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Carbon dioxide fixation by spermatozoa of cod
Comp. Bioch4nn. Phytiol., 1968, Vol. 2S, pp. 703 to 709. PerEamon Pre~#. Printed in Great Britain
CARBON DIOXIDE FIXATION BY SPERMATOZOA OF COD M. S. M O U N I B and J. S. EISAN Fisheries Research Board of Canada, Halifax Laboratory, P.O. Box 429, Halifax, Nova Scotia, Canada (Received 9 October 1967)
Abstractml. Sperm of cod, in the presence of 14COi, incorPorated label into
keto-, amino and other organic acids, lipids, proteins and nucleic acids. 2. Pyruvate enhanced ltCOi incorporation into the supematant of the incubation mixtttre, whereas malonate depressed it. 3. Extracts of frozen sperm and their acetone powder showed activity of malic enzyme, and of phosphoenolpyruvate and propionyl CoA carboxyhtses. INTRODUCTION THERE has been considerable evidence in the literature indicating that carbon dioxide plays an important part in the metabolism of spermatozoa. Humphrey & Mann (1949) observed that respiration of sperm of ram was higher in the presence of 5 per cent CO2+ 95 per cent 02 than with either air or pure O s. Also Salisbury & Kinney (1957) found a marked stimulation of aerobic glycolysis of sperm, in the presence of metabolic CO s. Under anaerobic conditiom, fructolysis of bull spermatozoa was more with 5 per cent COs+ 95 per cent N s than with N s alone (Lodge & Salisbury, 1963). Recently Afzelins & Mohri (1966) reported that when spermatozoa of sea urchin were incubated in the presence of KOH to remove respiratory COs, there was a reduction in the cristae of the mitochondrlal midpiece, and the reduction paralleled a decrease of endogenous phospholipids of the sperm. They also noticed that the disappearance of cristae was more marked after 1-hr incubation with KOH present than after 6-hr incubation without. Despite the above-mentioned reports, the mechanism of action of COs in the metabolism of sperm is still obscure. However, we have recently suggested that a CO 2 fixation with pyruvate takes place in sperm of cod and salmon (Mounib & Eisan, 1966; Mounib, 1967a). This suggestion stems from two observations: (1) the incorporation of all three carbon atoms of pyruvate, particularly C-1, into the total lipids biosynthesized by salmon sperm (Mounib & Eisan, 1966). (2) Incubation of sperm of cod or salmon with 14C-pyruvate labeled in any position yielded radioactive malate, oxaloacetate, ~-ketoglutarate and aspartate (Mounib, 1967a). These observations strengthened the suggestion that CO s fixation operates in sperm, and the present work was undertaken to examine this proposition by direct methods. A preliminary account of the present investigation has already been reported (Mounib & Eisan, 1967a). 703
704
M.S. Motmm Am) J. S. Exsx~r MATERIAL AND METHODS
Incubation of sperm in the presence of x~70 2 Sperm of cod, Gadus morhua, were collected as previously described (Mounib, 1967a). Sperm were washed by suspending them in cold saline (1.15% NaC1, w/v), followed by centrifugation (1500g at 4°C) for 20 min. The supernatant was discarded, the washing procedure was repeated, the packed sperm were resuspended in cold saline, and penicillin was added (5000 international units/ml of sperm suspension). A special flask was used for incubation studies. The flask was made by American Instrument Co., Silver Spring, Maryland, according to our specifications, and is essentially that of Dixon & Keilin (Dixon, 1952) except that it has one single and one double side-arm instead of one single-arm. One milliliter of sperm suspension + 1.5 ml of saline buffered with phosphate, pH 7.4-, were placed in the main chamber of the flask, and pyrnvate and/or malonate were added to the incubation mixture when required. 