SUBCELLULAR DISTRIBUTION OF TAURINE AND CYSTEINE SULPHINATE DECARBOXYLASE ACTIVITY IN OX RETINA S. MACAIONE, G. Tuccr, G. DE LUCAand R. M. DI GIORGIO Department of Biochemistry. University of Messina. Italy (Received 16 April 1916. Accepted 31 M a y 1976) Abstract-Taurine levels have been determined in primary and secondary subcellular fractions of ox retina and pigment epithelium. About the 79.57; of recovered taurine is located in the soluble fraction (S,). while the remainder is associated with the particulate components. In the secondary subcellular fractions, taurine is primarily associated with the synaptosomal fraction. Cysteine sulphinate decarhoxylase is predominantly associated with particulate components of retinal cells. About the 50% of the recovered enzyme activity of crude mitochondria is present in the synaptosoma1 fraction.
ALTHOUGH taurine is the predominant amino acid in demonstrates that this enzyme activity is present also mature retina (KUBICEK & DOLENEK, 1958; PASANTES- in the synaptosomal fraction. et al., 1972a; MACAIONE et al., 1974) its phyMORALES MATERIALS AND METHODS siological role remains uncertain; however taurine has a depressant effect on mammalian spinal and cortical Chemicals. In general AR grade chemicals were used. neurons (CURTIS& WATKINS,1961) and reversibly The sources of certain additional chemicals were as folabolishes the b-wave of the electroretinogram in a lows: L-cysteinesulphinic acid and L-cysteic acid (Serva. perfused retina preparation (PASANTES-MOKALES et a/., Entwicklungslabor, Heidelberg. Germany): dithiothreitol (Fluka AG, Chemische Fabrik. Buchs, Scweiz); pyridox1973). Uptake systems for taurine have been demon- al-5-phosphate (PLP) and Triton X-100 (Koch-Light strated in retina (STARR& VOADEN,1972; PASANTES- Laboratories Ltd, Colnbrook. England); ninhydrin and MORALESet al.. 1972h; NEALet al., 1973) and also taurine (Schuchardt, GmbH Hohenbrunn. Germany). Aiiirnals. Ox eyes were obtained from the local slaughterin pigment epithelium (LAKEet al., 1975). In brain house and used immediately after the animal's death. tissue the accumulation of [35S]taurine by a specific The eyes were enucleated and dissected just behind the synaptosomal population was shown by SIEGHART & ora serrata. The posterior halves of the eye balls were KAROBATH (1974). Both pre-loaded [35S]taurine and placed in ice-cold saline and everted. The vitreous. if it endogenous amino acid are released from chicken was still adherent to the exposed retina, was removed with retina in response to intensive light stimulation forceps. Retinae were prepared free from underlying choroid, carefully washed to remove pigment epithelium and (PASANTES-MORALES et al., 1973). In frog retina, approx 7.5;; of the total taurine con- immediately frozen at - IO'C. The retinal pigment epithe& PoTTs (1962). tent is associated with photoreceptor cells (KENNEDY lium was isolated according to GLOCKLIN The complete operation was carried out in a cold room & VOADEN,1974a.h). Our previous work on rat retina has shown that maintained at O"4"C and with normal light. Subcellularfracrionatiori The pooled retinae were honiotaurine levels increase during postnatal growth and genized in 0.32 M-sucrose (9 ml/g tissue) in a glass homthat most of the taurine present in the retinal crude ogenizer with a Teflon pestle at 20o0 rev./min for 3 min mitochondria1 fraction is associated with synaptic at 4°C and centrifuged at 800g for 5 min to obtain pellet endings (MACAIONE et ul., 1975). P and supernatant S. Pellet P was then suspended in The presence of the cysteine oxidase activity in 0.32 M-sucrose (4 ml/g tissue), homogenized at 1500 re\.,' cytoplasm and in crude mitochondria of rat retina min and sedimented at 8009 for 5 min to obtain pellet (Dr GIORGIO et al., 1975) and the observations of PI (nuclear fraction) and supernatant S,. The crude mitoHAYESet a/. (1975) in cat retina and of LAKEet al. chondrial fraction (P2) was obtained according to PAPA (1975) in frog retina, have raised the question as to et al. (1965) from the combined supernatants S and S , . whether taurine is formed in retina or whether it is The microsomal (P,) and the soluble fractions (S3) were prepared at 55,000 g for 90 min. taken up from the blood stream. The P, pellet was resuspended in 0.32 M-sucrose ( I ml/g The present study establishes the distribution of the tissue) and layered on a discontinuous density gradient cysteine sulphinate decarboxylase and the levels of consisting of 6 ml each of 0.8 and 1.2 M-sucrose. The grataurine in the ox retina subcellular fractions and dient was prepared at room temperature and then kept 141 1
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S. MACAIOSE. C. TLCCI.G . DE LUCA and R. M. DIGIORGIO
