- Email: [email protected]
Metabolism of pyruvate by the ovarian tissue of cod
M. S. Nounib
610
operations can be carried out at a much faster rate than with the conventionaI chamber. While the above description has been concerned with free-living amoebae, the modified chamber could also be used for micrurgy on other cells.
Fig. 1.
Fig. 2.
Fig. l.-Top view of the modified oil chamber. A glass or plastic frame is placed on a slide and the enclosed space is filled with mineral oil. The circles are droplets of medium containing cells (dots) to be operated on. Fig. P.-Side view of a micro-tool as applied to an amoeba in the open oil chamber.
The author wishes to thank Prof. J. F. Danielli and Dr I. J. Larch for their interest and help in the work. The work has been supported by U.S.P.H.S. Grant No. GM 11603 of N.I.H. REFERENCES 1. COKANDON, J. and DE FONBRUNE, P., Bnn. Inst. Pasteur 60, 113 (1938). 2. -Compf. Rend. Sot. BioZ. 130, 740 (1939). 3. GOLDSTEIN, L., in D. IN. PRESCOTT (ed.), Methods in Cell Physiology, Vol. I, p. 97. Academic Press, New York, 1964. 4. __ ProfopIasmafoZogia 5, 52 (1964). 5. HAWKINS, S. E. and COLE, R. J., ExpfZ CeIZ Res. 37, 26 (1965). 6. LORCH, I. J. and DANIELLI, J. F., Quarf. J. Micr. Sci. 94, 461 (1953).
METABOLISM
OF PYRUVATE
BY THE OVARIAN TISSUE OF COD
M. S. MOUNIB Fisheries
Research Board of Canada, Halifax Laboratory, Halifax, Nova Scotia, Canada Received
November
Endocrinology
Section,
3, 1966
Pmwom work from this laboratory has shown that pyruvate, when added to the incubation medium of the testicular tissue of cod or rabbit, undergoes: (1) reduction to lactate, and (2) oxidative decarboxylation [6]. The stoichiometry of the reaction, however, did not follow the dismutation equation of Krebs and Johnson [3]: 2 pyruvate + H,O+l lactate + I acetate + 1 CO,; and the evidence was in suppo~% of a CO,-fixation with pyruvate in the testicular tissue [6]. This communication presents Experimenfcrl
Cell Research
46
Pyruvate
metabolism
by ovarian
611
tissue
evidence that the ovarian tissue of cod metabolizes pyruvate essentially via the same routes. Live cod, Gadus morhua, were kept in tanks supplied with running sea water. To avoid contamination of ovarian tissue with eggs, mature-but not ripe-fish were used. The animal was killed and immediately the ovaries were placed in Petri dishes TABLE
I. Metabolkm
of pyruvate
by the ovarian ‘TOi:
Experiment
Condition
Pyruvate utilized
Lactate produced
1
Anaerobic Anaerobic Aerobic Aerobic
12.80 17.75 13.48 7.80
6.0s 10.53 4.66 4.18
2 3 4
tissue of cod.a
released from
l&C-lPyruvate
14c-‘& Pyruvate
l&C-3Pyruvate
02 uptake
0.3421 0.6056 1.2965 2.6610
0.0239 0.5812 0.8367
0.0246 0.7596 0.6295
30.7534 3S.3199
a all values are expressed as pcmoles/g dry matter of tissue/2 hr. Tissue was suspended in 2.5 ml saline (1.15 per cent NaCl) buffered with phosphate (pH 7.4). Substrate was added as I%-pyruvate labelled in carbon number 1, 2 or 3. Total amount of substrate at the beginning of the incubation was 3.2 pmoles that had a total activity of 1 ,UC. The gas phase was air for aerobic experiments, and 100 per cent N, for anaerobic work. In experiment 2, 3 and 4, three incubations were required to determine the WOz produced from the three C positions of pyruvate: in all three incubations the utilization of pyruvate, and the production of lactate due to the addition of pyruvate 131, were estimated [5, 71, and average values are reported. Other experimental details have already been given [5, 6, 7, 81.
