Juurnul of Neurucheinisiry. 1976. Vol 21. pp. 813 X15. Pergamon Press Printed
in
Great Britain
SHORT COMMUNICATION Subcellular localization of enzymes oxidizing citrate in the rat brain (Received 12 February 1976. Accepted 5 April 1976) THEUTlLlZATlON of citrate in brain, as in h e r , in addition to its tkr.eo-o,-isocitrate: NAD' oxidoreductase (IDH NAD)mediated oxidation in the Krebs cycle, is dependent on two other enzyme systems: (1) ATP: Citrate oxaloacetatelyase (ATP citrate lyase), which has been reported to be present in brain cytosol ( T u ~ E K ,1967a). (2) of T/,reo-D,-Isocitrate: NADP' oxidoreductase (IDH NADP) (GROMEK& RAFALOWSKA,1972), found in both the cytoplasmic and mitochondria1 fraction of the brain (RAFALOWSKA et ol., 1 9 7 4 ~ WATANABE ; et al., 1974). In contrast to the data on liver enzymes, studies on the subcellular localization of those enzymes in the nerve cells are fragmentary; no direct comparison has been made of the degree of citrate oxidation in various subcellular fractions involving either of the two metabolic pathways. Therefore in the present work the activities and the distribution of both enzymes responsible for citrate oxidation were determined in different brain subcellular fractions in order to establish which of the two routes of citrate oxidation plays an essential role in the metabolism of the CNS.
and bromophenol blue were layered into 7:0 polyacrylamide gels. Current intensity was 4 mA per 5 mm thick gel. Electrophoresis was run for 1.5 h. Gels were stained with the NBT method according to HENDERSON (1965). RESULTS AND DISCUSSION
In the subcellular fractions protein recovery was about 100a& The distribution of the subcellular marker activities (Fig. la,b) indicates a high purity of the fractions. The results presented in Fig. l e confirm the report of T U ~ E K (1967a) that the ATP citrate lyase is localized in the soluble fractions of the brain: S3 and W,. The presence of small amounts of the enzyme in fractions C, M and W, may be attributed instead to contamination with the soluble fraction, since the distribution of ATP citrate lyase in the former is consistent with that of the LDH activity (Fig. lb). Comparison of the RSA' values for fractions B (1.55) and S, (1.82) shows that the ATP citrate lyasedependent oxidation of citrate is greater in cytosol than in the synaptosomal fraction. IDH NADP is present in both soluble fractions S3 and W,, as well as in the particulate fractions C and W, (Fig. Ic), its specific activity in MATERIAL AND METHODS mitochondria being about 3 times higher than in cytosol Wistar rats. weighing about 200 g were used. Brain sub- (Fig. Id). It is worth mentioning that the values reported here are cellular fractions were obtained by the method of WHIThigher than those obtained in the previous work TAKER (1972) by differential centrifugation in sucrose graet al., 1974~).This difference is due to dients. Synaptosomes were subjected to hypo-osmotic (RAFALOWSKA shock and were separated into fractions W, and W, by changed conditions of disruption of mitochondria. In the centrifugation for 30 min at 17,OOOg. The cross contami- present study it has been shown that optimal disruption nation of the fractions were estimated by assaying the sub- of mitochondria with respect to NADP penetration to the cellular marker enzymes 1-mnlate hydro-lyase (fumarase) enzyme is achieved by using 0.2";, Triton X-100. Lower by the method of RACKER(1950) and L-lactate: NAD' Triton concentrations and sonification do not release the oxidoreductase (LDH) according to JOHNSON(1960). ATP total enzyme activity. Triton X-100 in concentrations up citrate lyase was determined at room temp. (22°C) accord- to 0.2;/, does not affect the cytoplasmic IDH NADP. A et trl. (19740) and IDH NADP by the higher IDH NADP activity in mitochondria than in cytoing to SZUTOWICZ (1963) (1955). Protein was estimated by the sol has also been observed by BAKER& NEWBURCH procedure of OCHOA method of LOWRYe t al. (1951). Prior to electrophoresis in chick brain and liver but not in heart. where the oppo& SMITH the subcellular fractions were diluted 1 : l with a solution site was found. Data reported by LOWENSTEIN containing: 0.02 M-Tris buffer, pH 7.75. 