for 1 h at 4 T . After centrifugation at 53.000 g for 30 min the following subcellular fractions were isolated: myelin (P,A). synaptosomal (PZB)and mitochondria1 IP2C)frac60 tions. The P,A, P,B. P,C fractions were diluted with 1.2 vol of water and sedimented at 22.000 g for 10 min. Tauriue assa!. Retinae and pigment epithelium were homogenized in ice-cold 0.067 M-Sorensen's phosphate buffer pH 6.8 containing 0.2"" Triton X-100 (9 ml g tissue). The extracts were immediately deproteinized by adding 200; (wiv) trichloracetic acid to make the final concentration 5%. After 2&30 min at 0 C the samples were centri- FIG. 2. Taurine distribution in the secondary subcellular fuged at 20.000g for 30min and the taurine content was fractions obtained from the crude mitochondria of ox assayed on aliquots of the protein-free supernatant on a retina (see the text for details). Distribution calculated as 55 x 0.9 cm 3AR ?,'A, 55 resin column. using a Carlo Erba " o of total recovered taurine. Aminoacid Analyzer Mod. G.P. Columns were washed with 0.3 N-lithium hydroxide. Elution was started at 55-C were dehydrated in progressively concentrated solutions of with lithium citrate buffer. pH 4.1 5 (90 m1.h flow rate): nin- ethanol and embedded in Durcopan. sectioned at 6 5 n m hydrin flow rate 45 m1.h. By this method taurine was and then stained with uranyl acetate and lead citrate mixture. separated from cysteic acid and cysteine sulphinate. The subcellular fractions were homogenized in RESULTS 0.067 M-Sorensen's phosphate buffer containing 0.2", Triton X-100, while Sorensen's phosphate buffer (1 M) was The subcellular distribution of taurine is shown in added to the S3 fraction to make the final concentration equal to 0.067 M. Samples were deproteinized and centri- Figs. 1 a n d 2. In the primary subcellular fractions taurfuged: aliquots of supernatant were used for taurine assay. ine follows the distribution pattern of a soluble amino & WHITTAKER, 1966). About 79.57; of € ~ y n i eassay. Cysteine sulphinic acid decarboxylrtse was acid (MANGAN assayed using the conditions of AGRAWALrf al. (1971) with recovered taurine has been found in the soluble fracslight modifications. in Warburg flasks equipped with a tion &). while 20.50, was associated with the particuside arm. late components of retina. Taurine associated with The reaction mixture in the main compartment con- the particulate fractions was released by adding Trilo-' wdithiothreitol and enzqme tained lO-'w-PLP. ton X-100. preparation in 0.067 M-Sorensen's phosphate buffer pH 6.8 In the secondary subcellular fractions, separated with O.l:o Triton X-100. Cysteine sulphinate or cqsteic acid in 0.067 M-phosphate buffer 10.2 ml) was in the side arm from P, by centrifugation on a sucrose density grato give a final concentration of I ~ - ' M rind the final dient and examined by electron microscopy (Figs. 3 4 ) , taurine is primarily associated with the synaptovolume was 3.25 ml. Control samples (0.2nil of 0.067 M-phosphate buffer soma1 fraction. Taurine is the predominant free amino acid in solution in the side arm) were also included. The samples were gassed with N, for 15 min and after a 15-20 min pre- o x retina. where it is present at a concentration of incubation at 37-C. the evolution o f C 0 2 was measured 2C-22 jtmo1;g wet wt. of tissue and of 1&18 pmoI/ by taking readings every 10min up to 1 h. 100 mg protein. Protein dvrernii~iario~i. Proteins were determined b! the In the pigment epithelium taurine is present at a procedure of LOWRY('r LII. (19511. concentration of 1.85-2.20 pmo1/100mg protein; the Electroti itiicroscopy. Specimens were sedimented at 55.000g for 60min. pre-fixed in glutaraldehyde (pH 7.2) retinaipigment epithelium ratio is about 8.5: 1. Cysteine sulphinate decarboxylase activity is priand fixed in Millonig's OsO, solution. Fived specimeiis
marily associated with the particulate components of 90
50
FIG. 1. Taurine distribution in the primary subcellular fractions (see the text for details) isolated from ox rctina. The recovered amount was 100.18", of homopenate. Results are the means of 2 experiments.
FIG. 7. Distribution of cystcinc' ~ulpl~inatt: decarboxylase activity in the primary subcellular fractions of ox retina. Distribution calculated as O 0 of total recovered activity.
FIG.3. The fraction called 'Crude Mitochondria' is rich in various structures. There can be seen mitochondria of dilTerent morphology and size (Fig. 3a. magnification 6000-fold). processes of Muller cells (Fig. 3b. magnification 20.000-fold) and synaptic zones characterized by vesicles of rather regular size (Fig. 3a-c). Magnification of Fig. 3c is 20.000-fold. C: cylium: S : synaptic zones; M P : process of Muller cell.
FIG.4. The fraction designatcd ’MLelin’ ma! br conbidered homogcnt.ous. Magnification is 10.000-Told.
FIG. 5. (a) The fraction called ‘S>naptosomes. ma! he considered honiogcncous. e ~ e i iif one can see some mitochondria of different size and morpliolog) (H.000x 1. (hl The insert shows the morphology of one 0 1 the so called ‘s!naptosonies’ (2O.OO(l x
).