14C-sodium bicarbonate (80/,mole that had 20 pC) was placed in the main compartment of the double side-arm, and 0"6 ml of 2 N HCI in the side-compartment. The single side-arm contained 0.2 ml of 3 N perchlorie acid. Hyamine hydroxide 10-X (0.5 ml) was placed in the cup of the stopcock plug, which was turned to isolate the alkaline solution from the atmosphere of the flask. Incubation was carried out in the conventional Warburg bath at 25°C. Ten minutes were allowed for equilibrium, then the 14CO2 was released into the atmosphere of the flask by adding HC1 to the 14C-bicarbonate. The incubation lasted for 2 hr and the reaction was stopped by tipping perchloric acid into the incubation mixture. The plug of the stopcock was then turned to allow the Hyamine hydroxide to be exposed to the atmosphere of the flask to absorb x4CO2. The temperature in the water bath was then raised to 42°C for 2 hr to liberate any CO~ from the medium. The reaction mixture was transferred to a centrifuge tube, gassed with CO2 and then with air, and kept in the cold. The supernatant was separated by centrifugation, neutralized, and incorporation of radioactivity was determined on suitable aliquots. The remaining supernatant was subjected to radiochromatography for keto-, organic and amino acids (Mounib, 1967b). The residue was washed and lipids were extracted (Mounib, 1967b), and the lipid-free residue was extracted with cold then hot trichloroacetie acid, TCA (Cohen, 1963). The remaining residue, "protein fraction", was washed, dried and hydrolysed as previously described (Mounib, 1967b). Counts of radioactivity were performed on suitable samples of lipid extract, cold TCA, hot TCA (nucleic acid fraction-Cohen, 1963) and hydrolysed protein fractions (Mounib, 1967b). Enzyme assays Sperm of cod were frozen at -20°C, and acetone powder was prepared as required (Prescott & Campbell, 1965). Extracts were made by grinding frozen sperm or their acetone powder, with distilled water, in an all-glass homogenizer, at 4°C. The supernatant was separated by centrifugation (20,000g) for 20 rain
705
CARBON DIOXIDE FIXATION BY SPERM
in the cold. Appropriate aliquots of the supernatant were used for measuring the activity of malic enzyme (Ochoa, 1955), pyruvate carboxylase (Utter & Keech, 1963), phosphoenolpyruvate carboxylase (PEP carboxylase) (Utter & Kurahashi, 1962) and propionyl CoA carboxylase (Tietz & Ochoa, 1962). Concentration of protein in the extract was estimated according to Lowry et al. (1951). RESULTS When spermatozoa of cod were incubated in the presence of 14COs, radioactivity was recovered in both the supematant and the residue of the incubation mixture (Table 1). Spermatozoa of cod incorporated 14COs into keto-, amino and other organic acids (Table 2). It is observed (Table 3) that pyruvate promoted the fixation of CO s by cod sperm. This observation, coupled with our previous finding that incubation of fish sperm with 14C-pyruvate labelled in any position resulted in radioactive malate, oxaloacetate, a-ketoglutarate and aspartate (Mounib, 1967a), TABLE1--INcoRPORATION
OF LABEL FROM l a C O j BY COD SPERM*
Fraction
14C incorporation (counts/min)
Supematant Total hpids Cold TCA extract Hot TCA extract (nucleic acids) Protein
760,702 1,471 18 92 930
* Incubation was carried out in a modified Dixon-Keilin flask for 2 hr at 25°C. Incubation mixture: 1 ml washed sperm (7"7 x 109 sperm) +1"5 ml 1.15% NaCI buffered with phosphate. The double side-arm contained 0"2 ml l~C-bicarbonate (80 /~mole that had 20/~C) in its main compartment, and 0"6 ml 2N HCI in its side-compartment; 14COs was released by tipping HC1 into the 14C-bicarbonate. TABLE
2--INCORPORATION
O F 1 4 C O 2 I N T O KETO, A M I N O A N D O R G A N I C ACIDS B Y C O D S P E R M
Label from t~COz incorporated in: Keto acids cz-Ketoglutaric Oxaloacetic
Pyruvic
Amino acids
Other organic acids
Aspartic Glutamic Alanine
Malic Succinic Fumaric
Glycine Serine Ethanolamine Urea Methioninc sulphoxide
Citric Aconitic GlycoHc Uric Malonic Lactic
706