4 nlM-DL-sodium (1962) and HENDERSON(1965) suggest that in liver, the isocitrate, 10 mwmagnesium chloride and 0.01 M-sodium mitochondria1 and cytoplasmic IDH NADP represent two sulphate. Samples containing 100 pg of protein, glycerol isoenzymes. Polyacrylamide gel electrophoresis of the cerebral enzymes revealed that the same holds true for the Abbrecintiorts used: IDH NADP. thrro-o,-isocitrate: brain mitochondrial and cytoplasmic IDH NADP (Fig. 2). The results obtained in the present study suggest that, NAD' oxidoreductase (EC 1.1.1.42); I D H NAD, threoi>,-isocitrate:NAD' oxidoreductase (EC 1.1.1.41); ATP as in liver mitochondria, citrate oxidation in brain mitocitrate lyase, ATP:citrate oxaloacetate-lyase (EC 4.1.3.8); chondria is dependent on the presence of the mitochonfumarase, L-malate hydro-lyase (EC 4.2.1.2); LDH, L-lact- drial IDH NADP isoenzyme, by-passing the ATP citrate lyase. In synaptosomes and in cytosol, citrate is oxidized ate:NAD+ oxidoreductase (EC 1.1.1.27). 'RSA was calculated as the ratio: percentage of total with the participation of two enzymes: cytoplasmic IDH recovered activity found in fraction to percentage of total NADP and ATP citrate lyase. The existence of two different routes of citrate oxidation in the soluble fractions of protein found in the same fraction. 813 Y C . 27.7- K
Short communication
di 2
I
3
IDH-NADP
I
"
ATP citrate lyme
2
FIG. I . Distribution of total and specific activities of enzymes oxidizing citrate i n subcellular fractions of brain tissue and total activities of enzyme markers. The particulate fractions were treated with 0.2". Triton X-100 prior to determinations. Assays were performed in the presence of 0.5 pwrotenone. Values presented in Fig. la. b. c. c refer to total activity ( = IOO",) of: I-initial homogenate; 2-P, fraction. 3-B. fraction. IDH NADP to ATP citrate lyase specific activity ratios i n particular fractions B-7.4. Data are the results from one typical experiment were: S , 4 . 9 ; W , - 4 . l : C 34.4: W,-9.4: similar to the four performed. P , : Crude. first pellet (nuclei. tissue debris); P2: Crude mitochondria containing: myelin. synaptosomes and mitochondrii: S, : cytosol; M. microsomes; A. myelin; B, synaptosomes: C: Mitochondria: W,. slnaptosomal particulate fraction; W,: Synaptosomal soluble fraction: H Homogenate the brain is related to the supply of different substrates. GATFIELD er 01.. 1966). the citrate content practically Acetyl-CoA produced i n the presence of ATP citrate Iyase. remains at a constant level (DUFFY et al., 1972; and NADPH as a product of the IDH NADP-mediated RAFAIO\VSLA er ( I / . . 1975). oxidation, may lead 10 acet)lcholine ( T L t t K . 1967tr) and fatty acid (BRADY.1960) synthesis; 2-oxoglutarate. formed DepLirtnirnr of Nt.itrochemisrr-y, URSZULA RAFALOWSKA in the reaction catalysed by IDH NADP. may enter the M d i c a l Rtsetirch Crntre. HANNAK S I ~ Z A K transaniinase reaction or may penetrate mitochondria in Polish k t i r f t V i J of Scietices, the exchange reaction with malate. at the same time parti00-784 Warsaw. 3 Dworkowci Str., cipating in the transfer of reduction equivalents ( Q ~ ~ A G - Polontf LIARELLO & PAPA.1969). The higher specific activity of IDH NADP than that of ATP citrate lyase (Fig. 1d.f) indicates that in brain. the citrate utilization is greater in REFERENCES the IDH NADP-dependent reaction. R. W. (1963) Biochem. J. 89, BAKERW. W. & NEWBURGH The activity of the two enzjmes is due to regulation 51&515. by pyridine and adenine nucleotides and a number of BRADYR. 0. (1960) J. hiol. Chem. 235, 3099-3103. amino acids ( R A t A L O L V S K A t't (11. iY74a.h 1975: SZUTOWICZ DUFFYT. E.. NELSONS. R. &: LOWRY0. H. (1972) J. Neuret d.. 1974h. 1975). ocheni. 19, 959-977. Taking into account the results of the present work and P. D.. LOWRY0. H., SCHULZD. & PASSONNEAU the facts that: ( I ) ATP in concentrations activating ATP GATFIELD J. V. (1966) J. Nrirrochem. 13, 185-195. citrate lyase inhibits IDH NADP activity and (2) 2-oxogluA. & RAFALOWSKA U. (1972) J . Nrirrochem. 19, tarate and glutamate--the products of IDH NADP- GROMEK & RAFALOWSKA. 268772695, mediated citratr oxidation (GROMEK ~ S. (1965) J . cup. Zoo/. 158, 263-274. 1 9 7 2 t a r e inhibitors of ATP citrate lyase (SLCTOWIT7 et H E N D E R WN. ul.. 1974h. 1975). it may be assumcd that these t n o meta- JOHNWNM. K . (1960) Biocltrrn. J . 77, 61@618. bolic routes complement each other. This may be of par- LOWrNSTElU J. M. & SMITHs. R. (1962) Biochini. hiophys. ticular importance during brain hypoxia, when notwithActu 56, 385-387. standing changes in the levels of a number of factors regu- LOWRY0. H.. ROSEBRUUGHN. J., FARRA. L. & R A N D A I . ~ . lating the activities of these enzymes (DGFFYt't nl.. 1972: R. J. (19511 J . h i d Chenr. 193, 265-275.