FIG.6. Fraction called ‘Purified Mitochondria’. Mitochondria of various size and morphology can be seen: some are well-preserved. while some are swollen with dilated ‘christae’. Magnification is 16,000-f0ld.
Taurine and cysteine levels in ox retina 90
30
1313
-
25 60
i
3 c
01 \
u
15-
FIG. 8. Distribution of cysteine sulphinate decarboxylase activity in the secondary subcellular fractions of ox retina. Distribution calculated as 7; of total recovered activity. 0
the primary subcellular fractions of ox retina and a major portion of the enzyme activity is present in the crude mitochondria1 fraction (Fig. 7). Further localization of cysteine sulphinate decarboxylase activity in the subcellular fractions of crude mitochondria has shown that about 46% of the recovered enzyme activity is present in the synaptosoma1 fraction (Fig. 8). The synaptosomal cysteine sulphinate decarboxylase is released only by adding Triton X-100, suggesting that it may be occluded in synaptosomes. The results on the decarboxylation of both cysteine sulphinate and cysteic acid in the homogenate and the primary subcellular fractions are reported in Fig. 9 and Fig. 10. The ratio between the decarboxylation of cysteine sulphinate and of cysteate (See Table 1) was constant throughout all the steps of our fractionation procedure. This ratio is approx 6.6 and is similar to that obtained by GUION-RAIN & CHATAGNER (1 972) for rat liver.
10
20
30 rnin
40
50
FIG.10. Decarboxylation of cysteine sulphinate (M-H supernatant, A-A microsomal fraction) and of cysteate (0-0 supernatant, A--G microsomal fraction) in ox retina. Each flask contains 2 ml of subcellular fraction corresponding to 1 g of tissue. For enzyme activity assay, see Methods. DISCUSSION
The subcellular distribution of taurine (Figs. 1 and 2) gives preliminary evidence for the cellular compartmentation of this amino acid and suggests that taurine has a different functional role in the soluble and particulate fractions. The synaptosomal fraction contains a relatively high concentration of taurine and this could be consistent with its neurotransmitter function. Our results show that, in ox retina, there are high taurine levels; this is in agreement with the results of KUBICEK& DOLENEK (1958) for retina of several species and with the data of PASANTES-MORALES et al. (19720) for chicken and rat retina. The taurine concentration in ox retina is higher than that reported by PASANTES-MORALES et al. (1972n) and lower than found in rat retina by MACAIONE ef al. (1974). These differences could be related to the different methods employed in amino acid extraction. The data relative to the taurine distribution in the secondary subcellular fractions are similar to those found in our previous work on rat retina (MACAIONE TABLE1. RATIO OF CYSTEINE SLJLPHINATEDECARBOXYLATION (A) TO CYSTEATE DECARBOXYLATION (B) IN THE PRIMARY SUBCELLULAR FRACTIONS
Fraction !O
30 rnin
1
L
40
50
FIG.9. Decarboxylation of cysteine sulphinate (+a crude mitochondria, A-A nuclear fraction) and of cyscrude mitochondria, G-G nuclear fracteate (W tion) in ox retina. Each flask contains 2 m l of subcellular fraction corresponding to 1 g of tissue. The composition of the incubation mixture is described in the text.
Nuclear fraction Mitochondria1 fraction Microsomal fraction Supernatant
A/B
5.5 6.9
6.3 7.9
Details o n the preparation of the primary subcellular fractions and on enzyme activity assay are described under Methods. Values represent the ratio of six experiments averages.
1414
S.
MATAIONE.
G.
TCcC'l.
G. DE LL~CAand R . M. DI
r't ul.. 1975); they are in agreement with the results of AGKAWAL~ ' ul. t (1971) who found that most of
GlORGlO
regulatory effect on oxygen uptake (SICUTERIet ul., 1970). The high rates of respiration and aerobic glycolysis in retina and, in particular, in photoreceptor cells are well recognised (COHEN & NOELL,1960; GRAYMORE. 1959). Such a role for taurine might therefore be of importance and should be considered.
