails were fed abundantI) wit’h let,tuce leaves and fish food, with the occasional addition of calcium car’ Presented in part at the forty-third annual meeting of Biological Chemists at Nea York City (la). 2 With the technical assistance of Rachel Al. Dudley. 3 T,nt)oratory of Tropical Diseases. 231
of the
American
Societ>
232
EUGENE
C.
WEINBACH
bonate to the water.4 Experiments with the intact, living snails were carried out under the same conditions described by von Brand, Nolan, and Mann (1). Minces and homogenates were prepared in the following manner: Specimens were transferred from aquaria to a chilled Petri dish, the shell was carefully cracked open, and the entire soft tissues were removed. After sufficient material had been collected (the tissues were kept moist during the collection by a dampened piece of filter paper attached to the underside of an inverted watch glass), it was minced freehand by means of two scalpels, one held in each hand. Aliquots of the tissue were weighed immediately on a torsion balance and transferred to Warburg vessels which had been prepared previously to contain all necessary components except the tissue; or if the homogenates were desired, weighed amounts of the minced tissue were homogenized in the Potter-Elvehjem apparatus (3). All metabolic activities are expressed in terms of the initial dry weights calculated from the initial wet weights. During the course of these experiments numerous samples were taken to dryness to determine the initial moisture content. This value usually averaged 30% of the total weight; hence in those manometric experiments where a separate dry weight determination was not made, an initial moisture content of 80% was assumed. Early experiments demonstrated that the rate of endogenous metabolic activity was dependent upon the amount of minced tissue present in each vessel; thus enzyme activity was proportional to tissue concentration. Unless noted otherwise, each vessel contained approximately 200 mg. (wet weight) of tissue. This amount of tissue was adopted as a working standard because it usually provided sufficient metabolic activities which could be measured conveniently. Oxygen consumption was measured manometrically in Warburg respirometers at a standard temperature of 3O”C., except where otherwise indicated. In all aerobic experiments atmospheric air served as the gas phase, except when the influence of oxygen tension was studied. Anaerobic conditions were obtained by passing a stream of nitrogen (Linde 99.990/,) over heated copper and through the shaking respirometers for 20 min. Preliminary experiments employing a variety of suspending media (ranging from simple dechlorinated tap water to KrebsRinger phosphate) indicated little dependence of the metabolic activities under study upon the media. Except as noted, each vessel contained 1.0 ml. of 0.1 111 phosphate buffer (pH 7.4) plus other components as indicated to make a final volume of 3.0 ml. Protein-free aliquots were obtained for chemical analysis after the addition of trichloroacetic acid to the vessel contents and centrifugation (except when determining volatile fatty acids, where phosphotungstic acid was used as the deproteinizing agent). Lactic acid was estimated chemically by the method of Barker and Summerson (4). Pyruvate formation was followed routinely by the Friedemann-Haugen procedure (5). The identity of pyruvic acid and its formation in all critical experiments was established by the more specific Slavik-Michalec (6) modification of the Friedemann-Haugen method. Volatile fatty acid formation was estimated by the method of Conway (7), adapted for the Gilmont ultramicroburet. The identity of acetic acid and/or propionic acid as the major 4 We are indebted
to Mrs. M. 0. Nolan for her generous contribution
of snails.
INTERMEDIARY
METABOLISM
OF
SNAIL
233
part of the volatile acids was confirmed by treatment of the sample, after microdiffusion (7), with the lanthanum reagent of Hutchens and Kass (8). The substrates and inhibitors used were commercial products of reagentgrade quality and were added usually in the form of their sodium salts, adjusted to pH 7. Oxalacetic acid,5 prepared 1,~ the method of Krampitz and Werkman (O), was found to be 85% pure when standardized by the method of Greville (10). trans.Aconitic acid was synthesized by the method of Bruce (11). Iodoacetamide was prepared from the corresponding rhloramidc following the procedure of Anson (12). All of the cofactors used in these experiments \I-ere commercial products. Cytochrome c (Sigma) was standardized spectrophotometrically (13). Diphosphopyridine nucleotide (DPN) was approximately 40y0 pure as determined by reduction with hydrosulfite or by the method of Colowick, Kaplan, and C’iott’i (11). RESULTS
Comparison
of Metabolic Activities of Minced Snail Tissue with the Intact, Living Organism
Table I summarizes the data concerning some metabolic activities of the intact, living snail compared to minced tissues of the whole organism. The rate of endogenous respiration of the minced tissues approaches that of the living snail. This rate was constant for 2 hr., the maximum duration of any one experiment. The usual experimental period was limit,ed to 1 hr. to avoid any possible effects of bacterial contamination.6 The rate of endogenous respiration of the minced tissues from different lots of snails displayed some variation, the Qo, ranging from 1.1 to 3.3, with a mean value of 2.4. Some of the variation observed undoubtedly was due to the nutritional state of the snail as has been reported for studies with living snails (1). A few experiments with minces prepared from starving snails indicated a small but definite lowering of the Q+. Eflect of Oxygen Tension. Experiments conducted in a gas phase of pure oxygen revealed little or no increase (range: 0 to +28%) of respiration over those conducted in air. Following lowering of the tension to 11% oxygen, the decrease in respiration was 20%; at 5% oxygen, the decrease was 41y0. These results, while fundamentally similar to Ohose reported for living snails (1)) indicate that the dependency of the oxygen consumption on the tension is somewhat more pronounced in the mince t’han
in
the
whole
animal.
j We are indebted to Dr. H. D. Baernstein for a gift of this compound. 6 Additional evidence that these metabolic activities are not due t,o bacterial a&on was the observation that added sugars (glucose, mannose, and galactose) had no significant effect upon any of the mct,abolic activit,ies under study (see test).
234
EUGENE
C. WEINBACH
Under aerobic incubation the minced tissues produced small amounts of volatile fatty acids (expressed here as acetate), larger quantities of lactic acid, and even larger quantities of pyruvic acid, none of which are produced in detectable amounts by the living organism (Table I). Of particular interest is the metabolic block in the minced tissues that causes pyruvate to accumulate. This point will be discussed in a later section. When incubated anaerobically, both the living snail and the minced tissues produced lactic and acetic acids, but in different proportions. The production of volatile fatty acids in living snails under anaerobiosis has been reported in some detail (15). TABLE I Metabolic Activities of the Intact vs. Minced Snail Values expressed per milligram dry tissue per hour, representing of at least six determinations. All experiments with intact snails vessels containing 2.0 ml. dechlorinated water. Acetate production lowed in vessels containing at least 400 mg. fresh weight tissue. All mental conditions as given in Methods. Duration of experiments, 2 Aerobic
the average performed in in mince folother experihr.
Anaerobic
Metabolite
Oxygen uptake, cu. mm. 01. . Acetate produced, pg.. . . . Lactate produced, pg.. . . . Pyruvate produced, pg.. .
