Neurochemical Research (2) 671--680 (1977)
REGIONAL AND SUBCELLULAR DISTRIBUTION OF T A U R I N E S Y N T H E S I Z I N G E N Z Y M E S IN THE RAT C E N T R A L N E R V O U S SYSTEM H. PASANTES-MORALES,1 C , LORIETTE, AND F. CHATAGNER Laboratoire de Chimie Biologique 96 Boulevard Raspail, 75006, Paris, France
Accepted June 6, 1977
The distribution of cysteine oxidase (CO) and cysteine sulfinate decarboxylase (CSD) was examined in 12 regions of the rat central nervous system (CNS). The distribution of CO activity, expressed as ~mol of cysteine sulfinate formed per h per g, was the following: hypothalamus, superior and inferior colliculi, 94-99 tzmol/h/g; olfactory bulbs, cerebral cortex, striatum, and hippocampus, 44-51 ~mol/h/g; cerebellum, 71 tzmol/h/g; pons-medula and spinal cord, 94 and 60 /xmol/h/g, respectively. The distribution of CSD activity expressed as /xmol of cysteine sulfinate decarboxylated per h per g was the following: hypothalamus and colliculi, 14-21 /~mol/h/g; olfactory bulbs, cerebral cortex, striatum, hippocampus, and cerebellum, 8-13/zmol/h/g; pons-medulla, 7.3; and spinal cord, 3.6 /~mol/h/g. No CSD activity was detected in sciatic nerve. The subcellular distribution of CO and CSD activities was studied in hypothalamus, coUiculi, and cerebral cortex. CO activity was localized in synaptosomes, mitochondria, and microsomes. CSD was primarily confined to the crude mitochondrial fraction and after subfraction, recovered mainly in the synaptosomal fraction.
INTRODUCTION Taurine is unevenly distributed in the CNS. The taurine content of nervous areas considerably differs, showing a variation of over sixfold between regions showing the lowest and the highest taurine concentration (1-3). 1 On leave from Instituto de Biologia, Universidad Nacional Autonoma de Mrxico, Apartado Postal 70-600, M6xico 20, D.F., M6xico.
671 This journal is copyrighted by Plenum. Each article is available for $7.50 from Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011,
672
PASANTES-MORALES ET AL.
The process(es) regulating taurine levels in the nervous system is (are) still unknown. A correlation between the mechanisms presumably involved in maintaining taurine levels in brain areas and its regional distribution has not been clearly established (1,2). However, it has been recently described (2) that taurine formation from intracisternally injected cysteine appears to be proportional to the endogenous taurine concentration in rat brain regions. Therefore, we thought it of interest to determine the regional distribution of taurine-synthesizing enzymes in brain. The main pathway for taurine synthesis in brain appears to be that involving the oxidation of cysteine to cysteine sulfinic acid, followed by decarboxylation to form hypotaurine and the subsequent oxidation of hypotaurine to taurine (4). The first two reactions are catalyzed by cysteine oxidase (CO), (L-cysteine:oxygen oxidoreductase, EC 1.13.11.20) and cysteine sulfinate decarboxylase (CSD) (L-cysteine sulfinate carboxylyase, EC 4.1.1.29), respectively. The activity of these enzymes has been demonstrated in brain (5-10); the enzymatic activity responsible for the last step in taurine formation, namely the hypotaurine oxidation, has not yet been found in nervous tissue. Systematic studies on the regional distribution of CSD have not been carried out. Previous work of Agrawal et al. (9) has shown only small differences in CSD activity in four regions of rat CNS. More significant differences were found by Piha and Saukkonen (11) in regions of the calf brain. The distribution of CO activity in seven regions of rat brain has been recently reported by Misra and Olney (7). This paper describes the distribution of CO and CSD activities in 12 regions of rat CNS. Studies on the subcellular distribution of CSD activity in the whole brain have consistently revealed that a high proportion of the enzyme activity is associated with the isolated nerve endings (6,9,10). In contrast, the subcellular distribution of CO activity is controversial, the enzymatic activity being either observed associated with the synaptosomal fraction (6) or recovered in the soluble fraction associated with microsomes (7). In the present study, we have measured the activity of CO and CSD in fractions and subfractions obtained from four regions of rat brain.
EXPERIMENTAL PROCEDURE Male, adult rats (200-300 g), Sherman strain, were used in this study. After decapitation, cervical spinal cord and brain were removed and regions of brain dissected according to Glowinski and Iversen (12). Tissue was pooled when necessary and homogenized in 0.4% Triton-X-100 or in 0.32 M sucrose for fractionation studies. Sciatic nerves were cut into small pieces and homogenized in a glass-glass homogenizer.
TAURINE-SYNTHESIZING ENZYMES IN RAT CNS
673
Subcellular Fractionation Primary fractions were separated from sucrose homogenates (10:1, voUwt) according to the procedure of Gray and Whittaker (13). The homogenate was centrifuged at 800g for 10 rain to obtain the crude nuclear fraction and then at 12,000g for 20 rain to separate the crude mitochondrial fraction. The supernatant was centrifuged at 105,000g for 60 min to separate the microsomal fraction. For preparation of subfractions, the crude mitochondrial fraction was resuspended in 0.32 M sucrose, and ! ml of this suspension was layered over a discontinuous density gradient consisting of 2 ml each of 0.8 M and 1.2 M sucrose. The gradient was centrifuged at 50,000g for 120 vain in a SW-50-1 rotor in a Beckman-Spinco ultracentrifuge. The subcellular fractions myelin, synaptosomal, and mitochondrial were obtained, Triton-X-100 (0.4% final concentration) was added to all fractions before enzyme assays.
Enzyme Assays CO activity was assayed by the method of Yamaguchi et al. (14) as modified by Misra and Olney (7). Homogenates were prepared in 0.02 M phosphate buffer, pH 6.8, or in 0.32 M sucrose for fractionation experiments. The homogenate or fractions (0.4 ml) were incubated for 30 rain in a mixture containing 0.05 M phosphate buffer, pH 6.8, 0.25 mM Fe(NH4)2(SO4)~'6H20, 5 mM hydroxylamine-HC1, 2 mM NAD, 77 mM neutralized Lcysteine, and 1/zCi of D,L-[14C]-3-cysteine in a final volume of 2 ml. After incubation, the reaction was stopped by addition of 1 ml of 8% TCA. Samples were centrifuged and the acid-soluble fraction was applied to a Dowex-50X4H + column. The column was washed with 10 ml of water, and the radioactivity of aliquots of the eluate was estimated after the addition of Unisolve (Koch-Light). Values of blanks, in which the tissue was added only after the addition of TCA, were subtracted from the sample values. CSD was assayed by following the decarboxylation of L-l-[14C]-cysteine sulfinic acid as previously described (10). The incubation mixture contained 0.067 M phosphate buffer, pH 6.8, 33 mM neutralized unlabeled L-cysteine sulfinic acid, 0.1 /zCi of labeled 1-[14C]cysteine sulfinic acid, 0.1 mM pyridoxal phosphate, and 50-100 mg of homogenate (1:5 wt/ vol) in a final volume of 1.1 ml. After 60-rain incubation at 37~ the reaction was stopped by the addition of 0.5 ml of 6 N sulfuric acid and incubation continued for 30 rain. The labeled CO2 produced by the decarboxylation of the substrate was trapped in 0.1 ml of hyamine hydroxide. Radioactivity was estimated by dissolving hyamine in 10 ml of scintillation fluid (4 g PPO, 0.4 g POPOP in 1 liter of toluene). Radioactivity of blanks without tissue was subtracted from sample values in each experiment. Lactate dehydrogenase (LDH) activity was estimated by measuring the rate of decrease in absorbancy at 340 nm as NADH2 is oxidized. The assay mixture contained 0.01 M sodium pyruvate, 0.002 M NADH2, and 0.03 M sodium phosphate buffer, pH 7.4 (15).