M.S. Motn~xnANDJ. S. E[s,~
leaves little doubt that pyruvate is involved in COg fixation by sperm. Malonate depressed the CO2 fixation by sperm, whereas pyruvate counteracted the depressive effect of malonate (Table 3). T~LS 3--E~CT OFPYRUVATE AND
MALONATE ON THE INCORPORATION OF LABEL FROM 1 4 C O | BY COD SPERM INTO THE SUPERNATANT OF THE INCUBATION MIXTURE
Total incorporation (counts/min) Substrate added N o substrate
Pyruvate (3"2/~mole)
Without malonate
With malonate*
803,360 1.060,990
350,120 514,830
* Malonate concentration in the incubation mixture is 0"074 M. The participation of CO~ in the formation of uric acid indicates a de novo biosynthesis of purine (Buchanan et al., 1948). Also the presence of radioactive label in urea, glycolic acid and glycine (Table 2) is evidence for the operation of the purine cycle (Brown et al., 1966) and in accord with the presence of uricase, allantoinase and allantoicase in cod sperm (Mounib & Eisan, 1967b). Most of the incorporation of radioactivity in the residue of the incubation mixture was observed in the lipid extract, although small amounts of radioactivity were detectable in cold TCA, hot TCA (nucleic acids) and protein fractions. Extracts of sperm showed activity of malic enzyme, and it was observed that the enzyme depended on the presence of T P N and manganese (Table 4). Search for pyruvate carboxylase, by applying the 14CO2 or the spectrophotometric assay (Utter & Keech, 1963), was without success. TABLE 4
M A L I C ENZYME ACTIVITY IN EXTRACT OF FROZEN SPERM OF COD
System
Specific activity (mpmole TPNH/mg protein per rain)
Complete* - TPN
142"0 0
- MnCls
0
* Complete system contained in pmoles per 3 mh "Iris buffer, pH 7"4, 100; MnCls, 3; T P N , 1; L-malate, 10; and sperm extract (100y of protein). Measurements took place
at room temperature (approx. 22°C). Extracts of the acetone powder of sperm showed activities of PEP and propionyl CoA carboxylase (Table 5). A non-radioactive PEP was also isolated by chromatography from incubations of sperm with pyruvate in the presence of 14COs. Furthermore, when cod sperm were incubated with 14C-pyruvate, labeled in any position, radioactive PEP was produced. In view of the lability of this compound, special care must be taken in its isolation immediately after incubation.
CARBON DIOXIDE FIXATION BY SPERM
707
T~u~ 5--A~rzvn+Y oF PHOSPHORNOLPYRUVATE(PEP) AND OF PROPIONYLCOENZYMBA CARBOXYLASB I N ACETONE POWDER OF COD SPERM
Carboxylase*
Specific activity t (mtzmole/mg protein per min)
PEP Propionyl CoA
14"0 14"5
* Complete system for PEP and propionyl CoA carboxylase was according to Utter & Kurahashi (1962) and Tietz & Ochoa (1962) respectively. Measurements took place at 25°C for PEP carboxylase and at room temperature (approx. 22°C) for propionyl CoA carboxylase. t For PEP carboxylase--l*COi derived from oxaloacetate as a result of the exchange reaction: PEP+IDp+I*COI ~oxaJoacetate+ITP. For propionyl CoA carboxylase-ADP produced according to the reaction: Propionyl CoA + ATP .+- methylmaJonyl CoA + ADP + P. DISCUSSION The present investigation has shown unequivocally that sperm of cod are able to fix CO~. Spermatozoa of cod incorporated CO2 into keto-, amino and other organic acids, lipids, proteins and nucleic acids. The incorporation of t4COa into pyruvate and lactate by sperm (Table 2) could be explained by a COz fixation with pyruvate followed by the formation of a symmetrical intermediate, and the reversibility of these reactions. As malonate is a specific inhibitor to succinic dehydrogenase, and as pyruvate counteracted the depressive effect of malonate on CO2 fixation by sperm, it would appear that a considerable amount of succinate is formed from pyruvate. The succinate originating from pyruvate could be the result of its fixation with COa, followed by a reduction of the product of fixation to succinate (Saz & Vidrine, 1959). This latter possibility is supported by the finding that malonate enhanced incorporation from all carbon atoms of pyruvate into lipids by salmon sperm (Mounib & Eisan, 1966). It is noteworthy that the addition of malonate to treatments that had no pymvate did not bring the incorporation of t4CO++to a complete stop (Table 3). This might be explained by the presence of a natural counteracting mechanism, such as propionyl coenzyme A carboxylase, according to the following scheme: Pyruvofe CHflcodd tide