Intact snail
Mince
Intact snail
Mince
3.2 0 0 0
2.4 0.2 1.2 1.8
0.3 0.12 0
0.8 4.0 0
The effect of temperature on the endogenous respiration and production of pyruvate by the minced tissues was studied in the range 0.440%. The effect of temperature was as anticipated: a gradual increase in the rate of oxygen consumption paralleled by an increase in the rate of pyruvate production to maximal levels at 37”C., followed by a drop in both rates beyond this temperature. There is a striking parallelism between the effect of temperature upon the respiration of minced tissues as observed here’ and upon the respiration of living snails as reported by von Brand, Nolan, and Mann (1). 7 When these values were used to calculate the energy of activation according to the Arrhenius equation for the range 0.43O”C., a single, straight line was obtained, and p = 1.39 X lo4 (cf. von Brand, lo4 from experiments with living snails).
Nolan,
and Mann
(1) where
fi = 1.74 X
INTERMEDIARY
METABOLISM
OF
235
8N.4IL
Effect of Added Substrates on Endogenous
Respiration
The rate of endogenous respiration of the minced tissue was relatively unaffected by the addition of a variety of substrates. As shown in Table II, the addition of glucose, mannose, galactose, glucose l-phosphate, acids of the tricarboxylic acid cycle, and glubamate had little effect on the respiration of the tissues and the pyruvate production. TABLE
II
Effect of Added Substrates on Endogenous Metabolism Substrates present initially. All experiments conducted in phosphate buffer, pH 7.4. Values represent the average per cent change of metabolism compared to the endogenous rate. Figures in parentheses refer to the number of determinations for oxygen consumption. Pyruvate values based on three determinations for each substrate. Per cent change Substrate,
Oxygen
Glucose (9) ............... hlannose (3). ............. Galactose (3). .............. Glucose l-phosphate (3). .... Fructose diphosphate (3). .. Lactate (3). .......... Pyruvate (9) .......... Acetate (18). .......... Citrate (12). ........... Succinate (6). ........ Fumarate (6). ........... hIalat,e (9). .............. L(+)-Glutamate (3). ...... * Pyruvate
of
0.05 df consumption
+5 0
-8 -8 0 -18 -13 0
+16 +26 +2
Pynwate
production
-20 -12 +fJ 0 +50 0 -a
+25 +18 -17
+8
+13
levels not determined.
The failure of glucose and mannose to stimulate respiration parallels the results obtained by Baldwin (16) using the hepatopancreas of bhe large terrestrial snail, Helix pomatiu, while the lack of response to galacatosediffered from its stimulating effect on Helix respiration. The failure of the acids of the tricarboxylic acid cycle to stimulate respiration markedly might be attributed t,o nonpenetration of the cellular membrane, since these are in their anionic form at pH 7.4. Experiment’s performed
in a Inww
pH region
indicated
t,hat added
tricarhoxylk
acid
236
EUGENE
C.
WEINBACH
cycle intermediates now definitely increase the respiration beyond the lower base rate under these conditions; these data are summarized in Table III. Similar permeability effects were observed by Barron, Ardao, and Hearon (17) with Corynebacterium creatinovorans. As will be shown later in this paper (Table VI), the addition of cytochrome c or methylene blue to the minced tissuesstimulated the endogenousrespiration. Addition of glucose, acetate, citrate, succinate, and malate to the minced tissues.in the presence of cytochrome c or methylene blue did not augment the stimulated respiration beyond the increases observed in the absence of the added electron carriers. TABLE III Effect of pH on Rate of Oxidation of Tricarboxylic Acid Cycle Substrates Substrate present initially; phosphate buffer; pH determined at end of incubation period with glass electrode. Substrate concentration, 0.025 M. Values represent the average of at least six determinations. Figures in parentheses refer to per cent increase over endogenous rate. Substrate
Qo,PH
None (endogenous) Citrate a-Ketoglutarate Succinate Fumarate Malate
1.5
2.2 2.2 (0) 2.4 (+%a
2.6 (+18%‘,) 2.4 (+gm 2.2 (0)
Qoz PH 6.1
E 1.8 2.5 1.9 1.9
(+57%) (+29%0) (+78%) (+86%)
(+36%)
Effect of Inhibitors on EndogenousRespiration The effect of various inhibitors upon the rates of respiration and pyruvate production are summarized in Table IV. The marked suppression of the endogenous respiration by cyanide, and its failure to inhibit in the presence of methylene blue, suggeststhat electron transport to molecular oxygen in the minced snail tissues is via the cytochrome-cytochrome oxidase system. If azide is acting here in its metal-binding role (18), its inhibition of the endogenous respiration also indicates the presence of such a system. The inhibitions obtained with arsenite, p-chloromercuribenzoate, and iodoacetamide indicate the presence of sulfhydryl enzymes (19,20). The effect of cinnamate on mammalian tissues has been interpreted as denying endogenous fatty and amino acids to the metabolic pool (21). If the minced snail tissues are utilizing carbohydrate as their chief endogenous substrate,a the mar* Respiratory quotient studies and total carbohydrate utilization experiments with living snails indicated that the chief endogenous substrate of well-fed snails is carbohydrate (1,21a).
INTERMEDIARY
METABOLISM
OF
237
SPiAIL
ginal effect of cinnamate on respirat.ion would be expected. If fluoroacetate, trans-aconitate, and malonate were cell-permeable under the experimental conditions employed, t.heir failure to inhibit markedly the respiration suggests a slowing down of the tricarboxylic acid cycle in the minced tissue. Fluoride in a concentration of 0.01 JI had no effect upon the endogenous respiration. TABLE Efect
of Inhibitors
on
Rates
of Endogenow Minced
Inhibitors present the mean of at least more than ~1O~c).
IV Respiration Tissues
and Pyrwate
Per cent Final concentration
methylene
Azide. Arsenite. p-Chloromercuribenzoate.. Iodoacetamide Cinnamate. Fluoroacetate trans-Aconitate Malonate. CLThe (22).
2.2 2.2 1.0 10 1.0 1.0 10 10 10 IO 50
blue.
~
. . . . . . . ..._..._’ i
with
cyanide
inhibition 1
~~~
of Pyruvate production
x10-aM
.’
experiments
oxygen consumption
__~.-
i+
of
initially. Phosphate buffer, pH 7.4. Each value represents six determinations (which did not differ from each other b>
Inhibitor
Cyanidea. Cyanide
Production
were
conducted
-
70 0 43 57 34 71 21 10 0
~ i I after
l7 the
! 66 39 0 0 : - -2 manner of Robbie
Pyruvate Production by the Minced Tissues As shown in Table I, the minced tissues, when incubated aerobically, produced pyruvic acid at an average rate of 1.8 pg./mg. dry tissue/hr. Pyruvate accumulated during the mincing of the tissues, and its accumulation continued at a steady rate for several hours. The addition of a variety of substrates with the exception of fructose diphosphate had little effect on the pyruvate production (Table II). The stimulation of pyruvate production by fructose diphosphate indicates its utilization in snail tissuesin spite of its lack of effect upon the endogenous respiration. Lit. low oxygen tensions (Effect of Oxygen Tension), the lowered re-
238 spiratory however, production
EUGENE
C.