the taurine, localized in the brain crude mitochondria. was recovered in the two synaptosomal fractions obtained according to Dt ROBERTISet ul. (1962). Recent electroph~siologicaland biochemical evidence suggests that taurine might act as an inhibitory transmitter in brain. spinal cord and retina (CURTIS Ac~riorr,/edger?ierits-The authors are grateful to Dr. & WATKINS,1965: PASANTES-MORALES et ti1 19726; PUZZOLO. Assistant-professor of Histology and DOMENICO 1973). The action of taurine might be located in the Ernbriology Department of Messina University for invaluinner plexiform layer: in rabbit retina a remarkable able help in performing and explaining electron microuptake of labelled taurine into the retinal glial cells photographs. was observed by EHISGER(1973). All these data can confirm the possible neurotransmitter role of taurine. nevertheless they d o not explain the high taurine retinal levels and the consistent presREFERENCES ence of this amino acid in photoreceptor cells (KENNEDY & VOADEN.1974u.h; Ktt?; & YATES. 1974). AGRAWALH . C.. DAMSONA. N. & KACZMAREK L. K. Frog pigment epithelium has a particularly active ( 1 971) Biockrnr. J . 122. 759-763. mechanism for accumulating taurine; LAKEet ul. COHENL. H. & NOELLW. K. (1960) J . NcUrOChcfJl. 5, (1975) suggest that i i i r i r o a major portion of taurine. 253-276. J. C. (1961) Nature. Lorid. 191, present in retina. is taken up by the epithelium from CURTIS D. R. & WATKINS 101@IO1 I . the blood stream and is then transferred to photoCL'RTISD. R. & WATKINSJ. C. (1965) Pharmuc. Rtr. 17, receptor cells. 347-39 1 . In the ox, the retina/pipent epithelium ratio of DE ROBERTIS E.. PFLLFCiHlNO DE IRALIX A,. RODRIGUEZ DE taurine levels is about 8.5 : I . LORESA R N A I Z G. & SALGANICOFF L. (1962) J . NeuroIn the cat, photoreceptor cell degeneration is aschcrti. 9, 13-35. sociated with a decrease of taurine in plasma and DI GlORClo R. M.. TuCCI G. & MACAIONE S. (1975) Life et ul.. 1975). The decrease in the retinal retina (HAYES Sci. 16. 429436. taurine content may be due to a reduced synthesis EHISCERB. (1973) Brctiri Res. 60. 512-516. V . C. & POTTSA. M. (1962) I r i w s t . Ophrhul. in retina or a reduced taurine uptake from plasma. GLOCKLIK 1. I l l . In growing kittens the retinal degeneration is preC. N . (1959) Br. J . Ophrlia/. 43. 34-39. vented by taurine administration. but not by meth- GRAYMORE F. (1972) Biochini. bioGUION-RAIN M. C . & CHATACNER ionine or cysteine: HAYESet trl. (1975) suggested that phys. ..lctil 276. 272-276. the taurine synthetic pathway from sulphur amino HAYESK. C.. CAREY R . E. & SCHMIDT S. Y. (1975) Science, acids may be inadequate or limited during developR'.Y. 188. 919-951. ment. K t t s P. & YATM R. A. (1974) Br. J . Phurrnuc. 52. 118P. The data reported in this study demonstrate that K ~ S S E D YA. J. & VOADENM. J. (19740) J . N~wocheni. taurine is synthesized in ox retina. In the homogenate 23. 1093 1095. and the primary subcellular fractions both cysteine K E I ~ E D YA. J. & V O A D ~ N M. J. (19736) Biochrm. Soc. Trtrris. 2. I256~-1258. sulphinate and cysteic acid are decarboxylated. A. (1958) J . Ckrornar. 1. 266-268. The rate of decarboxylation for cysteine sulphinate K c ~ i c cR.~& DOLLNEK J. & VOADENM. J. (1975) Biocl~em. is higher than for cysteic acid (Figs. 9 and 10) and LAKEN.. MARSHALL Soc. Trans. 3. 524525. the ratio of the two decarboxylating activities. approx L O W R 0. ~ H.. ROSEBROUGHN. J.. FAKKA. L. & RANDALL 6.6. is constant (Table I ) ; this uniformity may exclude R. J. (1951) J . hid. Chcwi. 193. 765-275. the existence of two separate enzymes. M A C A I O \S.. I Rl'c;(;FRI P.. DE LUCA F. & Tuccr G. (1974) The distribution of the cysteine sulphinate decarJ . .\ t , u r i ) J i ~ , r i r .22. 887-891. boxylase activity gives evidence for the compartmen- MACAIOKE S.. T L C U G. & DI GloRGIo R. M. (1975) Ital. tation of this enzyme in ox retina (Fig. 7). J . Biochtvn. 24. 162-174. Synaptosomal fraction contains a relatively high MANGASJ . L. & WHITTAKER V. P. (1966) Biochrrn. J . 98. 128-1 37. concentration of enzyme activity (Fig. 8) and this suggests that the synaptosomal taurine can be synthe- NEALM. J.. PEACCKKD. G. & WHITER. D. (1973) Br. J . Pharrnuc. 41. 656-657P. sized ;ti situ. N. E., D'ERMO F. & These results are in agreement with the data of PAP.* S.. SECCHl A . G.. LOFRUMENTO QL.AGLIARIELLO E. (1965) Ira/. J . Biorhnn. 14, 175-183. AGRAWALet a/. (1971) and RASSIN& GALLL(1975) PASANTES-MORALES H.. K L ~ T HJ.,I LEDIC M. & MANUEL who have shown, in rat brain. a different subcellular P. ( 1 9 7 2 ~ Bruiri ) Rus. 41. 494497. distribution of enzymes involved in the taurine syn- PASANTES-MORALE, H., KLETHr J.. URBANP. F. & MANDEL thesis. P. (19721) Physiol. Chsn. Phys. 4. 339-348. 111 retina other functions for taurine are possible. PASANTES-MORALES H.. URBANP. F.. KLETHIJ. & MANDLL P. (1973) Bruiri Rrs. 51, 375-378. It has been proposed that taurine may also exert a
Taurine and cysteine levels in ox retina
RASSIN D. K. &
GALILL G. E. (1975) J . Ncurodwm. 24. 969-918. SICUTERI F.. FANCIULLACCI M., FRANCHI G., GlOTTl A. & GulDorTl A. (1970) C h i . m d . ital. 77, 21.