~C02r=oo iln
J Propionyl CoA
J
methyltool molonylCoAcorbo~y~ose Bio~nthelb
708
M. S. Mo~q~B ~
J. S. EISAN
In the light of the above-mentioned evidence for the presence of a mechanism of COs fixation with pyruvate, and in view of the absence of pyruvate carboxylase, it remains to be seen whether or not the fixation of COs into pyruvate is catalyzed by the malic enzyme. The fixation of CO S with pyruvate by sperm of cod provides an anaplerotic means for the replenishment of oxaloacetate (directly and/or via malate) that is required for the operation of the citric acid cycle, and for the synthesis of amino acids (Kornberg, 1965). This contention is in agreement with the present finding of 14CO2 participation in the synthesis of the intermediates of the citric acid cycle, and also in the formation of amino acids. Our results, thus, could offer an explanation to the stimulatory effect of CO s on the O s uptake of spermatozoa (Humphrey & Mann, 1949; Hamner & Williams, 1964). T h e presence of PEP carboxylase denotes the ability of sperm to synthesize PEP, one of the key metabolites in biosynthesis (Krebs, 1954). REFERENCES A~z~Ius B. A. & MOHRI H. (1966) Mitochondria respiring without exogenous substrate. A study of aged sea urchin spermatozoa. Expl Cell Res. 42, 10-17. BROWN G. W., JR.,JAMES J.,I-Im~m~soN R. J.,THOMAS W. N., ROBZNSON R. O., THOMPSOn A. L., BROWN E. & BROWN S. G. (1966) Uricolytic enzymes in liver of the dipnoan
Protopterus aethiopicus. Science. 153, 1653-1654. BUC~AN J. M., SONNEJ. C. & DELLUVAA. M, (1948) Biological precursors of uric acid-II. The role of lactate, glycine and carbon dioxide as precursors of the carbon chain and nitrogen atom 7 of uric acid. 3. biol. Chem. 173, 81-98. COHENS. (1963) 14CO, fixation and the accumulation of malonic acid in amphibian hybrids (R. pipiens ~ x R. sylvatica ~). Expl Cell Res. 29, 207-211. DIxoN M. (1952) Manometric Methods. pp. 105-128. Cambridge University Press. C. E. & WILLIAMSW. L. (1964) Identification of sperm stimulating factor of rabbit oviduct fluid. Proc. Soc. exp. biol. Med. 117, 240-243. HvMP~mm" G. F. & MANN T. (1949) Studies on the metabolism of semen--5. Citric acid in semen. Bioclwm. J. 44, 97-105. K o ~ E R o H. L. (1965) Anaplerotic sequences in microbial metabolism. Angew. Chem. 4, 558-565. KRm~ H. A. (1954) Considerations concerning the pathways of synthesis in living matter. Synthesis of glycogen from non-carbohydrate precursors. Bull..~ohns Hopkins Hosp. 95, 19-33. LODOE J. R. & SALXSBLrRyG. W. (1963) Factors influencing metabolic activity of bull spermatozoa--VI. Metabolic COs and fructose. ~t. Dairy Sd. 46, 140-144. LowRY O. H., ROS~nnOUOHN. L., FARRA. L. & RANDALLR. J. (1951) Protein measurement with Folin phenol reagent. 3. biol. Chem. 193, 265-275. Mo~n~m M. S. (1967a) Metabolism of pyruvate, acetate and glyoxylate by fish sperm. Comp. Bioct~'m. Physiol. 20, 987-992. Mo~rNIn M. S. (1967b) Metabolism of pyruvate in testes of fish and rabbits with particular reference to p-nitrophenol and 2,4-dinitrophenol. Comp. Biochem. Physiol. 22, 539-548. MouNm M. S. & EmANJ. S. (1966) Lipid biosynthesis from acetate, pyruvate and glyoxylate by salmon sperm. Fed. Proc. 25, 314. MOUNm M. S. & EmAN J. S. (1967a) Fixation of carbon dioxide by cod sperm. Fed, Proc. 26, 645. MOUNIB M. S. & EISANJ. S. (1967b) Uricolytic enzymes in cod sperm. Can. ~. Physiol. Pharraacol. (In press.)