FIG.2. Electrophoresis of IDH NADP in subcellular fractions of the rat brain. Details as in Fig. 1.
vr-814
Short communication
OCHOA s. (19.55) in M t , f l i ~ t l if1~ E/iZ)'t?IfJlOgJ'(COl-OWlrK S. P. & KAPLANN. 0. eds.) Vol. 1, pp. 699-704. Academic Press, New York. E. & PAPAS. (1969) in hfitochotirlrirr SrriicQUAGLIARIELLO lure und Funclioti (ERNSTERL. & DRAHOTA Z., eds.) pp. 335-346. Academic Press, New York. RACER E. (1950) Biochirm hioph!,s. Acru 4, 21 1-214. RAFALOWSKA U., PASTUSZKO A. & GROMEIC A. ( 1 9 7 4 ~Bull. ) 4 c d . pol. Sci. CI. If S i r . Sci. h i d . 22, 453459. RAFALOWSKAU., PASTLISZKO A. & GROMEKA. (1974h) FEBS Leu. 42, 1&22. RAFALOWSKAU., ERECINSKA M. & CHANCE B. (1975) J . Nezirocliem. 25. 497-501.
8 1
SZL!TCIWICZ A,, S T E P I EM., ~ LYSIAKW. SC A ~ G I I L S K S.~ (19744) Actii hiochim. pol. 21, N o 3. 331-338. SZUTOWICZ A,. STcPltk M. & ANGIELSKI s. (1974h) J . N
in
Great Britain
SHORT COMMUNICATION Subcellular localization of enzymes oxidizing citrate in the rat brain (Received 12 February 1976. Accepted 5 April 1976) THEUTlLlZATlON of citrate in brain, as in h e r , in addition to its tkr.eo-o,-isocitrate: NAD' oxidoreductase (IDH NAD)mediated oxidation in the Krebs cycle, is dependent on two other enzyme systems: (1) ATP: Citrate oxaloacetatelyase (ATP citrate lyase), which has been reported to be present in brain cytosol ( T u ~ E K ,1967a). (2) of T/,reo-D,-Isocitrate: NADP' oxidoreductase (IDH NADP) (GROMEK& RAFALOWSKA,1972), found in both the cytoplasmic and mitochondria1 fraction of the brain (RAFALOWSKA et ol., 1 9 7 4 ~ WATANABE ; et al., 1974). In contrast to the data on liver enzymes, studies on the subcellular localization of those enzymes in the nerve cells are fragmentary; no direct comparison has been made of the degree of citrate oxidation in various subcellular fractions involving either of the two metabolic pathways. Therefore in the present work the activities and the distribution of both enzymes responsible for citrate oxidation were determined in different brain subcellular fractions in order to establish which of the two routes of citrate oxidation plays an essential role in the metabolism of the CNS.