WEINBACH
rate was accompanied by diminished pyruvate production; it, was only in the complete absence of oxygen that pyruvate ceased, with a concomitant increase in lactate. TABLE
V
Assay of Snail Tissues for Certain Key Enzymes Ten per cent aqueous” homogenates were used in all experiments. Cytochrome oxidase and succinic dehydrogenase activity were determined essentially after the technique of Schneider and Potter (23). Malic dehydrogenase activity was measured according to the method of Potter (24). Fumarase activity was determined by measuring the rate of oxidation of malate (malate system as above) formed enzymatically from added fumarate. Thunberg tubes contained the same components as required in manometric experiments except for replacement of cytochrome c by 1 ml. of 2.6 X 10e4 M methylene blue, added from the hollow stopper; total volume 4.0 ml., Thunberg technique same as given by Burris (25). Manometric values based on first 30-min. readings. Each value represents the mean of at least six determinations. Type of assay System (fortified homogenates)
-
Substrate concn.
ManoQmetric: 02
Cytochrome oxidase 2 x IO-3 iv).........................
(+
8 X 10-S M
Fumarase
..
18.6 (0)
5 X lo+ M
37 >60
oxalacetate, . . . . . . . . . 5 X lo-* M
(fumarate-malate
~1Preliminary results. Although accumulate
(+
experiments
min.
cyanide,
Succinic dehydrogenase (+ malonate, 5 x 1W M). . .. .. . Malic dehydrogenase 1 X 10-Z M)
_-
system). with isotonic
-
5.2 (0) 3.6
saline gave no significantly
10 >60 15 different
the intact, living snail does not excrete pyrnvic acid, nor any in its tissues, the pyruvate blood level of 0.5 mg. ‘?&
indicates its importance as a metabolite. Evidence that pyruvate removal in the intact, organism is via a mechanism suggestive of the Krebs cycle was obtained with experiments employing fortified, aqueous homogenates. These results are summarized in Table V.
INTERMEDIARY
METABOLISM
OF
239
SNAIL
Experiments with Aqueous, Fortified Homogenates. In these experiments no attempt was made to determine all of the conditions necessary for optimal activity of the various enzyme systems studied.g Qualitative confirmation of the results obtained from manometric experiments was obtained by means of the Thunberg technique wherever possible (Table V). Cytochrome oxidase was found to be the most active enzyme of those tested. Thus, it was not a limiting factor in determining the activity of the dehydrogenases which are mediated through the cytochrome system. The powerful inhibition by cyanide of the oxidase could be “bypassed” by the addition of methylene .blue. These results provide additional evidence that electron transport to molecular oxygen is via the cytochrome system in snail tissues (E$ect of Inhibitors on Endogenous Respiration). Succinic and malic dehydrogenase, in common with those dehydrogenases found in vertebrate tissues, are sensitive to the action of selective inhibitors. The strong inhibition of the succinic dehydrogenase system by malonate contrasts with the weak malonate inhibition of the endogenous respiration of the minced tissues (Table IV). Similar to Potter’s (26) observation with mammalian tissues, methylene blue was much less effective for hydrogen transport than cytoc*hrome c in the succinic dehydrogenase system. Added oxalacetate was a powerful inhibitor of the malate system. Fumarase activiby, as measured indirectly here, undoubtedly represenbs minimal activity for this enzyme. Assay of a 24hr. aged (at 4°C.) homogenate for enzymatic activity revealed no significant loss of cytochrome oxidase, while succinic and malic dehydrogenase activities were diminished by more than 507&. Finequivocal experiments with citrate and a-ketoglutarate oxidation in homogenates were not obtained. Citrate oxidation in snail homogenates appears to require added DPN and triphosphopyridine nucleotide (TPN). As measured by the Thunberg technique, a-ketoglutarate was slowly oxidized in snail homogenates. E$ect of Added Cofactors upon the Removal of Pyruvate. As shown in Table VI, the more obvious cofactors which are associated with pyruvate oxidation in many vertebrate tissues, such as adenosine triphosphate (ATP), cocarboxylase, and DPN, when added either alone or in combination to the minced tissues did not expedite removal of endogenously formed pyruvate. -4dded fumarate, which in addition to its possible “sparking action” on a sluggish tricarboxylic acid cycle (27) should 9 However, in t,he case of cytochrome chrome r necessa.r,v for maximal activity
osidsse, the was determined
amount
of added
cyt,o-
240
EUGENE
C. WEINBACH
supply oxalacetic acid in situ, did not aid in the removal of pyruvate. The addition of the No. 4 group of cofactors in Table VI, ivhich were patterned after some recent work with pyruvate oxidation in tumor mitochondria (28), revealed increased amounts of pyruvate, rather than its removal. This increase is due primarily to the added cytochrome c TABLE VI E#ect of CofaczoW on Rates of Endogenous Respiration and Pyruuate Production of Minced Tissues Phosphatebuffer, pH 7.4. Cofactor concentrations listed are based on their assayed purities (see Methods). Values expressed per milligram dry tissue per hour, representing the mean value of at least six determinations. Cofactors
No additions (endogenous) 1. ATP,
1 X lO+‘M
2.3
1.9
1 X lo+ M (+Mg++)
2.2
1.8
3. DPN, 5 X 10e4M (nicotinamide)
2.5
2.2
4. DPN, 2 x 10-a M, ATP, 2 x lo+ M cyto c, 4 x 1O-6 M, fumarate, 7 X lO+ M, pantothenate, 3 X lo-* M
2.8
3.6
5. Cytochrome
c, 2 X 10e4 M
2.9
3.6
blue, 1 X 1OmJ M
3.5
8.3
2. Cocarboxylrtse,
ATP, 1 x 1O-3 M; fumarate, 5 X 10-2 M
6. Methylene
a The effect of coenzyme A (CoA) has not been studied thoroughly. Addition of a crude preparation of CoA to the minced tissues altered neither the accumulation of pyruvate nor the rate of endogenous respiration.
(Table VI, No. 5). Methylene the same response.
blue when added to minced tissues elicits DISCUSSION
The observations reveal that minced tissues prepared from the whole snail retain many of the metabolic activities of the living organism. The
INTERMEDIARY
METABOLISM
OF
SN.4IL
241
rate of endogenous respiration remains high, approaching that of the intact, living snail (Table I). The failure of some of the substrates listed in Table II to stimulate respiration when added to the mince most probably is attributable to their inability to penetrate the cellular membrane (i.e., see Table III). However, the presence in the minced tissues of large quantities of endogenous substrateslO which are not exhausted during the short experimental period also would limit the effects of added substrates. Other points of similarity between the respiratory behavior of the minced tissues and of the whole animal are reflected in their dependence upon oxygen tension and their response to temperature changes. The greater dependency of the respiratory rate of the minced tissues upon the oxygen tension probably is related to the destruction of the circulatory system. Nevertheless, at atmospheric oxygen pressure, the diffusion of oxygen into the minced tissues is not a limiting factor, since the respiration of these tissues is unaffected by increasing the oxygen pressure to 760 mm. Hg. On the other hand, some aspects of the anaerobic as well as the aerobic metabolism of the minced tissues differ from those of the living snail. Although both the living snail and the minced tissues produced lactic and acetic acid+ under anaerobic incubation, greater amounts of these acids were produced by the minced tissues and in an inverted ratio with respect to t’he observations in the whde snail (Table I). This increased production of lactate (and acetate) indicated a faster rate of carbohydrat,e breakdown (via glycolysis) in the minced tissues than in t.he whole organism. The finding of increased lactate production in tissue minces when compared with that of the whole organism is not unusual. The observations of Fischer (29) concerning the helminth, Parascuris ep?dorUm, are similar. Since the regulatory mechanism controlling such processes is unknown at present, any explanation would be tentative. The accumulation aerobically of pyruvic acid in the minced tissues also reflects metabolic disturbance. Undoubtedly, pyruvic acid or its precursors in the living snail are utilized at such a rate that none accumulate in the tissues or in the incubation medium. The stimulation of the respiration by added intermediates of the triI” Ekperiments with starving snails indicated a large reservoir of endogenous carboh,vdratc (1). I1 The term “acetic acid” is used here as a convenience. As stated in Methods, it is not possible definitely to distinguish between acetic and/or propionic acids as the volatile acid involved.