SlECHART
1415
W. & KARORATH M. (1974) J. A’c~rochem.23.
911-915.
STARRM. S. & VOADEN M. J. (1972) 126 1- 1269.
Irsiojr
Rtzs. 12.
ALTHOUGH taurine is the predominant amino acid in demonstrates that this enzyme activity is present also mature retina (KUBICEK & DOLENEK, 1958; PASANTES- in the synaptosomal fraction. et al., 1972a; MACAIONE et al., 1974) its phyMORALES MATERIALS AND METHODS siological role remains uncertain; however taurine has a depressant effect on mammalian spinal and cortical Chemicals. In general AR grade chemicals were used. neurons (CURTIS& WATKINS,1961) and reversibly The sources of certain additional chemicals were as folabolishes the b-wave of the electroretinogram in a lows: L-cysteinesulphinic acid and L-cysteic acid (Serva. perfused retina preparation (PASANTES-MOKALES et a/., Entwicklungslabor, Heidelberg. Germany): dithiothreitol (Fluka AG, Chemische Fabrik. Buchs, Scweiz); pyridox1973). Uptake systems for taurine have been demon- al-5-phosphate (PLP) and Triton X-100 (Koch-Light strated in retina (STARR& VOADEN,1972; PASANTES- Laboratories Ltd, Colnbrook. England); ninhydrin and MORALESet al.. 1972h; NEALet al., 1973) and also taurine (Schuchardt, GmbH Hohenbrunn. Germany). Aiiirnals. Ox eyes were obtained from the local slaughterin pigment epithelium (LAKEet al., 1975). In brain house and used immediately after the animal's death. tissue the accumulation of [35S]taurine by a specific The eyes were enucleated and dissected just behind the synaptosomal population was shown by SIEGHART & ora serrata. The posterior halves of the eye balls were KAROBATH (1974). Both pre-loaded [35S]taurine and placed in ice-cold saline and everted. The vitreous. if it endogenous amino acid are released from chicken was still adherent to the exposed retina, was removed with retina in response to intensive light stimulation forceps. Retinae were prepared free from underlying choroid, carefully washed to remove pigment epithelium and (PASANTES-MORALES et al., 1973). In frog retina, approx 7.5;; of the total taurine con- immediately frozen at - IO'C. The retinal pigment epithe& PoTTs (1962). tent is associated with photoreceptor cells (KENNEDY lium was isolated according to GLOCKLIN The complete operation was carried out in a cold room & VOADEN,1974a.h). Our previous work on rat retina has shown that maintained at O"4"C and with normal light. Subcellularfracrionatiori The pooled retinae were honiotaurine levels increase during postnatal growth and genized in 0.32 M-sucrose (9 ml/g tissue) in a glass homthat most of the taurine present in the retinal crude ogenizer with a Teflon pestle at 20o0 rev./min for 3 min mitochondria1 fraction is associated with synaptic at 4°C and centrifuged at 800g for 5 min to obtain pellet endings (MACAIONE et ul., 1975). P and supernatant S. Pellet P was then suspended in The presence of the cysteine oxidase activity in 0.32 M-sucrose (4 ml/g tissue), homogenized at 1500 re\.,' cytoplasm and in crude mitochondria of rat retina min and sedimented at 8009 for 5 min to obtain pellet (Dr GIORGIO et al., 1975) and the observations of PI (nuclear fraction) and supernatant S,. The crude mitoHAYESet a/. (1975) in cat retina and of LAKEet al. chondrial fraction (P2) was obtained according to PAPA (1975) in frog retina, have raised the question as to et al. (1965) from the combined supernatants S and S , . whether taurine is formed in retina or whether it is The microsomal (P,) and the soluble fractions (S3) were prepared at 55,000 g for 90 min. taken up from the blood stream. The P, pellet was resuspended in 0.32 M-sucrose ( I ml/g The present study establishes the distribution of the tissue) and layered on a discontinuous density gradient cysteine sulphinate decarboxylase and the levels of consisting of 6 ml each of 0.8 and 1.2 M-sucrose. The grataurine in the ox retina subcellular fractions and dient was prepared at room temperature and then kept 141 1
1412
S. MACAIOSE. C. TLCCI.G . DE LUCA and R. M. DIGIORGIO