RESULTS
Distribution of CO and CSD Activities in Regions of Rat CNS CO activity in brain regions varied from 41 to 99 /zmol of cysteine sulfinate formed per h per g, with hypothalamus, colliculi, and ponsmedulla having the highest activity and olfactory bulbs, posterior cortex, and striatum the lowest (Table I). CSD activity, measured in the
674
P A S A N T E S - M O R A L E S ET AL.
TABLE I REGIONAL DISTRIBUTION OF CYSTEINE OXIDASE AND CYSTEINE SULFINATE DECARBOXYLASE ACTIVITIES IN RAT C N S
Region Olfactory bulbs Anterior cortex Posterior cortex Striatum Hippocampus
Hypothalamus Superior colliculi Inferior colliculi Cerebellum
Pons-medulla Spinal cord Sciatic nerve
CO ~ (txmol/h/g) 45.1 51.2 44.2 47.3 48.7 94.1 99.3 97.0 72.2 95.0 60.3
• 3.1 _+ 2.7 + 1.5 • 3.4 • 2.8 • 3.1 • 2.9 • 3.9 • 5.9 +- 5.7 _+ 3.4
(4) (4) (4) (4) (4) (6) (6) (6) (4) (5) (4)
CSD b (t~mol/h/g) 13.37 9.79 8.16 11.94 8.51 20.48 21.51 14.80 9.12 7.30 3.66
• 0.51 (5) • 0.34 (8) • 0.99 (4) -4- 1.23 (4) • 0~96 (4) • 0.2l (5) • 1.16 (5) -+ 1.92 (5) • 0.76 (7) • 0.82 (4) -+ 0.16 (9)
Not detected
a CO activity is expressed as/xmol of cysteine sulfinate formed per h per g tissue. b CSD activity is expressed as t~mol of cysteine sulfinate decarboxylated per h per g tissue. Samples of tissue were homogenized in 0.02 M phosphate buffer, p H 6.8 (1:10 wt/vol), for CO assays and in 0.4% Triton-X-100 for CSD assays. E n z y m e activities were assayed as described in the "'Experimental P r o c e d u r e " section. The results are means • The number of determinations is indicated in parentheses.
presence of pyridoxal phosphate (PLP), ranged from 3.6 /zmol of substrate decarboxylated per h per g in spinal cord to 14-21/zmol/h/g in hypothalamus and colliculi (Table I).
CSD Activation by PLP The effect of 10 -4 M PLP on CSD activity was studied in 12 regions of rat CNS. Exogenous PLP considerably enhanced CSD activity in all brain regions. The activation by PLP varied from 191 to 219% in spinal cord, pons-meduUa, and cerebellum and from 300 to 400% in supraspinal regions. The highest activation by PLP was observed in hypothalamus. Previously reported activation of the whole brain CSD by PLP of only 100% probably reflect the presence of somewhat high amounts of dithiothreitol in the incubation mixture (10).
Subcellular Distribution The distribution of CO and CSD activities in subcellular fractions from pons-medulla, cortex, hypothalamus, and coUiculi was examined. No
675
T A U R I N E - S Y N T H E S I Z I N G E N Z Y M E S IN RAT CNS
T A B L E II DISTRIBUTION OF CYSTEINE OXIDASE ACTIVITY IN PRIMARY SUBCELLULAR FRACTIONS FROM FOUR REGIONS OF RAT BRAIN a Fraction Total homogenate Nuclear Mitochondrial Microsomal Supernatant
Pons-medulla 95.0 10.6 47.3 27.1 9.0
• • • • •
7.3 I.I 4.0 2.2 2.3
(4) (4) (4) (3) (3)
Cortex 51.2 4.2 20.1 12.3 6.2
Colliculi
• 4.2 • 0.4 • 1.9 • 1.2 --+- 1.6
(4) (4) (4) (3) (3)
99.3 12.6 56.6 23.2 I 1.3
Hypothalamus
--- 8.5 ___ 1.2 _+ 5.7 + 3.3 --- 2.8
(4) (4) (4) (5) (5)
94.1 12.5 53.0 27.5 12.2
_+ 4.1 • 1.5 • 3.9 --_ 3.4 - 3.5
(4) (4) (4) (4) (4)
Cysteine oxidase activity is expressed as/zmol o f cysteine sulfinate formed per h per g o f original tissue. Brain fractions were obtained by centrifugation o f homogenates prepared in 0.32 M sucrose as described in the "'Experimental P r o c e d u r e " section. Cysteine oxidase activity was assayed after addition o f Triton-X-100 (0.4% final concentration). Recoveries varies from 83 to 11 I%. The results are means +SEM. The number of determinations is indicated in parentheses.