CARBON DIOXIDE F I X A T I O N BY SPERM
709
OCHOAS. (1955) Malic enzyme. In Methods in Ertzyrnology (Edited by COLOWICKS. P. and KAI'L~ N. O.) Vol. 1, pp. 739-753. Academic Press, New York. PamCOTT L. M. & CAMPBELLJ. W. (1965) Phosphoenolpyruvate carboxylase activity and glycogenesis in the flatworm, Hymenolepis diminuta. Comp. Biochem. Physiol. 14, 491-511. SALXSBURYG. W. &KtNNEY W. C., JR. (1957) Factors influencing metabolic activity of bull spermatozoa--III, pH. ~t. Dairy Sd. 40, 1343-1349. S~z H. J. & VmmNE A., JR. (1959) The mechanism of formation of succinate and propionate by z~scaris lumbricoides muscle. ~7. biol. Chem. 234, 2001-2005. Turrz A. & OCHOA S. (1962) Propionyl CoA carboxylase from pig heart. In Methods in Enzymology (Edited by COLOWICK S. P. and KAPLAN N. O.) Vol. V, pp. 570-575. Academic Press, New York. U r r ~ M. F. & K~CH D. B. (1963) Pyruvate carboxylase---I. Nature of the reaction. ~. biol. Chem. 238, 2603-2608. U t-t~a M. F. & K ~ H I K. (1962) Oxalacetic synthesizing enzyme. In Methods in Enzymology (Edited by COLOWlCK S. P. and KAPL~ N. O.) Vol. V, pp. 758-763. Academic Press, New York.
CARBON DIOXIDE FIXATION BY SPERMATOZOA OF COD M. S. M O U N I B and J. S. EISAN Fisheries Research Board of Canada, Halifax Laboratory, P.O. Box 429, Halifax, Nova Scotia, Canada (Received 9 October 1967)
Abstractml. Sperm of cod, in the presence of 14COi, incorPorated label into
keto-, amino and other organic acids, lipids, proteins and nucleic acids. 2. Pyruvate enhanced ltCOi incorporation into the supematant of the incubation mixtttre, whereas malonate depressed it. 3. Extracts of frozen sperm and their acetone powder showed activity of malic enzyme, and of phosphoenolpyruvate and propionyl CoA carboxyhtses. INTRODUCTION THERE has been considerable evidence in the literature indicating that carbon dioxide plays an important part in the metabolism of spermatozoa. Humphrey & Mann (1949) observed that respiration of sperm of ram was higher in the presence of 5 per cent CO2+ 95 per cent 02 than with either air or pure O s. Also Salisbury & Kinney (1957) found a marked stimulation of aerobic glycolysis of sperm, in the presence of metabolic CO s. Under anaerobic conditiom, fructolysis of bull spermatozoa was more with 5 per cent COs+ 95 per cent N s than with N s alone (Lodge & Salisbury, 1963). Recently Afzelins & Mohri (1966) reported that when spermatozoa of sea urchin were incubated in the presence of KOH to remove respiratory COs, there was a reduction in the cristae of the mitochondrlal midpiece, and the reduction paralleled a decrease of endogenous phospholipids of the sperm. They also noticed that the disappearance of cristae was more marked after 1-hr incubation with KOH present than after 6-hr incubation without. Despite the above-mentioned reports, the mechanism of action of COs in the metabolism of sperm is still obscure. However, we have recently suggested that a CO 2 fixation with pyruvate takes place in sperm of cod and salmon (Mounib & Eisan, 1966; Mounib, 1967a). This suggestion stems from two observations: (1) the incorporation of all three carbon atoms of pyruvate, particularly C-1, into the total lipids biosynthesized by salmon sperm (Mounib & Eisan, 1966). (2) Incubation of sperm of cod or salmon with 14C-pyruvate labeled in any position yielded radioactive malate, oxaloacetate, ~-ketoglutarate and aspartate (Mounib, 1967a). These observations strengthened the suggestion that CO s fixation operates in sperm, and the present work was undertaken to examine this proposition by direct methods. A preliminary account of the present investigation has already been reported (Mounib & Eisan, 1967a). 703