for 1 h at 4 T . After centrifugation at 53.000 g for 30 min the following subcellular fractions were isolated: myelin (P,A). synaptosomal (PZB)and mitochondria1 IP2C)frac60 tions. The P,A, P,B. P,C fractions were diluted with 1.2 vol of water and sedimented at 22.000 g for 10 min. Tauriue assa!. Retinae and pigment epithelium were homogenized in ice-cold 0.067 M-Sorensen's phosphate buffer pH 6.8 containing 0.2"" Triton X-100 (9 ml g tissue). The extracts were immediately deproteinized by adding 200; (wiv) trichloracetic acid to make the final concentration 5%. After 2&30 min at 0 C the samples were centri- FIG. 2. Taurine distribution in the secondary subcellular fuged at 20.000g for 30min and the taurine content was fractions obtained from the crude mitochondria of ox assayed on aliquots of the protein-free supernatant on a retina (see the text for details). Distribution calculated as 55 x 0.9 cm 3AR ?,'A, 55 resin column. using a Carlo Erba " o of total recovered taurine. Aminoacid Analyzer Mod. G.P. Columns were washed with 0.3 N-lithium hydroxide. Elution was started at 55-C were dehydrated in progressively concentrated solutions of with lithium citrate buffer. pH 4.1 5 (90 m1.h flow rate): nin- ethanol and embedded in Durcopan. sectioned at 6 5 n m hydrin flow rate 45 m1.h. By this method taurine was and then stained with uranyl acetate and lead citrate mixture. separated from cysteic acid and cysteine sulphinate. The subcellular fractions were homogenized in RESULTS 0.067 M-Sorensen's phosphate buffer containing 0.2", Triton X-100, while Sorensen's phosphate buffer (1 M) was The subcellular distribution of taurine is shown in added to the S3 fraction to make the final concentration equal to 0.067 M. Samples were deproteinized and centri- Figs. 1 a n d 2. In the primary subcellular fractions taurfuged: aliquots of supernatant were used for taurine assay. ine follows the distribution pattern of a soluble amino & WHITTAKER, 1966). About 79.57; of € ~ y n i eassay. Cysteine sulphinic acid decarboxylrtse was acid (MANGAN assayed using the conditions of AGRAWALrf al. (1971) with recovered taurine has been found in the soluble fracslight modifications. in Warburg flasks equipped with a tion &). while 20.50, was associated with the particuside arm. late components of retina. Taurine associated with The reaction mixture in the main compartment con- the particulate fractions was released by adding Trilo-' wdithiothreitol and enzqme tained lO-'w-PLP. ton X-100. preparation in 0.067 M-Sorensen's phosphate buffer pH 6.8 In the secondary subcellular fractions, separated with O.l:o Triton X-100. Cysteine sulphinate or cqsteic acid in 0.067 M-phosphate buffer 10.2 ml) was in the side arm from P, by centrifugation on a sucrose density grato give a final concentration of I ~ - ' M rind the final dient and examined by electron microscopy (Figs. 3 4 ) , taurine is primarily associated with the synaptovolume was 3.25 ml. Control samples (0.2nil of 0.067 M-phosphate buffer soma1 fraction. Taurine is the predominant free amino acid in solution in the side arm) were also included. The samples were gassed with N, for 15 min and after a 15-20 min pre- o x retina. where it is present at a concentration of incubation at 37-C. the evolution o f C 0 2 was measured 2C-22 jtmo1;g wet wt. of tissue and of 1&18 pmoI/ by taking readings every 10min up to 1 h. 100 mg protein. Protein dvrernii~iario~i. Proteins were determined b! the In the pigment epithelium taurine is present at a procedure of LOWRY('r LII. (19511. concentration of 1.85-2.20 pmo1/100mg protein; the Electroti itiicroscopy. Specimens were sedimented at 55.000g for 60min. pre-fixed in glutaraldehyde (pH 7.2) retinaipigment epithelium ratio is about 8.5: 1. Cysteine sulphinate decarboxylase activity is priand fixed in Millonig's OsO, solution. Fived specimeiis
marily associated with the particulate components of 90
50
FIG. 1. Taurine distribution in the primary subcellular fractions (see the text for details) isolated from ox rctina. The recovered amount was 100.18", of homopenate. Results are the means of 2 experiments.
FIG. 7. Distribution of cystcinc' ~ulpl~inatt: decarboxylase activity in the primary subcellular fractions of ox retina. Distribution calculated as O 0 of total recovered activity.
FIG.3. The fraction called 'Crude Mitochondria' is rich in various structures. There can be seen mitochondria of dilTerent morphology and size (Fig. 3a. magnification 6000-fold). processes of Muller cells (Fig. 3b. magnification 20.000-fold) and synaptic zones characterized by vesicles of rather regular size (Fig. 3a-c). Magnification of Fig. 3c is 20.000-fold. C: cylium: S : synaptic zones; M P : process of Muller cell.
FIG.4. The fraction designatcd ’MLelin’ ma! br conbidered homogcnt.ous. Magnification is 10.000-Told.
FIG. 5. (a) The fraction called ‘S>naptosomes. ma! he considered honiogcncous. e ~ e i iif one can see some mitochondria of different size and morpliolog) (H.000x 1. (hl The insert shows the morphology of one 0 1 the so called ‘s!naptosonies’ (2O.OO(l x
).
FIG.6. Fraction called ‘Purified Mitochondria’. Mitochondria of various size and morphology can be seen: some are well-preserved. while some are swollen with dilated ‘christae’. Magnification is 16,000-f0ld.