242
EUGENE
C. WEINBACH
carboxylic acid cycle (Table III), and also the demonstration of the functioning of the key enzymes, cytochrome oxidase, succinic and malic dehydrogenase, and fumarase (Table V) suggest that some mechanism similar to the tricarboxylic acid cycle is operating in the living snail, permitting in a normal manner the efficient utilization of pyruvate. The demonstration of some of these enzymes in molluscan tissues had been observed previously. Baldwin (16) reported finding succinic dehydrogenase and the probable presence of cytochrome oxidase in the hepatopancreas of the terrestrial snail, Helix pomatiu, and Humphrey (30) found an efficient succinoxidase system in oyster muscle. That the oxidation of pyruvate does not proceed maximally in the minced snail tissues is not surprising. Their failure to utilize completely the endogenously formed pyruvic acid, as well as the inability of the/ inhibitors fluoroacetate, trans-aconitate, and malonate to affect significantly the respiration of the mince (Table IV), reflects the metabolic disturbance associated with the mincing process. Pertinent, also, is the failure of added cofactors, known to be associated with pyruvate metabolism, to expedite the utilization of pyruvate (Table VI). However, there is the possibility of loss (during mincing) of an unknown, labile cofactor necessary for efficient pyruvate utilization. The destruction of cellular organization apparently causes a shift in metabolism toward aerobic glycolysis with the formation in these tissues of pyruvate, lactate, and acetate (Table I). Lack of oxygen is not concerned here, since incubation of the minced tissues in 100% oxygen does not alter the pyruvate production. Presumably, some energy-producing metabolic pathways present in the intact organism are unavailable in the minced tissues. ACKNOWLEDGMENTS The author is grateful to Dr. T. von Brand and’Dr. Leslie Hellerman helpful guidance and counsel throughout the course of this work.
for their
SUMMARY 1. A study of certain aspects of metabolism of minced snail tissues as compared to the whole organism reveals similarities in respiratory activity with respect to rate, temperature effects, and dependency upon oxygen tension, and differences in the products of anaerobic and aerobic carbohydrate degradation. 2. Although differences in the anaerobic carbohydrate metabolism are present, the greatest disparity in the behavior of the mince and the
INTERMEDIARY
METABOLISM
243
OF SNAIL
whole organism is observed aerobically. Acetate, lactate, and pyruvate accumulate in the minced tissues,while none of these substancesaccumulate in the living organism. 3. The presence of cytochrome oxidase and of succinic and malic dehydrogenases as well as of fumarase has been demonstrated. This may be suggestive of the functioning in snail Gssuesof a mechanism similar to the tricarboxylic acid cycle, but the evidence is not yet complete. 4. The accumulation of acetate, lactate, and pyruvate in the minced tissues is explained tentatively in terms of a disturbance of the pyruvate metabolism. REFERENCES 1.
T-OS BRAND,
T.,
NOLAN,
M.,
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AND
MANN,
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WEINBACH, ITON BRAND,
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21~. VON BRAND, T., BAERNSTEIN, B. D., AND MEHLMAN, B., Biol. Bull. 98, 26676 (1950). 22. ROBBIE, W. A. Methods in Medical Research, Vol. I. Year Book Publishers Inc., Chicago, p. 307 (1948). 23. SCHNEIDER, W. C., AND POTTER, V. R., J. Biol. Chem. 149, 217-227 (1943). 24. POTTER, V. R., J. Biol. Chem. 166, 311-24 (1946). 26. BURRIS, R. H., in UMBREIT, W. W., BURRIS, R. H., AND STAUFFER, J. F., Manometric Techniques and Related Methods for the Study of Tissue Metabolism, 2nd Ed., p. 105. Burgess Publishing Co., Minneapolis, 1949. 26. POTTER, V. R., J. Biol. Chem. 141, 775-87 (1941). 27. GREEN, D. E., LOOMIS, W. F., AND AUERBACH, V. H., J. Biol. Chem. 173, 389403 (1948). 28. WENNER, C. E., SPIRTES, M. A., AND WEINHOUSE, S., Proc. Sot. Exptl. Biol. Med. 78, 416-21 (1951). 29. FISCHER, A., Biochem. 2. 144, 224-8 (1924). 30. HUMPHREY, G. F., J. Exptl. Biol. 24,352-60 (1947).
the Federal Security Agency, Service, National Institutes Microbiological Institute,3 Received
United States Public of Health, National Bethesda, Maryland
October
Health
7. 1952
INTRODUCTION
Fundamental studies relating to comparative cellular metabolism among a wide variety of organisms are of intrinsic interest for biochemistry and underlie the approach to many practical problems. Thus the importance of a rational approach to the problem of eradicating tremat’ode diseases by a systematic study of the physiology of their intermediate hosts has been pointed out by von Brand, Nolan, and Mann (1). Such an approach includes studies of metabolic pathways which, theoretically at least, may be found vulnerable to attack by chemical agents. Previous studies, employing intact, living snails to investigate the mode of action of certain molluscacides, indicated the need for investigation on the cellular level (2). The present paper reports the results of such an investigation. It is concerned mainly with aerobic metabolic activities of minced tissues of Australorbis glabratus, the intermediate snail host of the human blood fluke SchistosomamansoG, in the West Indies and South America. METHODS ~1~t.sfralorbi.s glabrafus, the only species of snail used for this study, u-as lab orat’org-reared from Venezuelan specimens and was kept in balanced aquaria in :t room with a minimum temperature of 21°C. The snails were fed abundantI) wit’h let,tuce leaves and fish food, with the occasional addition of calcium car’ Presented in part at the forty-third annual meeting of Biological Chemists at Nea York City (la). 2 With the technical assistance of Rachel Al. Dudley. 3 T,nt)oratory of Tropical Diseases. 231
of the
American
Societ>
232
EUGENE
C.