marked differences were observed in the CO distribution in primary subcellular fractions obtained from brain regions. Most of the enzyme activity was found associated with the crude mitochondrial fraction and with the microsomal fraction (Table II). CSD activity was differently distributed: in hypothalamus, 70% of the total enzyme activity was found to be associated with the crude mitochondrial fraction; in cerebral cortex and colliculi, this percentage was lower (61 and 64%, respectively), and in pons-medulla it was only 40%. The remaining CSD activity was mainly recovered in the soluble supernatant fraction. Only small CSD activity was found in the microsomal pellet (Table III). T A B L E 1II DISTRIBUTION OF CYSTEINE SULFINATE DECARBOXYLASE ACTIVITY IN PRIMARY SUBCELLULAR FRACTIONS FROM FOUR REGIONS OF RAT BRAIN a Fraction
Pons-medulla
Total homogenate 7.30 +-+-0.51 (4) Nuclear 1.26 • 0.09 (4) Mitochondrial 2.78 • 0.22 (4) Microsomal 0.28 • 0.06 (3) Supernatant 2.37 • 0.21 (3)
Cortex 9.79 1.18 6.49 0.49 1.61
• • + • •
0.42 0.08 0.47 0.08 0.38
CoUiculi (4) (4) (4) (3) (3)
21.51 2.63 12.85 1.33 3.90
+- 0.73 -+ 0.02 --- 1.16 --- 0.29 • 0.34
Hypothalamus (4) (4) (4) (5) (5)
20.48 1.96 14.26 0.79 2.81
• • • • •
0.69 0.16 1.29 0.26 0.30
(4) (4) (4) (5) (5)
Cysteine sulfinate decarboxylase activity is expressed a s / x m o l o f substrate decarboxylated per h per g o f original tissue. Brain fractions were obtained by centrifugation o f homogenates prepared in 0.32 M sucrose as described in the "'Experimental P r o c e d u r e " section. CSD was assayed after addition of Triton-X-100 (0.4% final concentration). Recoveries varied from 81 to 99%. The results are means __+-SEM. The number of determinations is indicated in parentheses.
676
PASANTES-MORALES ET AL.
TABLE IV DISTRIBUTION OF CYSTEINE OXIDASE AND CYSTEINE SULFINATE DECARBOXYLASE ACTIVITIES IN SUBFRACTIONS OF THE CRUDE MITOCHONDRIAL FRACTION OF RAT BRAIN REGIONS a Percent distribution Cortex Fraction Myelin Synaptosomes Mitochondria
Colliculi
Hypothatamus
CO
CSD
CO
CSD
CO
CSD
23.0 34.6 42.4
11.3 74.0 18.2
24.5 33.0 39.5
14.2 63.5 22.3
24.2 30.2 45.6
13.7 69.2 17.1
Subfractions were obtained from the crude mitochondrial fraction layered over a discontinuous gradient consisting of 2 ml each of 0.8 M and 1.2 M sucrose. After 120-min centrifugation at 50,000g, the myelin and synaptosomal fractions were recovered from the interphases, and mitochondria from the pellet. CO and CSD activities were assayed after addition of Triton-X-100. Recoveries with respect to the crude mitochondrial fraction varied from 78 to 86%. Results are means of three separate experiments. The SEMs were less than 12%.
Further subfractionation of the crude mitochondrial fraction showed CO activity similarly distributed between synaptosomal and mitochondrial fractions and CSD activity primarily localized in the synaptosomal fraction (Table IV). The relative distribution of CO and CSD activities in the crude mitochondrial fraction was compared with that of LDH, which is a marker of the cytoplasm occluded in the pinched-off nerve terminals. The CO-LDH ratios were 1.2-1.4 in the four examined regions. CSDLDH ratios were 1.7-1.8 in hypothalamus, colliculi, and cerebral cortex and 1.3 in pons-medulla.
DISCUSSION CO activity of rat brain areas, assayed under the conditions described by Misra and Olney (7), is considerably higher than that reported by Yamaguchi et al. (5) and by Rassin and Gaull (6). This observation is in agreement with the remark of Misra and Olney (7), according to which saturating concentrations of the substrate L-cysteine for the brain enzyme are higher than those required for the liver enzyme. Marked regional variations in CO and CSD activities were observed in rat brain, but the extent of these variations differs for the two enzymes. Values of CO activity in regions showing the highest activity, the
TAURINE-SYNTHESIZING ENZYMES IN RAT CNS
677
hypothalamus and colliculi, are not more than twofold those found in regions having the lowest activity. In contrast, CSD activity shows a variation of over fivefold between the highest and the lowest observed values in brain regions. A comparison of CO and CSD activities in supraspinal regions shows that, although differing in their absolute values, both enzymes have a similar pattern of regional distribution, showing intermediate activities in cerebral cortex, hippocampus, striatum, and olfactory bulbs and the highest activity in hypothalamus and colliculi. In spinal cord, cerebellum, and pons-medulla, CO activity is high, whereas in these regions, CSD shows the lowest activity. It is interesting to note that, in areas where the activity of both enzymes is particularly high, namely hypothalamus and colliculi, evidence for a physiological role of taurine has been provided. Indeed, recent pharmacological studies suggest that taurine may be involved in hypothalamic neuroendocrine functions (1618). On the other hand, the superior colliculi contain synaptic contacts coming from the optic pathway, yet evidence has been accumulated of a role for taurine in vision (19). The present results, showing that these regions possess a high capacity for taurine synthesis, in particular at synaptic levels, afford new support for a role for taurine in these areas. Our results on the subcellular distribution of CSD agree with those previously reported in the whole brain, showing a considerable enzyme activity associated with particles (7,9,10). This pattern of subcellular distribution of CSD was found in all the examined areas. With respect to the subcellular distribution of CO, our results agree with those of Rassin and Gaull (6), who found a considerable proportion of the enzymatic activity in the crude mitochondrial fraction and, after subfractionation, recovered most of the CO activity in the synaptosomal fraction. Misra and Olney (7) found most of the CO activity (80%) associated with microsomes sedimented from the soluble fraction. This discrepancy is difficult to explain, since our results on total enzyme activity values and regional distribution are similar to those of Misra and Olney. The method employed for the preparation of the primary fractions is identical to that used by these authors, although CO activity in our assays is determined after treating fractions and subfractions with Triton-X-100, whereas in assays of Misra and Olney, fractions are resuspended in isotonic sucrose; under these conditions, any enzyme occluded within synaptosomes might not be accessible to substrate and cofactors. The dilution of samples in the incubation medium, however, should be great enough to produce disruption of synaptosomes. It should be stated that the fractionation procedure used in this work corresponds to that originally described for brain cortex homogenates and that some differ-
678
PASANTES-MORALES ET AL.