Taurine and cysteine levels in ox retina 90
30
1313
-
25 60
i
3 c
01 \
u
15-
FIG. 8. Distribution of cysteine sulphinate decarboxylase activity in the secondary subcellular fractions of ox retina. Distribution calculated as 7; of total recovered activity. 0
the primary subcellular fractions of ox retina and a major portion of the enzyme activity is present in the crude mitochondria1 fraction (Fig. 7). Further localization of cysteine sulphinate decarboxylase activity in the subcellular fractions of crude mitochondria has shown that about 46% of the recovered enzyme activity is present in the synaptosoma1 fraction (Fig. 8). The synaptosomal cysteine sulphinate decarboxylase is released only by adding Triton X-100, suggesting that it may be occluded in synaptosomes. The results on the decarboxylation of both cysteine sulphinate and cysteic acid in the homogenate and the primary subcellular fractions are reported in Fig. 9 and Fig. 10. The ratio between the decarboxylation of cysteine sulphinate and of cysteate (See Table 1) was constant throughout all the steps of our fractionation procedure. This ratio is approx 6.6 and is similar to that obtained by GUION-RAIN & CHATAGNER (1 972) for rat liver.
10
20
30 rnin
40
50
FIG.10. Decarboxylation of cysteine sulphinate (M-H supernatant, A-A microsomal fraction) and of cysteate (0-0 supernatant, A--G microsomal fraction) in ox retina. Each flask contains 2 ml of subcellular fraction corresponding to 1 g of tissue. For enzyme activity assay, see Methods. DISCUSSION
The subcellular distribution of taurine (Figs. 1 and 2) gives preliminary evidence for the cellular compartmentation of this amino acid and suggests that taurine has a different functional role in the soluble and particulate fractions. The synaptosomal fraction contains a relatively high concentration of taurine and this could be consistent with its neurotransmitter function. Our results show that, in ox retina, there are high taurine levels; this is in agreement with the results of KUBICEK& DOLENEK (1958) for retina of several species and with the data of PASANTES-MORALES et al. (19720) for chicken and rat retina. The taurine concentration in ox retina is higher than that reported by PASANTES-MORALES et al. (1972n) and lower than found in rat retina by MACAIONE ef al. (1974). These differences could be related to the different methods employed in amino acid extraction. The data relative to the taurine distribution in the secondary subcellular fractions are similar to those found in our previous work on rat retina (MACAIONE TABLE1. RATIO OF CYSTEINE SLJLPHINATEDECARBOXYLATION (A) TO CYSTEATE DECARBOXYLATION (B) IN THE PRIMARY SUBCELLULAR FRACTIONS
Fraction !O
30 rnin
1
L
40
50
FIG.9. Decarboxylation of cysteine sulphinate (+a crude mitochondria, A-A nuclear fraction) and of cyscrude mitochondria, G-G nuclear fracteate (W tion) in ox retina. Each flask contains 2 m l of subcellular fraction corresponding to 1 g of tissue. The composition of the incubation mixture is described in the text.
Nuclear fraction Mitochondria1 fraction Microsomal fraction Supernatant
A/B
5.5 6.9
6.3 7.9
Details o n the preparation of the primary subcellular fractions and on enzyme activity assay are described under Methods. Values represent the ratio of six experiments averages.
1414
S.
MATAIONE.
G.
TCcC'l.
G. DE LL~CAand R . M. DI
r't ul.. 1975); they are in agreement with the results of AGKAWAL~ ' ul. t (1971) who found that most of
GlORGlO
regulatory effect on oxygen uptake (SICUTERIet ul., 1970). The high rates of respiration and aerobic glycolysis in retina and, in particular, in photoreceptor cells are well recognised (COHEN & NOELL,1960; GRAYMORE. 1959). Such a role for taurine might therefore be of importance and should be considered.
the taurine, localized in the brain crude mitochondria. was recovered in the two synaptosomal fractions obtained according to Dt ROBERTISet ul. (1962). Recent electroph~siologicaland biochemical evidence suggests that taurine might act as an inhibitory transmitter in brain. spinal cord and retina (CURTIS Ac~riorr,/edger?ierits-The authors are grateful to Dr. & WATKINS,1965: PASANTES-MORALES et ti1 19726; PUZZOLO. Assistant-professor of Histology and DOMENICO 1973). The action of taurine might be located in the Ernbriology Department of Messina University for invaluinner plexiform layer: in rabbit retina a remarkable able help in performing and explaining electron microuptake of labelled taurine into the retinal glial cells photographs. was observed by EHISGER(1973). All these data can confirm the possible neurotransmitter role of taurine. nevertheless they d o not explain the high taurine retinal levels and the consistent presREFERENCES ence of this amino acid in photoreceptor cells (KENNEDY & VOADEN.1974u.h; Ktt?; & YATES. 