WEINBACH
bonate to the water.4 Experiments with the intact, living snails were carried out under the same conditions described by von Brand, Nolan, and Mann (1). Minces and homogenates were prepared in the following manner: Specimens were transferred from aquaria to a chilled Petri dish, the shell was carefully cracked open, and the entire soft tissues were removed. After sufficient material had been collected (the tissues were kept moist during the collection by a dampened piece of filter paper attached to the underside of an inverted watch glass), it was minced freehand by means of two scalpels, one held in each hand. Aliquots of the tissue were weighed immediately on a torsion balance and transferred to Warburg vessels which had been prepared previously to contain all necessary components except the tissue; or if the homogenates were desired, weighed amounts of the minced tissue were homogenized in the Potter-Elvehjem apparatus (3). All metabolic activities are expressed in terms of the initial dry weights calculated from the initial wet weights. During the course of these experiments numerous samples were taken to dryness to determine the initial moisture content. This value usually averaged 30% of the total weight; hence in those manometric experiments where a separate dry weight determination was not made, an initial moisture content of 80% was assumed. Early experiments demonstrated that the rate of endogenous metabolic activity was dependent upon the amount of minced tissue present in each vessel; thus enzyme activity was proportional to tissue concentration. Unless noted otherwise, each vessel contained approximately 200 mg. (wet weight) of tissue. This amount of tissue was adopted as a working standard because it usually provided sufficient metabolic activities which could be measured conveniently. Oxygen consumption was measured manometrically in Warburg respirometers at a standard temperature of 3O”C., except where otherwise indicated. In all aerobic experiments atmospheric air served as the gas phase, except when the influence of oxygen tension was studied. Anaerobic conditions were obtained by passing a stream of nitrogen (Linde 99.990/,) over heated copper and through the shaking respirometers for 20 min. Preliminary experiments employing a variety of suspending media (ranging from simple dechlorinated tap water to KrebsRinger phosphate) indicated little dependence of the metabolic activities under study upon the media. Except as noted, each vessel contained 1.0 ml. of 0.1 111 phosphate buffer (pH 7.4) plus other components as indicated to make a final volume of 3.0 ml. Protein-free aliquots were obtained for chemical analysis after the addition of trichloroacetic acid to the vessel contents and centrifugation (except when determining volatile fatty acids, where phosphotungstic acid was used as the deproteinizing agent). Lactic acid was estimated chemically by the method of Barker and Summerson (4). Pyruvate formation was followed routinely by the Friedemann-Haugen procedure (5). The identity of pyruvic acid and its formation in all critical experiments was established by the more specific Slavik-Michalec (6) modification of the Friedemann-Haugen method. Volatile fatty acid formation was estimated by the method of Conway (7), adapted for the Gilmont ultramicroburet. The identity of acetic acid and/or propionic acid as the major 4 We are indebted
to Mrs. M. 0. Nolan for her generous contribution
of snails.
INTERMEDIARY
METABOLISM
OF
SNAIL
233
part of the volatile acids was confirmed by treatment of the sample, after microdiffusion (7), with the lanthanum reagent of Hutchens and Kass (8). The substrates and inhibitors used were commercial products of reagentgrade quality and were added usually in the form of their sodium salts, adjusted to pH 7. Oxalacetic acid,5 prepared 1,~ the method of Krampitz and Werkman (O), was found to be 85% pure when standardized by the method of Greville (10). trans.Aconitic acid was synthesized by the method of Bruce (11). Iodoacetamide was prepared from the corresponding rhloramidc following the procedure of Anson (12). All of the cofactors used in these experiments \I-ere commercial products. Cytochrome c (Sigma) was standardized spectrophotometrically (13). Diphosphopyridine nucleotide (DPN) was approximately 40y0 pure as determined by reduction with hydrosulfite or by the method of Colowick, Kaplan, and C’iott’i (11). RESULTS
Comparison
of Metabolic Activities of Minced Snail Tissue with the Intact, Living Organism
Table I summarizes the data concerning some metabolic activities of the intact, living snail compared to minced tissues of the whole organism. The rate of endogenous respiration of the minced tissues approaches that of the living snail. This rate was constant for 2 hr., the maximum duration of any one experiment. The usual experimental period was limit,ed to 1 hr. to avoid any possible effects of bacterial contamination.6 The rate of endogenous respiration of the minced tissues from different lots of snails displayed some variation, the Qo, ranging from 1.1 to 3.3, with a mean value of 2.4. Some of the variation observed undoubtedly was due to the nutritional state of the snail as has been reported for studies with living snails (1). A few experiments with minces prepared from starving snails indicated a small but definite lowering of the Q+. Eflect of Oxygen Tension. Experiments conducted in a gas phase of pure oxygen revealed little or no increase (range: 0 to +28%) of respiration over those conducted in air. Following lowering of the tension to 11% oxygen, the decrease in respiration was 20%; at 5% oxygen, the decrease was 41y0. These results, while fundamentally similar to Ohose reported for living snails (1)) indicate that the dependency of the oxygen consumption on the tension is somewhat more pronounced in the mince t’han
in
the
whole
animal.
j We are indebted to Dr. H. D. Baernstein for a gift of this compound. 6 Additional evidence that these metabolic activities are not due t,o bacterial a&on was the observation that added sugars (glucose, mannose, and galactose) had no significant effect upon any of the mct,abolic activit,ies under study (see test).
234
EUGENE
C. WEINBACH
Under aerobic incubation the minced tissues produced small amounts of volatile fatty acids (expressed here as acetate), larger quantities of lactic acid, and even larger quantities of pyruvic acid, none of which are produced in detectable amounts by the living organism (Table I). Of particular interest is the metabolic block in the minced tissues that causes pyruvate to accumulate. This point will be discussed in a later section. When incubated anaerobically, both the living snail and the minced tissues produced lactic and acetic acids, but in different proportions. The production of volatile fatty acids in living snails under anaerobiosis has been reported in some detail (15). TABLE I Metabolic Activities of the Intact vs. Minced Snail Values expressed per milligram dry tissue per hour, representing of at least six determinations. All experiments with intact snails vessels containing 2.0 ml. dechlorinated water. Acetate production lowed in vessels containing at least 400 mg. fresh weight tissue. All mental conditions as given in Methods. Duration of experiments, 2 Aerobic
the average performed in in mince folother experihr.
Anaerobic
Metabolite
Oxygen uptake, cu. mm. 01. . Acetate produced, pg.. . . . Lactate produced, pg.. . . . Pyruvate produced, pg.. .
Intact snail
Mince
Intact snail
Mince
3.2 0 0 0
2.4 0.2 1.2 1.8
0.3 0.12 0
0.8 4.0 0
The effect of temperature on the endogenous respiration and production of pyruvate by the minced tissues was studied in the range 0.440%. The effect of temperature was as anticipated: a gradual increase in the rate of oxygen consumption paralleled by an increase in the rate of pyruvate production to maximal levels at 37”C., followed by a drop in both rates beyond this temperature. There is a striking parallelism between the effect of temperature upon the respiration of minced tissues as observed here’ and upon the respiration of living snails as reported by von Brand, Nolan, and Mann (1). 7 When these values were used to calculate the energy of activation according to the Arrhenius equation for the range 0.43O”C., a single, straight line was obtained, and p = 1.39 X lo4 (cf. von Brand, lo4 from experiments with living snails).