ences might be obtained when these procedures are applied to subcellular fractionation in other brain regions. This work was performed when an abstract was published (20) in which Misra et al. reported that the use of more accurate techniques resulted in localization of CO activity to microsomal and synaptosornal fractions. Both CO and CSD are found to be associated with the nerve ending fraction. The subcellular distribution of CO activity in fractions and subfractions shows, however, that only about 15% of the total activity is present in synaptosomes. In contrast, CSD activity in synaptosomes accounts for more than 60% of the enzyme activity in the crude mitochondrial fraction and about 48% of the total CSD activity. The comparison between CSD and L D H distribution indicates that CSD activity is effectively concentrated in this fraction and that the observed distribution is not simply a result of the enzyme activity present in the cytoplasm of the sealed synaptic terminals. These results, taken together with the observation that variations in CSD activity in brain regions are greater than those of CO, suggest that the decarboxylase might play a role in the regulation of taurine biosynthesis, particularly at the synaptic levels. The different extent of CSD activation by PLP in nervous regions is also noteworthy. It has been demonstrated that the PLP activation of CSD in fractions obtained from brain homogenates is higher in the particulate fraction than in the soluble fraction (10,21). Our results, showing a different activation of CSD by PLP in brain regions, might thus be related to a different CSD subcellular distribution. In hypothalamus, the region in which CSD shows the highest activation by the cofactor, the enzyme is mainly localized in the particulate fraction, whereas the opposite is observed in pons-medulla. The present results show that the differences observed in the activity of taurine-synthesizing enzymes in brain regions do not correlate with the regional distribution of taurine (1-3). The maintenance of taurine concentration in the nervous system remains a puzzling question. In addition to the efficiency of the biosynthetic pathway, the processes responsible for taurine levels in CNS areas include the rate of the degradative reactions and the capacity of plasma-brain exchanges. The transport of taurine into brain regions, however, studied in vivo as well as in vitro, under conditions where the high-affinity or the low-affinity uptake is operative (1-3), does not correlate with the endogenous levels of taurine. The catabolism of taurine, on the other hand, is extremely low (22), and probably is of little significance in the regulation of brain taurine levels. A possible explanation would be the existence of two or more pools of taurine, with different turnover rates, independently
TAURINE-SYNTHESIZING ENZYMES IN RAT CNS
679
controlled by the aforementioned processes. The subcellular distribution of CO and CSD activities, suggesting a compartmentalization of taurine biosynthesis, is in keeping with this hypothesis.
ACKNOWLEDGMENTS This work was partially supported by the D6partement de Biologie du Commissariat l'Energie Atomique (for the purchase of labeled substrates) and by funds provided by the Institut National de la Sant6 et de la Recherche M6dicale (Contract 7-1-197-6).
REFERENCES I. KANDERA, J., LEVI, G., and LAJTHA, A. 1968. Control of cerebral metabolite levels. II. Amino acid uptake and levels in various areas of the rat brain. Arch. Biochem. Biophys. 126:249-260. 2. COLLINS, G. G. S. 1974. The rates of synthesis, uptake and disappearance of (14C)taurine in eight areas of the rat central nervous system. Brain Res. 76:447-459. 3. LOMBARDINI,J. B. 1976. Regional and subcellular studies on taurine in the rat central nervous system. Pages 311-326, in HUXTABLE, R., and BARBEAU, A. (eds.), Taurine, Raven Press, New York. 4. AWAPARA, J. 1976. The metabolism of taurine in the animal. Pages 1-19, in HUXTABLE, R., and BARBEAU, A. (eds.), Taurine, Raven Press, New York. 5. YAMAGUCHI, K., SAKAKIBARA,S., ASAMIZU, J., and UEDA, I. 1973. Induction and activation of cysteine oxidase of rat liver. II. The measurement of cysteine metabolism in vivo and the activation of the in vivo activity of cysteine oxidase. Biochim. Biophys. Acta 297:48-59. 6. RASSIN, D. K., and GAULL, G. E. 1975. Subcellular distribution of enzymes of transmethylation and transsulphuration in rat brain. J. Neurochem. 24:969-978. 7. M~SRA, C. H., and OLNEY, J. W. 1975. Cysteine oxidase in brain. Brain Res. 97:117126. 8. BERGERET,B., CHATAGNER,F., and FROMAGEOT,C. 1955. Quelques relations entre le phosphate de pyridoxal et la d6carboxylation de l'acide cyst6ine sulfinique par divers organes du rat normal ou du rat carenc6 en vitamine B6. Biochim. Biophys. Acta 17:128-135. 9. AGRAWAL, H. C., DAVISON, A. N., and KACZMAREK, L. K. 1971. Subcellular distribution of taurine and cysteine sulphinate decarboxylase in developing rat brain. Biochem. J. 122:759-763. 10. PASANTES-MORALES,H., MAPES, C., TAPIA, R., and MANDEL, P. 1976. Properties of soluble and particulate cysteine sulfinate decarboxylase of the adult and the developing rat brain. Brain Res. I07:575-589. 11. PmA, R. S., and SAUKKONEN, H. 1966. The L-cysteine sulphinic acid decarboxylase activity in various areas of the brain. Suom. KemistiL B39:112-114. 12. GLOWINS~I, J., and IVERSEN, L. L. 1966. Regional studies on catecholamines in the rat brain. I. The disposition of (SH)norepinephrine, (3H)dopamine and (~rI)DOPA in various regions of the brain. J. Neurochem. 19:655-669.
680
PASANTES-MORALES
ET A L .
13. GRAy, E. G., and WHITTAKER,V. P. 1962. The isolation of nerve endings from brain: An electron-microscope study of cell fragments derived by homogenization and centrifugation. J. Anat. (Lond.) 96:79-87. 14. YAMAGUCHI,K., SAKAKIBARA,S., KYOICHIRO,K., and UEDA, I. 1971. Induction and activation of cysteine oxidase and tyrosine transaminase activities of intact and adrenaiectomized rats. Biochim. Biophys. Acta 237:502-512. 15. BERGMEYER,H. U., BERNT, E., and HESS, B. 1963. Lactic dehydrogenase. Pages 736741, in BERGMEYER, H. U. (ed.), Methods of Enzymatic Analysis, Academic Press, New York. 16. THUT, P. D., HRUSKA, R. E., HUXTABLE, R., and BRESSLER, R. 1976. Effect of taurine on eating and drinking behavior. Pages 357-364, in HUXTABLE, R., and BARBEAU, A. (eds.), Taurine, Raven Press, New York. 17. HRUSKA, R. E., THUT, P. D,, HUXTABLE, R., and BRESSLER, R. 1973. Taurine: hypothermic effect in mice. Pharmacologist 15:301. 18. SGARAGLI,G. P., PAVAN, F., and GALLI, A. 1975. Effects of amino acid compounds injected into cerebrospinal fluid spaces, on colonic temperature, arterial blood pressure and behavior of the rat. Neuropharmacology l 1:45-56. 19. MANDEL, P., PASANTES-MORALES,H., and URBAN, P. F. 1976. Taurine, a putative transmitter in retina, Pages 89-105, in BONTING, L. (ed.), Transmitters in the Visual Process, Pergamon Press, Oxford, New York. 20. MISRA, C. H., MENA, E. E,, RHEE, V., and OLNEY, J. W. 1977. Intracellular distribution of cysteine oxidase in the rat central nervous system. Fed. Proc. 36:751. 21. RASS~N,D. K., and STURMAN,J. A. 1975. Cysteine sulfinic acid decarboxylase in rat brain: Effect of vitamin B6-deficiency on soluble and particulate components. Life Sci. 16:875-882. 22. PECK, E. J., and AWAPARA, J. 1967. Formation of taurine and isethionic acid in rat brain. Biochim. Biophys. Acta 141:499-506.