704
M.S. Motmm Am) J. S. Exsx~r MATERIAL AND METHODS
Incubation of sperm in the presence of x~70 2 Sperm of cod, Gadus morhua, were collected as previously described (Mounib, 1967a). Sperm were washed by suspending them in cold saline (1.15% NaC1, w/v), followed by centrifugation (1500g at 4°C) for 20 min. The supernatant was discarded, the washing procedure was repeated, the packed sperm were resuspended in cold saline, and penicillin was added (5000 international units/ml of sperm suspension). A special flask was used for incubation studies. The flask was made by American Instrument Co., Silver Spring, Maryland, according to our specifications, and is essentially that of Dixon & Keilin (Dixon, 1952) except that it has one single and one double side-arm instead of one single-arm. One milliliter of sperm suspension + 1.5 ml of saline buffered with phosphate, pH 7.4-, were placed in the main chamber of the flask, and pyrnvate and/or malonate were added to the incubation mixture when required. 14C-sodium bicarbonate (80/,mole that had 20 pC) was placed in the main compartment of the double side-arm, and 0"6 ml of 2 N HCI in the side-compartment. The single side-arm contained 0.2 ml of 3 N perchlorie acid. Hyamine hydroxide 10-X (0.5 ml) was placed in the cup of the stopcock plug, which was turned to isolate the alkaline solution from the atmosphere of the flask. Incubation was carried out in the conventional Warburg bath at 25°C. Ten minutes were allowed for equilibrium, then the 14CO2 was released into the atmosphere of the flask by adding HC1 to the 14C-bicarbonate. The incubation lasted for 2 hr and the reaction was stopped by tipping perchloric acid into the incubation mixture. The plug of the stopcock was then turned to allow the Hyamine hydroxide to be exposed to the atmosphere of the flask to absorb x4CO2. The temperature in the water bath was then raised to 42°C for 2 hr to liberate any CO~ from the medium. The reaction mixture was transferred to a centrifuge tube, gassed with CO2 and then with air, and kept in the cold. The supernatant was separated by centrifugation, neutralized, and incorporation of radioactivity was determined on suitable aliquots. The remaining supernatant was subjected to radiochromatography for keto-, organic and amino acids (Mounib, 1967b). The residue was washed and lipids were extracted (Mounib, 1967b), and the lipid-free residue was extracted with cold then hot trichloroacetie acid, TCA (Cohen, 1963). The remaining residue, "protein fraction", was washed, dried and hydrolysed as previously described (Mounib, 1967b). Counts of radioactivity were performed on suitable samples of lipid extract, cold TCA, hot TCA (nucleic acid fraction-Cohen, 1963) and hydrolysed protein fractions (Mounib, 1967b). Enzyme assays Sperm of cod were frozen at -20°C, and acetone powder was prepared as required (Prescott & Campbell, 1965). Extracts were made by grinding frozen sperm or their acetone powder, with distilled water, in an all-glass homogenizer, at 4°C. The supernatant was separated by centrifugation (20,000g) for 20 rain
705
CARBON DIOXIDE FIXATION BY SPERM
in the cold. Appropriate aliquots of the supernatant were used for measuring the activity of malic enzyme (Ochoa, 1955), pyruvate carboxylase (Utter & Keech, 1963), phosphoenolpyruvate carboxylase (PEP carboxylase) (Utter & Kurahashi, 1962) and propionyl CoA carboxylase (Tietz & Ochoa, 1962). Concentration of protein in the extract was estimated according to Lowry et al. (1951). RESULTS When spermatozoa of cod were incubated in the presence of 14COs, radioactivity was recovered in both the supematant and the residue of the incubation mixture (Table 1). Spermatozoa of cod incorporated 14COs into keto-, amino and other organic acids (Table 2). It is observed (Table 3) that pyruvate promoted the fixation of CO s by cod sperm. This observation, coupled with our previous finding that incubation of fish sperm with 14C-pyruvate labelled in any position resulted in radioactive malate, oxaloacetate, a-ketoglutarate and aspartate (Mounib, 1967a), TABLE1--INcoRPORATION