and bromophenol blue were layered into 7:0 polyacrylamide gels. Current intensity was 4 mA per 5 mm thick gel. Electrophoresis was run for 1.5 h. Gels were stained with the NBT method according to HENDERSON (1965). RESULTS AND DISCUSSION
In the subcellular fractions protein recovery was about 100a& The distribution of the subcellular marker activities (Fig. la,b) indicates a high purity of the fractions. The results presented in Fig. l e confirm the report of T U ~ E K (1967a) that the ATP citrate lyase is localized in the soluble fractions of the brain: S3 and W,. The presence of small amounts of the enzyme in fractions C, M and W, may be attributed instead to contamination with the soluble fraction, since the distribution of ATP citrate lyase in the former is consistent with that of the LDH activity (Fig. lb). Comparison of the RSA' values for fractions B (1.55) and S, (1.82) shows that the ATP citrate lyasedependent oxidation of citrate is greater in cytosol than in the synaptosomal fraction. IDH NADP is present in both soluble fractions S3 and W,, as well as in the particulate fractions C and W, (Fig. Ic), its specific activity in MATERIAL AND METHODS mitochondria being about 3 times higher than in cytosol Wistar rats. weighing about 200 g were used. Brain sub- (Fig. Id). It is worth mentioning that the values reported here are cellular fractions were obtained by the method of WHIThigher than those obtained in the previous work TAKER (1972) by differential centrifugation in sucrose graet al., 1974~).This difference is due to dients. Synaptosomes were subjected to hypo-osmotic (RAFALOWSKA shock and were separated into fractions W, and W, by changed conditions of disruption of mitochondria. In the centrifugation for 30 min at 17,OOOg. The cross contami- present study it has been shown that optimal disruption nation of the fractions were estimated by assaying the sub- of mitochondria with respect to NADP penetration to the cellular marker enzymes 1-mnlate hydro-lyase (fumarase) enzyme is achieved by using 0.2";, Triton X-100. Lower by the method of RACKER(1950) and L-lactate: NAD' Triton concentrations and sonification do not release the oxidoreductase (LDH) according to JOHNSON(1960). ATP total enzyme activity. Triton X-100 in concentrations up citrate lyase was determined at room temp. (22°C) accord- to 0.2;/, does not affect the cytoplasmic IDH NADP. A et trl. (19740) and IDH NADP by the higher IDH NADP activity in mitochondria than in cytoing to SZUTOWICZ (1963) (1955). Protein was estimated by the sol has also been observed by BAKER& NEWBURCH procedure of OCHOA method of LOWRYe t al. (1951). Prior to electrophoresis in chick brain and liver but not in heart. where the oppo& SMITH the subcellular fractions were diluted 1 : l with a solution site was found. Data reported by LOWENSTEIN containing: 0.02 M-Tris buffer, pH 7.75. 4 nlM-DL-sodium (1962) and HENDERSON(1965) suggest that in liver, the isocitrate, 10 mwmagnesium chloride and 0.01 M-sodium mitochondria1 and cytoplasmic IDH NADP represent two sulphate. Samples containing 100 pg of protein, glycerol isoenzymes. Polyacrylamide gel electrophoresis of the cerebral enzymes revealed that the same holds true for the Abbrecintiorts used: IDH NADP. thrro-o,-isocitrate: brain mitochondrial and cytoplasmic IDH NADP (Fig. 2). The results obtained in the present study suggest that, NAD' oxidoreductase (EC 1.1.1.42); I D H NAD, threoi>,-isocitrate:NAD' oxidoreductase (EC 1.1.1.41); ATP as in liver mitochondria, citrate oxidation in brain mitocitrate lyase, ATP:citrate oxaloacetate-lyase (EC 4.1.3.8); chondria is dependent on the presence of the mitochonfumarase, L-malate hydro-lyase (EC 4.2.1.2); LDH, L-lact- drial IDH NADP isoenzyme, by-passing the ATP citrate lyase. In synaptosomes and in cytosol, citrate is oxidized ate:NAD+ oxidoreductase (EC 1.1.1.27). 'RSA was calculated as the ratio: percentage of total with the participation of two enzymes: cytoplasmic IDH recovered activity found in fraction to percentage of total NADP and ATP citrate lyase. The existence of two different routes of citrate oxidation in the soluble fractions of protein found in the same fraction. 813 Y C . 27.7- K
Short communication
di 2
I
3
IDH-NADP
I
"
ATP citrate lyme
2