REGIONAL AND SUBCELLULAR DISTRIBUTION OF T A U R I N E S Y N T H E S I Z I N G E N Z Y M E S IN THE RAT C E N T R A L N E R V O U S SYSTEM H. PASANTES-MORALES,1 C , LORIETTE, AND F. CHATAGNER Laboratoire de Chimie Biologique 96 Boulevard Raspail, 75006, Paris, France
Accepted June 6, 1977
The distribution of cysteine oxidase (CO) and cysteine sulfinate decarboxylase (CSD) was examined in 12 regions of the rat central nervous system (CNS). The distribution of CO activity, expressed as ~mol of cysteine sulfinate formed per h per g, was the following: hypothalamus, superior and inferior colliculi, 94-99 tzmol/h/g; olfactory bulbs, cerebral cortex, striatum, and hippocampus, 44-51 ~mol/h/g; cerebellum, 71 tzmol/h/g; pons-medula and spinal cord, 94 and 60 /xmol/h/g, respectively. The distribution of CSD activity expressed as /xmol of cysteine sulfinate decarboxylated per h per g was the following: hypothalamus and colliculi, 14-21 /~mol/h/g; olfactory bulbs, cerebral cortex, striatum, hippocampus, and cerebellum, 8-13/zmol/h/g; pons-medulla, 7.3; and spinal cord, 3.6 /~mol/h/g. No CSD activity was detected in sciatic nerve. The subcellular distribution of CO and CSD activities was studied in hypothalamus, coUiculi, and cerebral cortex. CO activity was localized in synaptosomes, mitochondria, and microsomes. CSD was primarily confined to the crude mitochondrial fraction and after subfraction, recovered mainly in the synaptosomal fraction.
INTRODUCTION Taurine is unevenly distributed in the CNS. The taurine content of nervous areas considerably differs, showing a variation of over sixfold between regions showing the lowest and the highest taurine concentration (1-3). 1 On leave from Instituto de Biologia, Universidad Nacional Autonoma de Mrxico, Apartado Postal 70-600, M6xico 20, D.F., M6xico.
671 This journal is copyrighted by Plenum. Each article is available for $7.50 from Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011,
672
PASANTES-MORALES ET AL.
The process(es) regulating taurine levels in the nervous system is (are) still unknown. A correlation between the mechanisms presumably involved in maintaining taurine levels in brain areas and its regional distribution has not been clearly established (1,2). However, it has been recently described (2) that taurine formation from intracisternally injected cysteine appears to be proportional to the endogenous taurine concentration in rat brain regions. Therefore, we thought it of interest to determine the regional distribution of taurine-synthesizing enzymes in brain. The main pathway for taurine synthesis in brain appears to be that involving the oxidation of cysteine to cysteine sulfinic acid, followed by decarboxylation to form hypotaurine and the subsequent oxidation of hypotaurine to taurine (4). The first two reactions are catalyzed by cysteine oxidase (CO), (L-cysteine:oxygen oxidoreductase, EC 1.13.11.20) and cysteine sulfinate decarboxylase (CSD) (L-cysteine sulfinate carboxylyase, EC 4.1.1.29), respectively. The activity of these enzymes has been demonstrated in brain (5-10); the enzymatic activity responsible for the last step in taurine formation, namely the hypotaurine oxidation, has not yet been found in nervous tissue. Systematic studies on the regional distribution of CSD have not been carried out. Previous work of Agrawal et al. (9) has shown only small differences in CSD activity in four regions of rat CNS. More significant differences were found by Piha and Saukkonen (11) in regions of the calf brain. The distribution of CO activity in seven regions of rat brain has been recently reported by Misra and Olney (7). This paper describes the distribution of CO and CSD activities in 12 regions of rat CNS. Studies on the subcellular distribution of CSD activity in the whole brain have consistently revealed that a high proportion of the enzyme activity is associated with the isolated nerve endings (6,9,10). In contrast, the subcellular distribution of CO activity is controversial, the enzymatic activity being either observed associated with the synaptosomal fraction (6) or recovered in the soluble fraction associated with microsomes (7). In the present study, we have measured the activity of CO and CSD in fractions and subfractions obtained from four regions of rat brain.
EXPERIMENTAL PROCEDURE Male, adult rats (200-300 g), Sherman strain, were used in this study. After decapitation, cervical spinal cord and brain were removed and regions of brain dissected according to Glowinski and Iversen (12). Tissue was pooled when necessary and homogenized in 0.4% Triton-X-100 or in 0.32 M sucrose for fractionation studies. Sciatic nerves were cut into small pieces and homogenized in a glass-glass homogenizer.
TAURINE-SYNTHESIZING ENZYMES IN RAT CNS
673
Subcellular Fractionation Primary fractions were separated from sucrose homogenates (10:1, voUwt) according to the procedure of Gray and Whittaker (13). The homogenate was centrifuged at 800g for 10 rain to obtain the crude nuclear fraction and then at 12,000g for 20 rain to separate the crude mitochondrial fraction. The supernatant was centrifuged at 105,000g for 60 min to separate the microsomal fraction. For preparation of subfractions, the crude mitochondrial fraction was resuspended in 0.32 M sucrose, and ! ml of this suspension was layered over a discontinuous density gradient consisting of 2 ml each of 0.8 M and 1.2 M sucrose. The gradient was centrifuged at 50,000g for 120 vain in a SW-50-1 rotor in a Beckman-Spinco ultracentrifuge. The subcellular fractions myelin, synaptosomal, and mitochondrial were obtained, Triton-X-100 (0.4% final concentration) was added to all fractions before enzyme assays.
Enzyme Assays CO activity was assayed by the method of Yamaguchi et al. (14) as modified by Misra and Olney (7). Homogenates were prepared in 0.02 M phosphate buffer, pH 6.8, or in 0.32 M sucrose for fractionation experiments. The homogenate or fractions (0.4 ml) were incubated for 30 rain in a mixture containing 0.05 M phosphate buffer, pH 6.8, 0.25 mM Fe(NH4)2(SO4)~'6H20, 5 mM hydroxylamine-HC1, 2 mM NAD, 77 mM neutralized Lcysteine, and 1/zCi of D,L-[14C]-3-cysteine in a final volume of 2 ml. After incubation, the reaction was stopped by addition of 1 ml of 8% TCA. Samples were centrifuged and the acid-soluble fraction was applied to a Dowex-50X4H + column. The column was washed with 10 ml of water, and the radioactivity of aliquots of the eluate was estimated after the addition of Unisolve (Koch-Light). Values of blanks, in which the tissue was added only after the addition of TCA, were subtracted from the sample values. CSD was assayed by following the decarboxylation of L-l-[14C]-cysteine sulfinic acid as previously described (10). The incubation mixture contained 0.067 M phosphate buffer, pH 6.8, 33 mM neutralized unlabeled L-cysteine sulfinic acid, 0.1 /zCi of labeled 1-[14C]cysteine sulfinic acid, 0.1 mM pyridoxal phosphate, and 50-100 mg of homogenate (1:5 wt/ vol) in a final volume of 1.1 ml. After 60-rain incubation at 37~ the reaction was stopped by the addition of 0.5 ml of 6 N sulfuric acid and incubation continued for 30 rain. The labeled CO2 produced by the decarboxylation of the substrate was trapped in 0.1 ml of hyamine hydroxide. Radioactivity was estimated by dissolving hyamine in 10 ml of scintillation fluid (4 g PPO, 0.4 g POPOP in 1 liter of toluene). Radioactivity of blanks without tissue was subtracted from sample values in each experiment. Lactate dehydrogenase (LDH) activity was estimated by measuring the rate of decrease in absorbancy at 340 nm as NADH2 is oxidized. The assay mixture contained 0.01 M sodium pyruvate, 0.002 M NADH2, and 0.03 M sodium phosphate buffer, pH 7.4 (15).