Nolan,
and Mann
(1) where
fi = 1.74 X
INTERMEDIARY
METABOLISM
OF
235
8N.4IL
Effect of Added Substrates on Endogenous
Respiration
The rate of endogenous respiration of the minced tissue was relatively unaffected by the addition of a variety of substrates. As shown in Table II, the addition of glucose, mannose, galactose, glucose l-phosphate, acids of the tricarboxylic acid cycle, and glubamate had little effect on the respiration of the tissues and the pyruvate production. TABLE
II
Effect of Added Substrates on Endogenous Metabolism Substrates present initially. All experiments conducted in phosphate buffer, pH 7.4. Values represent the average per cent change of metabolism compared to the endogenous rate. Figures in parentheses refer to the number of determinations for oxygen consumption. Pyruvate values based on three determinations for each substrate. Per cent change Substrate,
Oxygen
Glucose (9) ............... hlannose (3). ............. Galactose (3). .............. Glucose l-phosphate (3). .... Fructose diphosphate (3). .. Lactate (3). .......... Pyruvate (9) .......... Acetate (18). .......... Citrate (12). ........... Succinate (6). ........ Fumarate (6). ........... hIalat,e (9). .............. L(+)-Glutamate (3). ...... * Pyruvate
of
0.05 df consumption
+5 0
-8 -8 0 -18 -13 0
+16 +26 +2
Pynwate
production
-20 -12 +fJ 0 +50 0 -a
+25 +18 -17
+8
+13
levels not determined.
The failure of glucose and mannose to stimulate respiration parallels the results obtained by Baldwin (16) using the hepatopancreas of bhe large terrestrial snail, Helix pomatiu, while the lack of response to galacatosediffered from its stimulating effect on Helix respiration. The failure of the acids of the tricarboxylic acid cycle to stimulate respiration markedly might be attributed t,o nonpenetration of the cellular membrane, since these are in their anionic form at pH 7.4. Experiment’s performed
in a Inww
pH region
indicated
t,hat added
tricarhoxylk
acid
236
EUGENE
C.
WEINBACH
cycle intermediates now definitely increase the respiration beyond the lower base rate under these conditions; these data are summarized in Table III. Similar permeability effects were observed by Barron, Ardao, and Hearon (17) with Corynebacterium creatinovorans. As will be shown later in this paper (Table VI), the addition of cytochrome c or methylene blue to the minced tissuesstimulated the endogenousrespiration. Addition of glucose, acetate, citrate, succinate, and malate to the minced tissues.in the presence of cytochrome c or methylene blue did not augment the stimulated respiration beyond the increases observed in the absence of the added electron carriers. TABLE III Effect of pH on Rate of Oxidation of Tricarboxylic Acid Cycle Substrates Substrate present initially; phosphate buffer; pH determined at end of incubation period with glass electrode. Substrate concentration, 0.025 M. Values represent the average of at least six determinations. Figures in parentheses refer to per cent increase over endogenous rate. Substrate
Qo,PH
None (endogenous) Citrate a-Ketoglutarate Succinate Fumarate Malate
1.5
2.2 2.2 (0) 2.4 (+%a
2.6 (+18%‘,) 2.4 (+gm 2.2 (0)
Qoz PH 6.1
E 1.8 2.5 1.9 1.9
(+57%) (+29%0) (+78%) (+86%)
(+36%)
Effect of Inhibitors on EndogenousRespiration The effect of various inhibitors upon the rates of respiration and pyruvate production are summarized in Table IV. The marked suppression of the endogenous respiration by cyanide, and its failure to inhibit in the presence of methylene blue, suggeststhat electron transport to molecular oxygen in the minced snail tissues is via the cytochrome-cytochrome oxidase system. If azide is acting here in its metal-binding role (18), its inhibition of the endogenous respiration also indicates the presence of such a system. The inhibitions obtained with arsenite, p-chloromercuribenzoate, and iodoacetamide indicate the presence of sulfhydryl enzymes (19,20). The effect of cinnamate on mammalian tissues has been interpreted as denying endogenous fatty and amino acids to the metabolic pool (21). If the minced snail tissues are utilizing carbohydrate as their chief endogenous substrate,a the mar* Respiratory quotient studies and total carbohydrate utilization experiments with living snails indicated that the chief endogenous substrate of well-fed snails is carbohydrate (1,21a).
INTERMEDIARY
METABOLISM
OF
237
SPiAIL
ginal effect of cinnamate on respirat.ion would be expected. If fluoroacetate, trans-aconitate, and malonate were cell-permeable under the experimental conditions employed, t.heir failure to inhibit markedly the respiration suggests a slowing down of the tricarboxylic acid cycle in the minced tissue. Fluoride in a concentration of 0.01 JI had no effect upon the endogenous respiration. TABLE Efect
of Inhibitors
on
Rates
of Endogenow Minced
Inhibitors present the mean of at least more than ~1O~c).
IV Respiration Tissues
and Pyrwate
Per cent Final concentration
methylene
Azide. Arsenite. p-Chloromercuribenzoate.. Iodoacetamide Cinnamate. Fluoroacetate trans-Aconitate Malonate. CLThe (22).
2.2 2.2 1.0 10 1.0 1.0 10 10 10 IO 50
blue.
~
. . . . . . . ..._..._’ i
with
cyanide
inhibition 1
~~~
of Pyruvate production
x10-aM
.’
experiments
oxygen consumption
__~.-
i+
of
initially. Phosphate buffer, pH 7.4. Each value represents six determinations (which did not differ from each other b>
Inhibitor
Cyanidea. Cyanide
Production
were
conducted
-
70 0 43 57 34 71 21 10 0
~ i I after
l7 the
! 66 39 0 0 : - -2 manner of Robbie
Pyruvate Production by the Minced Tissues As shown in Table I, the minced tissues, when incubated aerobically, produced pyruvic acid at an average rate of 1.8 pg./mg. dry tissue/hr. Pyruvate accumulated during the mincing of the tissues, and its accumulation continued at a steady rate for several hours. The addition of a variety of substrates with the exception of fructose diphosphate had little effect on the pyruvate production (Table II). The stimulation of pyruvate production by fructose diphosphate indicates its utilization in snail tissuesin spite of its lack of effect upon the endogenous respiration. Lit. low oxygen tensions (Effect of Oxygen Tension), the lowered re-
238 spiratory however, production
EUGENE
C.
WEINBACH
rate was accompanied by diminished pyruvate production; it, was only in the complete absence of oxygen that pyruvate ceased, with a concomitant increase in lactate. TABLE
V
Assay of Snail Tissues for Certain Key Enzymes Ten per cent aqueous” homogenates were used in all experiments. Cytochrome oxidase and succinic dehydrogenase activity were determined essentially after the technique of Schneider and Potter (23). Malic dehydrogenase activity was measured according to the method of Potter (24). Fumarase activity was determined by measuring the rate of oxidation of malate (malate system as above) formed enzymatically from added fumarate. Thunberg tubes contained the same components as required in manometric experiments except for replacement of cytochrome c by 1 ml. of 2.6 X 10e4 M methylene blue, added from the hollow stopper; total volume 4.0 ml., Thunberg technique same as given by Burris (25). Manometric values based on first 30-min. readings. Each value represents the mean of at least six determinations. Type of assay System (fortified homogenates)
-
Substrate concn.