OF LABEL FROM l a C O j BY COD SPERM*
Fraction
14C incorporation (counts/min)
Supematant Total hpids Cold TCA extract Hot TCA extract (nucleic acids) Protein
760,702 1,471 18 92 930
* Incubation was carried out in a modified Dixon-Keilin flask for 2 hr at 25°C. Incubation mixture: 1 ml washed sperm (7"7 x 109 sperm) +1"5 ml 1.15% NaCI buffered with phosphate. The double side-arm contained 0"2 ml l~C-bicarbonate (80 /~mole that had 20/~C) in its main compartment, and 0"6 ml 2N HCI in its side-compartment; 14COs was released by tipping HC1 into the 14C-bicarbonate. TABLE
2--INCORPORATION
O F 1 4 C O 2 I N T O KETO, A M I N O A N D O R G A N I C ACIDS B Y C O D S P E R M
Label from t~COz incorporated in: Keto acids cz-Ketoglutaric Oxaloacetic
Pyruvic
Amino acids
Other organic acids
Aspartic Glutamic Alanine
Malic Succinic Fumaric
Glycine Serine Ethanolamine Urea Methioninc sulphoxide
Citric Aconitic GlycoHc Uric Malonic Lactic
706
M.S. Motn~xnANDJ. S. E[s,~
leaves little doubt that pyruvate is involved in COg fixation by sperm. Malonate depressed the CO2 fixation by sperm, whereas pyruvate counteracted the depressive effect of malonate (Table 3). T~LS 3--E~CT OFPYRUVATE AND
MALONATE ON THE INCORPORATION OF LABEL FROM 1 4 C O | BY COD SPERM INTO THE SUPERNATANT OF THE INCUBATION MIXTURE
Total incorporation (counts/min) Substrate added N o substrate
Pyruvate (3"2/~mole)
Without malonate
With malonate*
803,360 1.060,990
350,120 514,830
* Malonate concentration in the incubation mixture is 0"074 M. The participation of CO~ in the formation of uric acid indicates a de novo biosynthesis of purine (Buchanan et al., 1948). Also the presence of radioactive label in urea, glycolic acid and glycine (Table 2) is evidence for the operation of the purine cycle (Brown et al., 1966) and in accord with the presence of uricase, allantoinase and allantoicase in cod sperm (Mounib & Eisan, 1967b). Most of the incorporation of radioactivity in the residue of the incubation mixture was observed in the lipid extract, although small amounts of radioactivity were detectable in cold TCA, hot TCA (nucleic acids) and protein fractions. Extracts of sperm showed activity of malic enzyme, and it was observed that the enzyme depended on the presence of T P N and manganese (Table 4). Search for pyruvate carboxylase, by applying the 14CO2 or the spectrophotometric assay (Utter & Keech, 1963), was without success. TABLE 4
M A L I C ENZYME ACTIVITY IN EXTRACT OF FROZEN SPERM OF COD
System
Specific activity (mpmole TPNH/mg protein per rain)
Complete* - TPN
142"0 0
- MnCls
0
* Complete system contained in pmoles per 3 mh "Iris buffer, pH 7"4, 100; MnCls, 3; T P N , 1; L-malate, 10; and sperm extract (100y of protein). Measurements took place
at room temperature (approx. 22°C). Extracts of the acetone powder of sperm showed activities of PEP and propionyl CoA carboxylase (Table 5). A non-radioactive PEP was also isolated by chromatography from incubations of sperm with pyruvate in the presence of 14COs. Furthermore, when cod sperm were incubated with 14C-pyruvate, labeled in any position, radioactive PEP was produced. In view of the lability of this compound, special care must be taken in its isolation immediately after incubation.