RESULTS
Distribution of CO and CSD Activities in Regions of Rat CNS CO activity in brain regions varied from 41 to 99 /zmol of cysteine sulfinate formed per h per g, with hypothalamus, colliculi, and ponsmedulla having the highest activity and olfactory bulbs, posterior cortex, and striatum the lowest (Table I). CSD activity, measured in the
674
P A S A N T E S - M O R A L E S ET AL.
TABLE I REGIONAL DISTRIBUTION OF CYSTEINE OXIDASE AND CYSTEINE SULFINATE DECARBOXYLASE ACTIVITIES IN RAT C N S
Region Olfactory bulbs Anterior cortex Posterior cortex Striatum Hippocampus
Hypothalamus Superior colliculi Inferior colliculi Cerebellum
Pons-medulla Spinal cord Sciatic nerve
CO ~ (txmol/h/g) 45.1 51.2 44.2 47.3 48.7 94.1 99.3 97.0 72.2 95.0 60.3
• 3.1 _+ 2.7 + 1.5 • 3.4 • 2.8 • 3.1 • 2.9 • 3.9 • 5.9 +- 5.7 _+ 3.4
(4) (4) (4) (4) (4) (6) (6) (6) (4) (5) (4)
CSD b (t~mol/h/g) 13.37 9.79 8.16 11.94 8.51 20.48 21.51 14.80 9.12 7.30 3.66
• 0.51 (5) • 0.34 (8) • 0.99 (4) -4- 1.23 (4) • 0~96 (4) • 0.2l (5) • 1.16 (5) -+ 1.92 (5) • 0.76 (7) • 0.82 (4) -+ 0.16 (9)
Not detected
a CO activity is expressed as/xmol of cysteine sulfinate formed per h per g tissue. b CSD activity is expressed as t~mol of cysteine sulfinate decarboxylated per h per g tissue. Samples of tissue were homogenized in 0.02 M phosphate buffer, p H 6.8 (1:10 wt/vol), for CO assays and in 0.4% Triton-X-100 for CSD assays. E n z y m e activities were assayed as described in the "'Experimental P r o c e d u r e " section. The results are means • The number of determinations is indicated in parentheses.
presence of pyridoxal phosphate (PLP), ranged from 3.6 /zmol of substrate decarboxylated per h per g in spinal cord to 14-21/zmol/h/g in hypothalamus and colliculi (Table I).
CSD Activation by PLP The effect of 10 -4 M PLP on CSD activity was studied in 12 regions of rat CNS. Exogenous PLP considerably enhanced CSD activity in all brain regions. The activation by PLP varied from 191 to 219% in spinal cord, pons-meduUa, and cerebellum and from 300 to 400% in supraspinal regions. The highest activation by PLP was observed in hypothalamus. Previously reported activation of the whole brain CSD by PLP of only 100% probably reflect the presence of somewhat high amounts of dithiothreitol in the incubation mixture (10).
Subcellular Distribution The distribution of CO and CSD activities in subcellular fractions from pons-medulla, cortex, hypothalamus, and coUiculi was examined. No
675
T A U R I N E - S Y N T H E S I Z I N G E N Z Y M E S IN RAT CNS
T A B L E II DISTRIBUTION OF CYSTEINE OXIDASE ACTIVITY IN PRIMARY SUBCELLULAR FRACTIONS FROM FOUR REGIONS OF RAT BRAIN a Fraction Total homogenate Nuclear Mitochondrial Microsomal Supernatant
Pons-medulla 95.0 10.6 47.3 27.1 9.0
• • • • •
7.3 I.I 4.0 2.2 2.3
(4) (4) (4) (3) (3)
Cortex 51.2 4.2 20.1 12.3 6.2
Colliculi
• 4.2 • 0.4 • 1.9 • 1.2 --+- 1.6
(4) (4) (4) (3) (3)
99.3 12.6 56.6 23.2 I 1.3
Hypothalamus
--- 8.5 ___ 1.2 _+ 5.7 + 3.3 --- 2.8
(4) (4) (4) (5) (5)
94.1 12.5 53.0 27.5 12.2
_+ 4.1 • 1.5 • 3.9 --_ 3.4 - 3.5
(4) (4) (4) (4) (4)
Cysteine oxidase activity is expressed as/zmol o f cysteine sulfinate formed per h per g o f original tissue. Brain fractions were obtained by centrifugation o f homogenates prepared in 0.32 M sucrose as described in the "'Experimental P r o c e d u r e " section. Cysteine oxidase activity was assayed after addition o f Triton-X-100 (0.4% final concentration). Recoveries varies from 83 to 11 I%. The results are means +SEM. The number of determinations is indicated in parentheses.