ManoQmetric: 02
Cytochrome oxidase 2 x IO-3 iv).........................
(+
8 X 10-S M
Fumarase
..
18.6 (0)
5 X lo+ M
37 >60
oxalacetate, . . . . . . . . . 5 X lo-* M
(fumarate-malate
~1Preliminary results. Although accumulate
(+
experiments
min.
cyanide,
Succinic dehydrogenase (+ malonate, 5 x 1W M). . .. .. . Malic dehydrogenase 1 X 10-Z M)
_-
system). with isotonic
-
5.2 (0) 3.6
saline gave no significantly
10 >60 15 different
the intact, living snail does not excrete pyrnvic acid, nor any in its tissues, the pyruvate blood level of 0.5 mg. ‘?&
indicates its importance as a metabolite. Evidence that pyruvate removal in the intact, organism is via a mechanism suggestive of the Krebs cycle was obtained with experiments employing fortified, aqueous homogenates. These results are summarized in Table V.
INTERMEDIARY
METABOLISM
OF
239
SNAIL
Experiments with Aqueous, Fortified Homogenates. In these experiments no attempt was made to determine all of the conditions necessary for optimal activity of the various enzyme systems studied.g Qualitative confirmation of the results obtained from manometric experiments was obtained by means of the Thunberg technique wherever possible (Table V). Cytochrome oxidase was found to be the most active enzyme of those tested. Thus, it was not a limiting factor in determining the activity of the dehydrogenases which are mediated through the cytochrome system. The powerful inhibition by cyanide of the oxidase could be “bypassed” by the addition of methylene .blue. These results provide additional evidence that electron transport to molecular oxygen is via the cytochrome system in snail tissues (E$ect of Inhibitors on Endogenous Respiration). Succinic and malic dehydrogenase, in common with those dehydrogenases found in vertebrate tissues, are sensitive to the action of selective inhibitors. The strong inhibition of the succinic dehydrogenase system by malonate contrasts with the weak malonate inhibition of the endogenous respiration of the minced tissues (Table IV). Similar to Potter’s (26) observation with mammalian tissues, methylene blue was much less effective for hydrogen transport than cytoc*hrome c in the succinic dehydrogenase system. Added oxalacetate was a powerful inhibitor of the malate system. Fumarase activiby, as measured indirectly here, undoubtedly represenbs minimal activity for this enzyme. Assay of a 24hr. aged (at 4°C.) homogenate for enzymatic activity revealed no significant loss of cytochrome oxidase, while succinic and malic dehydrogenase activities were diminished by more than 507&. Finequivocal experiments with citrate and a-ketoglutarate oxidation in homogenates were not obtained. Citrate oxidation in snail homogenates appears to require added DPN and triphosphopyridine nucleotide (TPN). As measured by the Thunberg technique, a-ketoglutarate was slowly oxidized in snail homogenates. E$ect of Added Cofactors upon the Removal of Pyruvate. As shown in Table VI, the more obvious cofactors which are associated with pyruvate oxidation in many vertebrate tissues, such as adenosine triphosphate (ATP), cocarboxylase, and DPN, when added either alone or in combination to the minced tissues did not expedite removal of endogenously formed pyruvate. -4dded fumarate, which in addition to its possible “sparking action” on a sluggish tricarboxylic acid cycle (27) should 9 However, in t,he case of cytochrome chrome r necessa.r,v for maximal activity
osidsse, the was determined
amount
of added
cyt,o-
240
EUGENE
C. WEINBACH
supply oxalacetic acid in situ, did not aid in the removal of pyruvate. The addition of the No. 4 group of cofactors in Table VI, ivhich were patterned after some recent work with pyruvate oxidation in tumor mitochondria (28), revealed increased amounts of pyruvate, rather than its removal. This increase is due primarily to the added cytochrome c TABLE VI E#ect of CofaczoW on Rates of Endogenous Respiration and Pyruuate Production of Minced Tissues Phosphatebuffer, pH 7.4. Cofactor concentrations listed are based on their assayed purities (see Methods). Values expressed per milligram dry tissue per hour, representing the mean value of at least six determinations. Cofactors
No additions (endogenous) 1. ATP,
1 X lO+‘M
2.3
1.9
1 X lo+ M (+Mg++)
2.2
1.8
3. DPN, 5 X 10e4M (nicotinamide)
2.5
2.2
4. DPN, 2 x 10-a M, ATP, 2 x lo+ M cyto c, 4 x 1O-6 M, fumarate, 7 X lO+ M, pantothenate, 3 X lo-* M
2.8
3.6
5. Cytochrome
c, 2 X 10e4 M
2.9
3.6
blue, 1 X 1OmJ M
3.5
8.3
2. Cocarboxylrtse,
ATP, 1 x 1O-3 M; fumarate, 5 X 10-2 M
6. Methylene
a The effect of coenzyme A (CoA) has not been studied thoroughly. Addition of a crude preparation of CoA to the minced tissues altered neither the accumulation of pyruvate nor the rate of endogenous respiration.
(Table VI, No. 5). Methylene the same response.
blue when added to minced tissues elicits DISCUSSION
The observations reveal that minced tissues prepared from the whole snail retain many of the metabolic activities of the living organism. The
INTERMEDIARY
METABOLISM
OF
SN.4IL
241
rate of endogenous respiration remains high, approaching that of the intact, living snail (Table I). The failure of some of the substrates listed in Table II to stimulate respiration when added to the mince most probably is attributable to their inability to penetrate the cellular membrane (i.e., see Table III). However, the presence in the minced tissues of large quantities of endogenous substrateslO which are not exhausted during the short experimental period also would limit the effects of added substrates. Other points of similarity between the respiratory behavior of the minced tissues and of the whole animal are reflected in their dependence upon oxygen tension and their response to temperature changes. The greater dependency of the respiratory rate of the minced tissues upon the oxygen tension probably is related to the destruction of the circulatory system. Nevertheless, at atmospheric oxygen pressure, the diffusion of oxygen into the minced tissues is not a limiting factor, since the respiration of these tissues is unaffected by increasing the oxygen pressure to 760 mm. Hg. On the other hand, some aspects of the anaerobic as well as the aerobic metabolism of the minced tissues differ from those of the living snail. Although both the living snail and the minced tissues produced lactic and acetic acid+ under anaerobic incubation, greater amounts of these acids were produced by the minced tissues and in an inverted ratio with respect to t’he observations in the whde snail (Table I). This increased production of lactate (and acetate) indicated a faster rate of carbohydrat,e breakdown (via glycolysis) in the minced tissues than in t.he whole organism. The finding of increased lactate production in tissue minces when compared with that of the whole organism is not unusual. The observations of Fischer (29) concerning the helminth, Parascuris ep?dorUm, are similar. Since the regulatory mechanism controlling such processes is unknown at present, any explanation would be tentative. The accumulation aerobically of pyruvic acid in the minced tissues also reflects metabolic disturbance. Undoubtedly, pyruvic acid or its precursors in the living snail are utilized at such a rate that none accumulate in the tissues or in the incubation medium. The stimulation of the respiration by added intermediates of the triI” Ekperiments with starving snails indicated a large reservoir of endogenous carboh,vdratc (1). I1 The term “acetic acid” is used here as a convenience. As stated in Methods, it is not possible definitely to distinguish between acetic and/or propionic acids as the volatile acid involved.