CARBON DIOXIDE FIXATION BY SPERM
707
T~u~ 5--A~rzvn+Y oF PHOSPHORNOLPYRUVATE(PEP) AND OF PROPIONYLCOENZYMBA CARBOXYLASB I N ACETONE POWDER OF COD SPERM
Carboxylase*
Specific activity t (mtzmole/mg protein per min)
PEP Propionyl CoA
14"0 14"5
* Complete system for PEP and propionyl CoA carboxylase was according to Utter & Kurahashi (1962) and Tietz & Ochoa (1962) respectively. Measurements took place at 25°C for PEP carboxylase and at room temperature (approx. 22°C) for propionyl CoA carboxylase. t For PEP carboxylase--l*COi derived from oxaloacetate as a result of the exchange reaction: PEP+IDp+I*COI ~oxaJoacetate+ITP. For propionyl CoA carboxylase-ADP produced according to the reaction: Propionyl CoA + ATP .+- methylmaJonyl CoA + ADP + P. DISCUSSION The present investigation has shown unequivocally that sperm of cod are able to fix CO~. Spermatozoa of cod incorporated CO2 into keto-, amino and other organic acids, lipids, proteins and nucleic acids. The incorporation of t4COa into pyruvate and lactate by sperm (Table 2) could be explained by a COz fixation with pyruvate followed by the formation of a symmetrical intermediate, and the reversibility of these reactions. As malonate is a specific inhibitor to succinic dehydrogenase, and as pyruvate counteracted the depressive effect of malonate on CO2 fixation by sperm, it would appear that a considerable amount of succinate is formed from pyruvate. The succinate originating from pyruvate could be the result of its fixation with COa, followed by a reduction of the product of fixation to succinate (Saz & Vidrine, 1959). This latter possibility is supported by the finding that malonate enhanced incorporation from all carbon atoms of pyruvate into lipids by salmon sperm (Mounib & Eisan, 1966). It is noteworthy that the addition of malonate to treatments that had no pymvate did not bring the incorporation of t4CO++to a complete stop (Table 3). This might be explained by the presence of a natural counteracting mechanism, such as propionyl coenzyme A carboxylase, according to the following scheme: Pyruvofe CHflcodd tide
~C02r=oo iln
J Propionyl CoA
J
methyltool molonylCoAcorbo~y~ose Bio~nthelb
708
M. S. Mo~q~B ~
J. S. EISAN
In the light of the above-mentioned evidence for the presence of a mechanism of COs fixation with pyruvate, and in view of the absence of pyruvate carboxylase, it remains to be seen whether or not the fixation of COs into pyruvate is catalyzed by the malic enzyme. The fixation of CO S with pyruvate by sperm of cod provides an anaplerotic means for the replenishment of oxaloacetate (directly and/or via malate) that is required for the operation of the citric acid cycle, and for the synthesis of amino acids (Kornberg, 1965). This contention is in agreement with the present finding of 14CO2 participation in the synthesis of the intermediates of the citric acid cycle, and also in the formation of amino acids. Our results, thus, could offer an explanation to the stimulatory effect of CO s on the O s uptake of spermatozoa (Humphrey & Mann, 1949; Hamner & Williams, 1964). T h e presence of PEP carboxylase denotes the ability of sperm to synthesize PEP, one of the key metabolites in biosynthesis (Krebs, 1954). REFERENCES A~z~Ius B. A. & MOHRI H. (1966) Mitochondria respiring without exogenous substrate. A study of aged sea urchin spermatozoa. Expl Cell Res. 42, 10-17. BROWN G. W., JR.,JAMES J.,I-Im~m~soN R. J.,THOMAS W. N., ROBZNSON R. O., THOMPSOn A. L., BROWN E. & BROWN S. G. (1966) Uricolytic enzymes in liver of the dipnoan
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