marked differences were observed in the CO distribution in primary subcellular fractions obtained from brain regions. Most of the enzyme activity was found associated with the crude mitochondrial fraction and with the microsomal fraction (Table II). CSD activity was differently distributed: in hypothalamus, 70% of the total enzyme activity was found to be associated with the crude mitochondrial fraction; in cerebral cortex and colliculi, this percentage was lower (61 and 64%, respectively), and in pons-medulla it was only 40%. The remaining CSD activity was mainly recovered in the soluble supernatant fraction. Only small CSD activity was found in the microsomal pellet (Table III). T A B L E 1II DISTRIBUTION OF CYSTEINE SULFINATE DECARBOXYLASE ACTIVITY IN PRIMARY SUBCELLULAR FRACTIONS FROM FOUR REGIONS OF RAT BRAIN a Fraction
Pons-medulla
Total homogenate 7.30 +-+-0.51 (4) Nuclear 1.26 • 0.09 (4) Mitochondrial 2.78 • 0.22 (4) Microsomal 0.28 • 0.06 (3) Supernatant 2.37 • 0.21 (3)
Cortex 9.79 1.18 6.49 0.49 1.61
• • + • •
0.42 0.08 0.47 0.08 0.38
CoUiculi (4) (4) (4) (3) (3)
21.51 2.63 12.85 1.33 3.90
+- 0.73 -+ 0.02 --- 1.16 --- 0.29 • 0.34
Hypothalamus (4) (4) (4) (5) (5)
20.48 1.96 14.26 0.79 2.81
• • • • •
0.69 0.16 1.29 0.26 0.30
(4) (4) (4) (5) (5)
Cysteine sulfinate decarboxylase activity is expressed a s / x m o l o f substrate decarboxylated per h per g o f original tissue. Brain fractions were obtained by centrifugation o f homogenates prepared in 0.32 M sucrose as described in the "'Experimental P r o c e d u r e " section. CSD was assayed after addition of Triton-X-100 (0.4% final concentration). Recoveries varied from 81 to 99%. The results are means __+-SEM. The number of determinations is indicated in parentheses.
676
PASANTES-MORALES ET AL.
TABLE IV DISTRIBUTION OF CYSTEINE OXIDASE AND CYSTEINE SULFINATE DECARBOXYLASE ACTIVITIES IN SUBFRACTIONS OF THE CRUDE MITOCHONDRIAL FRACTION OF RAT BRAIN REGIONS a Percent distribution Cortex Fraction Myelin Synaptosomes Mitochondria
Colliculi
Hypothatamus
CO
CSD
CO
CSD
CO
CSD
23.0 34.6 42.4
11.3 74.0 18.2
24.5 33.0 39.5
14.2 63.5 22.3
24.2 30.2 45.6
13.7 69.2 17.1
Subfractions were obtained from the crude mitochondrial fraction layered over a discontinuous gradient consisting of 2 ml each of 0.8 M and 1.2 M sucrose. After 120-min centrifugation at 50,000g, the myelin and synaptosomal fractions were recovered from the interphases, and mitochondria from the pellet. CO and CSD activities were assayed after addition of Triton-X-100. Recoveries with respect to the crude mitochondrial fraction varied from 78 to 86%. Results are means of three separate experiments. The SEMs were less than 12%.
Further subfractionation of the crude mitochondrial fraction showed CO activity similarly distributed between synaptosomal and mitochondrial fractions and CSD activity primarily localized in the synaptosomal fraction (Table IV). The relative distribution of CO and CSD activities in the crude mitochondrial fraction was compared with that of LDH, which is a marker of the cytoplasm occluded in the pinched-off nerve terminals. The CO-LDH ratios were 1.2-1.4 in the four examined regions. CSDLDH ratios were 1.7-1.8 in hypothalamus, colliculi, and cerebral cortex and 1.3 in pons-medulla.
DISCUSSION CO activity of rat brain areas, assayed under the conditions described by Misra and Olney (7), is considerably higher than that reported by Yamaguchi et al. (5) and by Rassin and Gaull (6). This observation is in agreement with the remark of Misra and Olney (7), according to which saturating concentrations of the substrate L-cysteine for the brain enzyme are higher than those required for the liver enzyme. Marked regional variations in CO and CSD activities were observed in rat brain, but the extent of these variations differs for the two enzymes. Values of CO activity in regions showing the highest activity, the
TAURINE-SYNTHESIZING ENZYMES IN RAT CNS
677
hypothalamus and colliculi, are not more than twofold those found in regions having the lowest activity. In contrast, CSD activity shows a variation of over fivefold between the highest and the lowest observed values in brain regions. A comparison of CO and CSD activities in supraspinal regions shows that, although differing in their absolute values, both enzymes have a similar pattern of regional distribution, showing intermediate activities in cerebral cortex, hippocampus, striatum, and olfactory bulbs and the highest activity in hypothalamus and colliculi. In spinal cord, cerebellum, and pons-medulla, CO activity is high, whereas in these regions, CSD shows the lowest activity. It is interesting to note that, in areas where the activity of both enzymes is particularly high, namely hypothalamus and colliculi, evidence for a physiological role of taurine has been provided. Indeed, recent pharmacological studies suggest that taurine may be involved in hypothalamic neuroendocrine functions (1618). On the other hand, the superior colliculi contain synaptic contacts coming from the optic pathway, yet evidence has been accumulated of a role for taurine in vision (19). The present results, showing that these regions possess a high capacity for taurine synthesis, in particular at synaptic levels, afford new support for a role for taurine in these areas. Our results on the subcellular distribution of CSD agree with those previously reported in the whole brain, showing a considerable enzyme activity associated with particles (7,9,10). This pattern of subcellular distribution of CSD was found in all the examined areas. With respect to the subcellular distribution of CO, our results agree with those of Rassin and Gaull (6), who found a considerable proportion of the enzymatic activity in the crude mitochondrial fraction and, after subfractionation, recovered most of the CO activity in the synaptosomal fraction. Misra and Olney (7) found most of the CO activity (80%) associated with microsomes sedimented from the soluble fraction. This discrepancy is difficult to explain, since our results on total enzyme activity values and regional distribution are similar to those of Misra and Olney. The method employed for the preparation of the primary fractions is identical to that used by these authors, although CO activity in our assays is determined after treating fractions and subfractions with Triton-X-100, whereas in assays of Misra and Olney, fractions are resuspended in isotonic sucrose; under these conditions, any enzyme occluded within synaptosomes might not be accessible to substrate and cofactors. The dilution of samples in the incubation medium, however, should be great enough to produce disruption of synaptosomes. It should be stated that the fractionation procedure used in this work corresponds to that originally described for brain cortex homogenates and that some differ-
678
PASANTES-MORALES ET AL.