kept in an ice bath; the outside capsule was removed and suitable samples of ovarian tissue were used for incubation purposes in the Warburg manometric apparatus at 25°C as previously reported [5, 6, 7, 81. The ovarian tissue of cod oxidized C-l of pyruvate in preference to C-2 or C-3; indicating that an oxidative decarboxylation of pyruvate is taking place. The reaction was more pronounced under aerobic than under anaerobic conditions (Table I>. Pyruvate was also reduced to lactate by the ovarian tissue (Table I). Further work has shown that as in the case of the testicular tissue [6], the amount of the radioactive lactate produced from laC-pyruvate, agreed with that calculated for lactate production due to the addition of pyruvate according to the method of Krebs and Johnson l.31. Both the oxidative decarboxylation and reduction reactions of pyruvate do not account for all the pyruvate utilized by the ovarian tissue (Table I); thus suggesting other reactions are taking place. In another series of experiments, the supernatant, at the end of the incubation period, was subjected to radiochromatography. It was found that W-pyruvate labelled in any position could incorporate its radioactivity into malate, oxalacetate, a-ketoglutarate, aspartate, and glutamate; thus indicating a CO,-fixation with pyruvate. Experiments in this laboratory have verified a direct CO,-fixation by the ovarian tissue of cod [9]. Experimental
Cell Research 46
J. F. Scaife and H. Brohe’e
612
The oxidative decarboxylation is the first step in pyruvate metabolism via the Krebs cycle [I]. Since it was observed in the ovary that C-l of pyruvate was oxidized in preference to C-2 or C-3, it is likely that the reaction is proceeding at a faster rate than is required by the Krebs cycle. The oxidative decarboxylation of pyruvate means the availability of acetyl CoA and CO,. Acetyl CoA may go into the tricarboxylic cycle after its condensation with oxalacetate to form citrate, participate in lipid biosynthesis, or may catalyze other reactions such as the carboxylation of pyruvate [IO, Ii]. Carbon dioxide, on the other hand, can participate in fixation reactions with pyruvate, leading to oxalacetate (directly and/or via malate) that is required for the operation of the Krebs cycle, and for the biosynthesis of amino acids [2]. In view of the above mentioned discussion it would appear that both the oxidative decarboxylation and the carbon dioxide-fixation of pyruvate play important roles in regulating the metabolism of the ovary. A similar suggestion has already been made for the testicular tissue [6]. Inasmuch as both testes and ovaries are considered to be target organs for gonadotropic hormones, it may be feasible to suggest that gonadotrophic hormones influence the gonadal activity through regulatory reactions such as the oxidative decarboxylation of pyruvate and CO,-fixation.
REFERENCES
1. 2. 3.
4. 5. 6. 7.
8. 9. 10. 11.
GREEN, D. E. and FLEISCHER, J., in D. ILL GREENBERG (ed.), Metabolic pathways, Vol. 1, p. 41. Academic Press, New York, London, 1960. KORNBERG, H. L., dngew. Chem. Infern. Ed. 4, 558 (1965). KREBS, H. A. and JOHNSON, W. R., Biochem. J. 31, 144 (1937). MELROSE, D. R. and TERNER, C., Biochem. J. 53, 296 (1953). MOWNIB, M. S., Acta EndocrinoZ. (Kobenhaun) 45, 631 (1964). __ Comp. Biochem. Physiol. In press. MOUNIB, M. S. and CHANG, III. C., Radiation Res. 22, 144 (1961). ~ Expfl Cell Res. 38, 201 (1965). MOUNIB, M. S. and EISAN, J. S., Unpublished results. Chem. 238, 2603 (1963). UTTER, &I. E. and KEECH; D. B., J.-Biol. UTTER, M. F., KEECH, D. B. and SCRUTTON, M. C., in G. \VEBER (ed.j, Advances in enzyme regulation, Vol. 11, p. 49. Pergamon Press, New York, 1964.
A QUANTITATIVE
MEASUREMENT
OF CELL
DAMAGE
BY MEANS
OF
Wr-BINDING J. F. SCAIFE Biologia,
Eurntom, Received
and H. BROHfiE C.C.R.,
November
zspra
(Va),
Italy
15, 19661
THE staining of cells with vital dyes has been used as a criterion of cell death, and as an indicator of mechanical damage to the cell membrane [5, 81. In the case of the thin-walled rat thymocyte staining with dilute solutions of Erythrosin B or Eosin Y 1 Revised
Experimental
version
received
Cell Research 46
January
IO, 1967.