1974). AGRAWALH . C.. DAMSONA. N. & KACZMAREK L. K. Frog pigment epithelium has a particularly active ( 1 971) Biockrnr. J . 122. 759-763. mechanism for accumulating taurine; LAKEet ul. COHENL. H. & NOELLW. K. (1960) J . NcUrOChcfJl. 5, (1975) suggest that i i i r i r o a major portion of taurine. 253-276. J. C. (1961) Nature. Lorid. 191, present in retina. is taken up by the epithelium from CURTIS D. R. & WATKINS 101@IO1 I . the blood stream and is then transferred to photoCL'RTISD. R. & WATKINSJ. C. (1965) Pharmuc. Rtr. 17, receptor cells. 347-39 1 . In the ox, the retina/pipent epithelium ratio of DE ROBERTIS E.. PFLLFCiHlNO DE IRALIX A,. RODRIGUEZ DE taurine levels is about 8.5 : I . LORESA R N A I Z G. & SALGANICOFF L. (1962) J . NeuroIn the cat, photoreceptor cell degeneration is aschcrti. 9, 13-35. sociated with a decrease of taurine in plasma and DI GlORClo R. M.. TuCCI G. & MACAIONE S. (1975) Life et ul.. 1975). The decrease in the retinal retina (HAYES Sci. 16. 429436. taurine content may be due to a reduced synthesis EHISCERB. (1973) Brctiri Res. 60. 512-516. V . C. & POTTSA. M. (1962) I r i w s t . Ophrhul. in retina or a reduced taurine uptake from plasma. GLOCKLIK 1. I l l . In growing kittens the retinal degeneration is preC. N . (1959) Br. J . Ophrlia/. 43. 34-39. vented by taurine administration. but not by meth- GRAYMORE F. (1972) Biochini. bioGUION-RAIN M. C . & CHATACNER ionine or cysteine: HAYESet trl. (1975) suggested that phys. ..lctil 276. 272-276. the taurine synthetic pathway from sulphur amino HAYESK. C.. CAREY R . E. & SCHMIDT S. Y. (1975) Science, acids may be inadequate or limited during developR'.Y. 188. 919-951. ment. K t t s P. & YATM R. A. (1974) Br. J . Phurrnuc. 52. 118P. The data reported in this study demonstrate that K ~ S S E D YA. J. & VOADENM. J. (19740) J . N~wocheni. taurine is synthesized in ox retina. In the homogenate 23. 1093 1095. and the primary subcellular fractions both cysteine K E I ~ E D YA. J. & V O A D ~ N M. J. (19736) Biochrm. Soc. Trtrris. 2. I256~-1258. sulphinate and cysteic acid are decarboxylated. A. (1958) J . Ckrornar. 1. 266-268. The rate of decarboxylation for cysteine sulphinate K c ~ i c cR.~& DOLLNEK J. & VOADENM. J. (1975) Biocl~em. is higher than for cysteic acid (Figs. 9 and 10) and LAKEN.. MARSHALL Soc. Trans. 3. 524525. the ratio of the two decarboxylating activities. approx L O W R 0. ~ H.. ROSEBROUGHN. J.. FAKKA. L. & RANDALL 6.6. is constant (Table I ) ; this uniformity may exclude R. J. (1951) J . hid. Chcwi. 193. 765-275. the existence of two separate enzymes. M A C A I O \S.. I Rl'c;(;FRI P.. DE LUCA F. & Tuccr G. (1974) The distribution of the cysteine sulphinate decarJ . .\ t , u r i ) J i ~ , r i r .22. 887-891. boxylase activity gives evidence for the compartmen- MACAIOKE S.. T L C U G. & DI GloRGIo R. M. (1975) Ital. tation of this enzyme in ox retina (Fig. 7). J . Biochtvn. 24. 162-174. Synaptosomal fraction contains a relatively high MANGASJ . L. & WHITTAKER V. P. (1966) Biochrrn. J . 98. 128-1 37. concentration of enzyme activity (Fig. 8) and this suggests that the synaptosomal taurine can be synthe- NEALM. J.. PEACCKKD. G. & WHITER. D. (1973) Br. J . Pharrnuc. 41. 656-657P. sized ;ti situ. N. E., D'ERMO F. & These results are in agreement with the data of PAP.* S.. SECCHl A . G.. LOFRUMENTO QL.AGLIARIELLO E. (1965) Ira/. J . Biorhnn. 14, 175-183. AGRAWALet a/. (1971) and RASSIN& GALLL(1975) PASANTES-MORALES H.. K L ~ T HJ.,I LEDIC M. & MANUEL who have shown, in rat brain. a different subcellular P. ( 1 9 7 2 ~ Bruiri ) Rus. 41. 494497. distribution of enzymes involved in the taurine syn- PASANTES-MORALE, H., KLETHr J.. URBANP. F. & MANDEL thesis. P. (19721) Physiol. Chsn. Phys. 4. 339-348. 111 retina other functions for taurine are possible. PASANTES-MORALES H.. URBANP. F.. KLETHIJ. & MANDLL P. (1973) Bruiri Rrs. 51, 375-378. It has been proposed that taurine may also exert a
Taurine and cysteine levels in ox retina
RASSIN D. K. &
GALILL G. E. (1975) J . Ncurodwm. 24. 969-918. SICUTERI F.. FANCIULLACCI M., FRANCHI G., GlOTTl A. & GulDorTl A. (1970) C h i . m d . ital. 77, 21.
SlECHART
1415
W. & KARORATH M. (1974) J. A’c~rochem.23.
911-915.
STARRM. S. & VOADEN M. J. (1972) 126 1- 1269.
Irsiojr
Rtzs. 12.