242
EUGENE
C. WEINBACH
carboxylic acid cycle (Table III), and also the demonstration of the functioning of the key enzymes, cytochrome oxidase, succinic and malic dehydrogenase, and fumarase (Table V) suggest that some mechanism similar to the tricarboxylic acid cycle is operating in the living snail, permitting in a normal manner the efficient utilization of pyruvate. The demonstration of some of these enzymes in molluscan tissues had been observed previously. Baldwin (16) reported finding succinic dehydrogenase and the probable presence of cytochrome oxidase in the hepatopancreas of the terrestrial snail, Helix pomatiu, and Humphrey (30) found an efficient succinoxidase system in oyster muscle. That the oxidation of pyruvate does not proceed maximally in the minced snail tissues is not surprising. Their failure to utilize completely the endogenously formed pyruvic acid, as well as the inability of the/ inhibitors fluoroacetate, trans-aconitate, and malonate to affect significantly the respiration of the mince (Table IV), reflects the metabolic disturbance associated with the mincing process. Pertinent, also, is the failure of added cofactors, known to be associated with pyruvate metabolism, to expedite the utilization of pyruvate (Table VI). However, there is the possibility of loss (during mincing) of an unknown, labile cofactor necessary for efficient pyruvate utilization. The destruction of cellular organization apparently causes a shift in metabolism toward aerobic glycolysis with the formation in these tissues of pyruvate, lactate, and acetate (Table I). Lack of oxygen is not concerned here, since incubation of the minced tissues in 100% oxygen does not alter the pyruvate production. Presumably, some energy-producing metabolic pathways present in the intact organism are unavailable in the minced tissues. ACKNOWLEDGMENTS The author is grateful to Dr. T. von Brand and’Dr. Leslie Hellerman helpful guidance and counsel throughout the course of this work.
for their
SUMMARY 1. A study of certain aspects of metabolism of minced snail tissues as compared to the whole organism reveals similarities in respiratory activity with respect to rate, temperature effects, and dependency upon oxygen tension, and differences in the products of anaerobic and aerobic carbohydrate degradation. 2. Although differences in the anaerobic carbohydrate metabolism are present, the greatest disparity in the behavior of the mince and the
INTERMEDIARY
METABOLISM
243
OF SNAIL
whole organism is observed aerobically. Acetate, lactate, and pyruvate accumulate in the minced tissues,while none of these substancesaccumulate in the living organism. 3. The presence of cytochrome oxidase and of succinic and malic dehydrogenases as well as of fumarase has been demonstrated. This may be suggestive of the functioning in snail Gssuesof a mechanism similar to the tricarboxylic acid cycle, but the evidence is not yet complete. 4. The accumulation of acetate, lactate, and pyruvate in the minced tissues is explained tentatively in terms of a disturbance of the pyruvate metabolism. REFERENCES 1.
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T.,
NOLAN,
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V. R., AND EI.VEHJEM, C. A., J. Biol. Chem. 114, 495-504 (1936). S. B., AND SUMMERSON, W. H., J. Biol. Chenz. X%,535-54 (1941). FRIEDEMANN, T. E., AND HAUGEN, G. E., J. Biol. Chem. 147, 415-42 (1943). SLAVIC, K., AND MICHALEC, C. Chemicke L&y 43, 102-104 (1949). CONWAY, E. J., Microdiffusion Analysis and Volumetric Error, 3rd I<;d., p, 246. D. Van Nostrand Co., Inc., New York, 1950. HUTC*HENS, J. O., AND KASS, B. M., J. Biol. C’henl. 177,571-5 (1949). KRAMPITZ, L. O., AND WERKMAN, C. H., Rio&em. J. 36, 595602 (1041). GREVILLE, G. D., Biochem. J. 33, 718-22 (1939). BRUCE, W. F., in Blatt, A. H., Organic Syntheses, ~011. Vol. II., p. 12. John Wiley and Sons, New York, 1943. ANSON, M. I,., J. Gen. Physiol. 23, 247-61 (1939). POTTER, V. R., in UMBREIT, W. W., BURRIS, I{. H., AND STAU~E.ER, J. F., Manometric Techniques and Related Methods for the Study of Tissue Metabolism, 2nd Ed., p. 213. Burgess Publishing Co., Minneapolis, 1949. COI~OI~ICK, S. P., KAPLAN, N. O., .~NI) CIOTTI, M. M., J. Bid. (‘hpm. 191, 447-59 (1951). MEHLM~N, B., AND VOX BRAND, T., Biol. Bull. 100, 199-205 (la51 ). BALDWIN, E., Biochem. J. 32, 1225-37 (1938). BARRON, R. S. G., ARDAO, M. I., AXD HEAROS, .\I.. .-I&. Rioche,,l. 29, 13Ob53 (1950). KEILIN, D., Proc. Roy. Sot. (London) Bl21, 165-i3 (1936). HELLERMAN, L., Physiol. Revs. 17, 454-84 (193i). HEI,LERMAN, L., CHINARD. F. P.. ANT) DEITZ, \‘. I<., .I. Rio/. Chetu. 147, 44362 (1943). WEINRACH, E. C., LOTTE, II. J.. FRISEI,I,, W. Ii.. ASI) HEI,LER~IAS, T,., J. Bid. (‘hem. 189, 779-92 (1951). POTTER,
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21~. VON BRAND, T., BAERNSTEIN, B. D., AND MEHLMAN, B., Biol. Bull. 98, 26676 (1950). 22. ROBBIE, W. A. Methods in Medical Research, Vol. I. Year Book Publishers Inc., Chicago, p. 307 (1948). 23. SCHNEIDER, W. C., AND POTTER, V. R., J. Biol. Chem. 149, 217-227 (1943). 24. POTTER, V. R., J. Biol. Chem. 166, 311-24 (1946). 26. BURRIS, R. H., in UMBREIT, W. W., BURRIS, R. H., AND STAUFFER, J. F., Manometric Techniques and Related Methods for the Study of Tissue Metabolism, 2nd Ed., p. 105. Burgess Publishing Co., Minneapolis, 1949. 26. POTTER, V. R., J. Biol. Chem. 141, 775-87 (1941). 27. GREEN, D. E., LOOMIS, W. F., AND AUERBACH, V. H., J. Biol. Chem. 173, 389403 (1948). 28. WENNER, C. E., SPIRTES, M. A., AND WEINHOUSE, S., Proc. Sot. Exptl. Biol. Med. 78, 416-21 (1951). 29. FISCHER, A., Biochem. 2. 144, 224-8 (1924). 30. HUMPHREY, G. F., J. Exptl. Biol. 24,352-60 (1947).