610
operations can be carried out at a much faster rate than with the conventionaI chamber. While the above description has been concerned with free-living amoebae, the modified chamber could also be used for micrurgy on other cells.
Fig. 1.
Fig. 2.
Fig. l.-Top view of the modified oil chamber. A glass or plastic frame is placed on a slide and the enclosed space is filled with mineral oil. The circles are droplets of medium containing cells (dots) to be operated on. Fig. P.-Side view of a micro-tool as applied to an amoeba in the open oil chamber.
The author wishes to thank Prof. J. F. Danielli and Dr I. J. Larch for their interest and help in the work. The work has been supported by U.S.P.H.S. Grant No. GM 11603 of N.I.H. REFERENCES 1. COKANDON, J. and DE FONBRUNE, P., Bnn. Inst. Pasteur 60, 113 (1938). 2. -Compf. Rend. Sot. BioZ. 130, 740 (1939). 3. GOLDSTEIN, L., in D. IN. PRESCOTT (ed.), Methods in Cell Physiology, Vol. I, p. 97. Academic Press, New York, 1964. 4. __ ProfopIasmafoZogia 5, 52 (1964). 5. HAWKINS, S. E. and COLE, R. J., ExpfZ CeIZ Res. 37, 26 (1965). 6. LORCH, I. J. and DANIELLI, J. F., Quarf. J. Micr. Sci. 94, 461 (1953).
METABOLISM
OF PYRUVATE
BY THE OVARIAN TISSUE OF COD
M. S. MOUNIB Fisheries
Research Board of Canada, Halifax Laboratory, Halifax, Nova Scotia, Canada Received
November
Endocrinology
Section,
3, 1966
Pmwom work from this laboratory has shown that pyruvate, when added to the incubation medium of the testicular tissue of cod or rabbit, undergoes: (1) reduction to lactate, and (2) oxidative decarboxylation [6]. The stoichiometry of the reaction, however, did not follow the dismutation equation of Krebs and Johnson [3]: 2 pyruvate + H,O+l lactate + I acetate + 1 CO,; and the evidence was in suppo~% of a CO,-fixation with pyruvate in the testicular tissue [6]. This communication presents Experimenfcrl
Cell Research
46
Pyruvate
metabolism
by ovarian
611
tissue
evidence that the ovarian tissue of cod metabolizes pyruvate essentially via the same routes. Live cod, Gadus morhua, were kept in tanks supplied with running sea water. To avoid contamination of ovarian tissue with eggs, mature-but not ripe-fish were used. The animal was killed and immediately the ovaries were placed in Petri dishes TABLE
I. Metabolkm
of pyruvate
by the ovarian ‘TOi:
Experiment
Condition
Pyruvate utilized
Lactate produced
1
Anaerobic Anaerobic Aerobic Aerobic
12.80 17.75 13.48 7.80
6.0s 10.53 4.66 4.18
2 3 4
tissue of cod.a
released from
l&C-lPyruvate
14c-‘& Pyruvate
l&C-3Pyruvate
02 uptake
0.3421 0.6056 1.2965 2.6610
0.0239 0.5812 0.8367
0.0246 0.7596 0.6295
30.7534 3S.3199
a all values are expressed as pcmoles/g dry matter of tissue/2 hr. Tissue was suspended in 2.5 ml saline (1.15 per cent NaCl) buffered with phosphate (pH 7.4). Substrate was added as I%-pyruvate labelled in carbon number 1, 2 or 3. Total amount of substrate at the beginning of the incubation was 3.2 pmoles that had a total activity of 1 ,UC. The gas phase was air for aerobic experiments, and 100 per cent N, for anaerobic work. In experiment 2, 3 and 4, three incubations were required to determine the WOz produced from the three C positions of pyruvate: in all three incubations the utilization of pyruvate, and the production of lactate due to the addition of pyruvate 131, were estimated [5, 71, and average values are reported. Other experimental details have already been given [5, 6, 7, 81.