FIG. I . Distribution of total and specific activities of enzymes oxidizing citrate i n subcellular fractions of brain tissue and total activities of enzyme markers. The particulate fractions were treated with 0.2". Triton X-100 prior to determinations. Assays were performed in the presence of 0.5 pwrotenone. Values presented in Fig. la. b. c. c refer to total activity ( = IOO",) of: I-initial homogenate; 2-P, fraction. 3-B. fraction. IDH NADP to ATP citrate lyase specific activity ratios i n particular fractions B-7.4. Data are the results from one typical experiment were: S , 4 . 9 ; W , - 4 . l : C 34.4: W,-9.4: similar to the four performed. P , : Crude. first pellet (nuclei. tissue debris); P2: Crude mitochondria containing: myelin. synaptosomes and mitochondrii: S, : cytosol; M. microsomes; A. myelin; B, synaptosomes: C: Mitochondria: W,. slnaptosomal particulate fraction; W,: Synaptosomal soluble fraction: H Homogenate the brain is related to the supply of different substrates. GATFIELD er 01.. 1966). the citrate content practically Acetyl-CoA produced i n the presence of ATP citrate Iyase. remains at a constant level (DUFFY et al., 1972; and NADPH as a product of the IDH NADP-mediated RAFAIO\VSLA er ( I / . . 1975). oxidation, may lead 10 acet)lcholine ( T L t t K . 1967tr) and fatty acid (BRADY.1960) synthesis; 2-oxoglutarate. formed DepLirtnirnr of Nt.itrochemisrr-y, URSZULA RAFALOWSKA in the reaction catalysed by IDH NADP. may enter the M d i c a l Rtsetirch Crntre. HANNAK S I ~ Z A K transaniinase reaction or may penetrate mitochondria in Polish k t i r f t V i J of Scietices, the exchange reaction with malate. at the same time parti00-784 Warsaw. 3 Dworkowci Str., cipating in the transfer of reduction equivalents ( Q ~ ~ A G - Polontf LIARELLO & PAPA.1969). The higher specific activity of IDH NADP than that of ATP citrate lyase (Fig. 1d.f) indicates that in brain. the citrate utilization is greater in REFERENCES the IDH NADP-dependent reaction. R. W. (1963) Biochem. J. 89, BAKERW. W. & NEWBURGH The activity of the two enzjmes is due to regulation 51&515. by pyridine and adenine nucleotides and a number of BRADYR. 0. (1960) J. hiol. Chem. 235, 3099-3103. amino acids ( R A t A L O L V S K A t't (11. iY74a.h 1975: SZUTOWICZ DUFFYT. E.. NELSONS. R. &: LOWRY0. H. (1972) J. Neuret d.. 1974h. 1975). ocheni. 19, 959-977. Taking into account the results of the present work and P. D.. LOWRY0. H., SCHULZD. & PASSONNEAU the facts that: ( I ) ATP in concentrations activating ATP GATFIELD J. V. (1966) J. Nrirrochem. 13, 185-195. citrate lyase inhibits IDH NADP activity and (2) 2-oxogluA. & RAFALOWSKA U. (1972) J . Nrirrochem. 19, tarate and glutamate--the products of IDH NADP- GROMEK & RAFALOWSKA. 268772695, mediated citratr oxidation (GROMEK ~ S. (1965) J . cup. Zoo/. 158, 263-274. 1 9 7 2 t a r e inhibitors of ATP citrate lyase (SLCTOWIT7 et H E N D E R WN. ul.. 1974h. 1975). it may be assumcd that these t n o meta- JOHNWNM. K . (1960) Biocltrrn. J . 77, 61@618. bolic routes complement each other. This may be of par- LOWrNSTElU J. M. & SMITHs. R. (1962) Biochini. hiophys. ticular importance during brain hypoxia, when notwithActu 56, 385-387. standing changes in the levels of a number of factors regu- LOWRY0. H.. ROSEBRUUGHN. J., FARRA. L. & R A N D A I . ~ . lating the activities of these enzymes (DGFFYt't nl.. 1972: R. J. (19511 J . h i d Chenr. 193, 265-275.
FIG.2. Electrophoresis of IDH NADP in subcellular fractions of the rat brain. Details as in Fig. 1.
vr-814
Short communication
OCHOA s. (19.55) in M t , f l i ~ t l if1~ E/iZ)'t?IfJlOgJ'(COl-OWlrK S. P. & KAPLANN. 0. eds.) Vol. 1, pp. 699-704. Academic Press, New York. E. & PAPAS. (1969) in hfitochotirlrirr SrriicQUAGLIARIELLO lure und Funclioti (ERNSTERL. & DRAHOTA Z., eds.) pp. 335-346. Academic Press, New York. RACER E. (1950) Biochirm hioph!,s. Acru 4, 21 1-214. RAFALOWSKA U., PASTUSZKO A. & GROMEIC A. ( 1 9 7 4 ~Bull. ) 4 c d . pol. Sci. CI. If S i r . Sci. h i d . 22, 453459. RAFALOWSKAU., PASTLISZKO A. & GROMEKA. (1974h) FEBS Leu. 42, 1&22. RAFALOWSKAU., ERECINSKA M. & CHANCE B. (1975) J . Nezirocliem. 25. 497-501.
8 1
SZL!TCIWICZ A,, S T E P I EM., ~ LYSIAKW. SC A ~ G I I L S K S.~ (19744) Actii hiochim. pol. 21, N o 3. 331-338. SZUTOWICZ A,. STcPltk M. & ANGIELSKI s. (1974h) J . N