ences might be obtained when these procedures are applied to subcellular fractionation in other brain regions. This work was performed when an abstract was published (20) in which Misra et al. reported that the use of more accurate techniques resulted in localization of CO activity to microsomal and synaptosornal fractions. Both CO and CSD are found to be associated with the nerve ending fraction. The subcellular distribution of CO activity in fractions and subfractions shows, however, that only about 15% of the total activity is present in synaptosomes. In contrast, CSD activity in synaptosomes accounts for more than 60% of the enzyme activity in the crude mitochondrial fraction and about 48% of the total CSD activity. The comparison between CSD and L D H distribution indicates that CSD activity is effectively concentrated in this fraction and that the observed distribution is not simply a result of the enzyme activity present in the cytoplasm of the sealed synaptic terminals. These results, taken together with the observation that variations in CSD activity in brain regions are greater than those of CO, suggest that the decarboxylase might play a role in the regulation of taurine biosynthesis, particularly at the synaptic levels. The different extent of CSD activation by PLP in nervous regions is also noteworthy. It has been demonstrated that the PLP activation of CSD in fractions obtained from brain homogenates is higher in the particulate fraction than in the soluble fraction (10,21). Our results, showing a different activation of CSD by PLP in brain regions, might thus be related to a different CSD subcellular distribution. In hypothalamus, the region in which CSD shows the highest activation by the cofactor, the enzyme is mainly localized in the particulate fraction, whereas the opposite is observed in pons-medulla. The present results show that the differences observed in the activity of taurine-synthesizing enzymes in brain regions do not correlate with the regional distribution of taurine (1-3). The maintenance of taurine concentration in the nervous system remains a puzzling question. In addition to the efficiency of the biosynthetic pathway, the processes responsible for taurine levels in CNS areas include the rate of the degradative reactions and the capacity of plasma-brain exchanges. The transport of taurine into brain regions, however, studied in vivo as well as in vitro, under conditions where the high-affinity or the low-affinity uptake is operative (1-3), does not correlate with the endogenous levels of taurine. The catabolism of taurine, on the other hand, is extremely low (22), and probably is of little significance in the regulation of brain taurine levels. A possible explanation would be the existence of two or more pools of taurine, with different turnover rates, independently
TAURINE-SYNTHESIZING ENZYMES IN RAT CNS
679
controlled by the aforementioned processes. The subcellular distribution of CO and CSD activities, suggesting a compartmentalization of taurine biosynthesis, is in keeping with this hypothesis.
ACKNOWLEDGMENTS This work was partially supported by the D6partement de Biologie du Commissariat l'Energie Atomique (for the purchase of labeled substrates) and by funds provided by the Institut National de la Sant6 et de la Recherche M6dicale (Contract 7-1-197-6).
REFERENCES I. KANDERA, J., LEVI, G., and LAJTHA, A. 1968. Control of cerebral metabolite levels. II. Amino acid uptake and levels in various areas of the rat brain. Arch. Biochem. Biophys. 126:249-260. 2. COLLINS, G. G. S. 1974. The rates of synthesis, uptake and disappearance of (14C)taurine in eight areas of the rat central nervous system. Brain Res. 76:447-459. 3. LOMBARDINI,J. B. 1976. Regional and subcellular studies on taurine in the rat central nervous system. Pages 311-326, in HUXTABLE, R., and BARBEAU, A. (eds.), Taurine, Raven Press, New York. 4. AWAPARA, J. 1976. The metabolism of taurine in the animal. Pages 1-19, in HUXTABLE, R., and BARBEAU, A. (eds.), Taurine, Raven Press, New York. 5. YAMAGUCHI, K., SAKAKIBARA,S., ASAMIZU, J., and UEDA, I. 1973. Induction and activation of cysteine oxidase of rat liver. II. The measurement of cysteine metabolism in vivo and the activation of the in vivo activity of cysteine oxidase. Biochim. Biophys. Acta 297:48-59. 6. RASSIN, D. K., and GAULL, G. E. 1975. Subcellular distribution of enzymes of transmethylation and transsulphuration in rat brain. J. Neurochem. 24:969-978. 7. M~SRA, C. H., and OLNEY, J. W. 1975. Cysteine oxidase in brain. Brain Res. 97:117126. 8. BERGERET,B., CHATAGNER,F., and FROMAGEOT,C. 1955. Quelques relations entre le phosphate de pyridoxal et la d6carboxylation de l'acide cyst6ine sulfinique par divers organes du rat normal ou du rat carenc6 en vitamine B6. Biochim. Biophys. Acta 17:128-135. 9. AGRAWAL, H. C., DAVISON, A. N., and KACZMAREK, L. K. 1971. Subcellular distribution of taurine and cysteine sulphinate decarboxylase in developing rat brain. Biochem. J. 122:759-763. 10. PASANTES-MORALES,H., MAPES, C., TAPIA, R., and MANDEL, P. 1976. Properties of soluble and particulate cysteine sulfinate decarboxylase of the adult and the developing rat brain. Brain Res. I07:575-589. 11. PmA, R. S., and SAUKKONEN, H. 1966. The L-cysteine sulphinic acid decarboxylase activity in various areas of the brain. Suom. KemistiL B39:112-114. 12. GLOWINS~I, J., and IVERSEN, L. L. 1966. Regional studies on catecholamines in the rat brain. I. The disposition of (SH)norepinephrine, (3H)dopamine and (~rI)DOPA in various regions of the brain. J. Neurochem. 19:655-669.
680
PASANTES-MORALES
ET A L .
13. GRAy, E. G., and WHITTAKER,V. P. 1962. The isolation of nerve endings from brain: An electron-microscope study of cell fragments derived by homogenization and centrifugation. J. Anat. (Lond.) 96:79-87. 14. YAMAGUCHI,K., SAKAKIBARA,S., KYOICHIRO,K., and UEDA, I. 1971. Induction and activation of cysteine oxidase and tyrosine transaminase activities of intact and adrenaiectomized rats. Biochim. Biophys. Acta 237:502-512. 15. BERGMEYER,H. U., BERNT, E., and HESS, B. 1963. Lactic dehydrogenase. Pages 736741, in BERGMEYER, H. U. (ed.), Methods of Enzymatic Analysis, Academic Press, New York. 16. THUT, P. D., HRUSKA, R. E., HUXTABLE, R., and BRESSLER, R. 1976. Effect of taurine on eating and drinking behavior. Pages 357-364, in HUXTABLE, R., and BARBEAU, A. (eds.), Taurine, Raven Press, New York. 17. HRUSKA, R. E., THUT, P. D,, HUXTABLE, R., and BRESSLER, R. 1973. Taurine: hypothermic effect in mice. Pharmacologist 15:301. 18. SGARAGLI,G. P., PAVAN, F., and GALLI, A. 1975. Effects of amino acid compounds injected into cerebrospinal fluid spaces, on colonic temperature, arterial blood pressure and behavior of the rat. Neuropharmacology l 1:45-56. 19. MANDEL, P., PASANTES-MORALES,H., and URBAN, P. F. 1976. Taurine, a putative transmitter in retina, Pages 89-105, in BONTING, L. (ed.), Transmitters in the Visual Process, Pergamon Press, Oxford, New York. 20. MISRA, C. H., MENA, E. E,, RHEE, V., and OLNEY, J. W. 1977. Intracellular distribution of cysteine oxidase in the rat central nervous system. Fed. Proc. 36:751. 21. RASS~N,D. K., and STURMAN,J. A. 1975. Cysteine sulfinic acid decarboxylase in rat brain: Effect of vitamin B6-deficiency on soluble and particulate components. Life Sci. 16:875-882. 22. PECK, E. J., and AWAPARA, J. 1967. Formation of taurine and isethionic acid in rat brain. Biochim. Biophys. Acta 141:499-506.