kept in an ice bath; the outside capsule was removed and suitable samples of ovarian tissue were used for incubation purposes in the Warburg manometric apparatus at 25°C as previously reported [5, 6, 7, 81. The ovarian tissue of cod oxidized C-l of pyruvate in preference to C-2 or C-3; indicating that an oxidative decarboxylation of pyruvate is taking place. The reaction was more pronounced under aerobic than under anaerobic conditions (Table I>. Pyruvate was also reduced to lactate by the ovarian tissue (Table I). Further work has shown that as in the case of the testicular tissue [6], the amount of the radioactive lactate produced from laC-pyruvate, agreed with that calculated for lactate production due to the addition of pyruvate according to the method of Krebs and Johnson l.31. Both the oxidative decarboxylation and reduction reactions of pyruvate do not account for all the pyruvate utilized by the ovarian tissue (Table I); thus suggesting other reactions are taking place. In another series of experiments, the supernatant, at the end of the incubation period, was subjected to radiochromatography. It was found that W-pyruvate labelled in any position could incorporate its radioactivity into malate, oxalacetate, a-ketoglutarate, aspartate, and glutamate; thus indicating a CO,-fixation with pyruvate. Experiments in this laboratory have verified a direct CO,-fixation by the ovarian tissue of cod [9]. Experimental
Cell Research 46
J. F. Scaife and H. Brohe’e
612
The oxidative decarboxylation is the first step in pyruvate metabolism via the Krebs cycle [I]. Since it was observed in the ovary that C-l of pyruvate was oxidized in preference to C-2 or C-3, it is likely that the reaction is proceeding at a faster rate than is required by the Krebs cycle. The oxidative decarboxylation of pyruvate means the availability of acetyl CoA and CO,. Acetyl CoA may go into the tricarboxylic cycle after its condensation with oxalacetate to form citrate, participate in lipid biosynthesis, or may catalyze other reactions such as the carboxylation of pyruvate [IO, Ii]. Carbon dioxide, on the other hand, can participate in fixation reactions with pyruvate, leading to oxalacetate (directly and/or via malate) that is required for the operation of the Krebs cycle, and for the biosynthesis of amino acids [2]. In view of the above mentioned discussion it would appear that both the oxidative decarboxylation and the carbon dioxide-fixation of pyruvate play important roles in regulating the metabolism of the ovary. A similar suggestion has already been made for the testicular tissue [6]. Inasmuch as both testes and ovaries are considered to be target organs for gonadotropic hormones, it may be feasible to suggest that gonadotrophic hormones influence the gonadal activity through regulatory reactions such as the oxidative decarboxylation of pyruvate and CO,-fixation.
REFERENCES
1. 2. 3.
4. 5. 6. 7.
8. 9. 10. 11.
GREEN, D. E. and FLEISCHER, J., in D. ILL GREENBERG (ed.), Metabolic pathways, Vol. 1, p. 41. Academic Press, New York, London, 1960. KORNBERG, H. L., dngew. Chem. Infern. Ed. 4, 558 (1965). KREBS, H. A. and JOHNSON, W. R., Biochem. J. 31, 144 (1937). MELROSE, D. R. and TERNER, C., Biochem. J. 53, 296 (1953). MOWNIB, M. S., Acta EndocrinoZ. (Kobenhaun) 45, 631 (1964). __ Comp. Biochem. Physiol. In press. MOUNIB, M. S. and CHANG, III. C., Radiation Res. 22, 144 (1961). ~ Expfl Cell Res. 38, 201 (1965). MOUNIB, M. S. and EISAN, J. S., Unpublished results. Chem. 238, 2603 (1963). UTTER, &I. E. and KEECH; D. B., J.-Biol. UTTER, M. F., KEECH, D. B. and SCRUTTON, M. C., in G. \VEBER (ed.j, Advances in enzyme regulation, Vol. 11, p. 49. Pergamon Press, New York, 1964.
A QUANTITATIVE
MEASUREMENT
OF CELL
DAMAGE
BY MEANS
OF
Wr-BINDING J. F. SCAIFE Biologia,
Eurntom, Received
and H. BROHfiE C.C.R.,
November
zspra
(Va),
Italy
15, 19661
THE staining of cells with vital dyes has been used as a criterion of cell death, and as an indicator of mechanical damage to the cell membrane [5, 81. In the case of the thin-walled rat thymocyte staining with dilute solutions of Erythrosin B or Eosin Y 1 Revised
Experimental
version
received
Cell Research 46
January
IO, 1967.