Life Sciences Vol . 20, pp . 1657-1662, 1977 . Printed in the U .S .A .

Pergamon Press

SUBCELLULÀR DISTRIBUTION OF HYPOTAURINE OXI DASS ACTIVITY IN OX RETINA Rosa M . DI Giorglo,Salvatore Macaione and Grazi a .De Luca Department of Biocheml stry, Un i versi t y of Messl na,Messi na, Italy (Received in final form April 18, 1977)

Sum mary Hypotaurine oxidase activity was determined In the primary and secondary subfra et I ons of ox ret I na . About 78% of enzyme actl vi ty was found In the soluble fraction while about 22% was associated with particulate components . In the secondary subcellular fractions about 55% of enzyme activity, recovered f rom crude mltochondri a, was present In the synaptos omal fraction . This finding suggests that t he conversi on of h ypotaurine to taurine Is possibl e in ox reti na . Recent studies have been carried out on the possi bl e rol e of t aurine and there Is now evidence that this amino acid may be i nvol ved as n eurotransmitter, or modulator, In the central nervous system (1, 2) . Taurine is present at high levels I n the ret I na of several specl es (3-7) . It has been demonstrated that I i ght or et act rl cal stimulation induces release of taurine from chicken ret I na ( 8, 9), that t suri ne 1 s actively taken up by the isolated retina but exerts a depressant action on the 15-wave amplitude of the electroretinogram ( 4) . These data lead to the hypothesis that taurine might act as Inhibitory transmitter also I n the retina . The discovery of the decarboxyl a t i on of cyst ei ne sul phi nat e to hypotaurine, which is then oxidized to taurine, led to the proposl t ion that this pathway represents the main blosynthetl c rout e f or t aurl ne In animal tissues (10, 11 ) . The finding of consist ent amounts of hypot aurl ne in organs and body fluids after injection or feeding of cyst ei ne (12-14) strongly supported the occurrence of this pathway In animals . Our earlier works (15, 16) on the metabol I sm of sul phur-containing amino acids In ox ret Ina have shown that cystel ne oxi dase and cystelne sulphinate decarboxylase are present i n thl s t i ssue and are mainly associated wi th the particulate components . Very little, however, Is known about the oxi dat i on of hypotaurine to taurlne .Sumizu (17) reported on the propert I es of parts al I y p url fled 1657

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hypotaurine oxidase, though Flori and Costa ( 18) were I at er unable to reproduce these resuIts . They suggested that hypotaurine maybe ox Idized by the small amounts of hydrogen peroxide present in t Issues, but It was demonstrated that NAD+ Is necessary as cof actor 1 n the oxi dat I on of hypotaurine (19) . No detectable conversion of hypot aura ne t o t aurl ne occurred In preparations from aduI t rat brain, nel they d I d et ect ricaI stimulation Induce the oxidation of hypotaurine in these preparations ( 19) . In order to complete the study of the enzymes concerned with the blosynthesis of taurine In ox retina we have examined the presence and the subcel lular distribution of hypotaurl ne oxi dase i n t his t issue . Experimental Section Materials - Hypotaurine and taurine were supplied by Calbl ochem, 2, 3-dimercapto-1-propanol (BAL ) by Koch Li ght, NAD + by Boehringer Mannheim Gm bH, Germany. AI I othe r reagent s u sed were of analytical grade . Methods - Ox eyes were obtal ned from the and used Immediately after the animal's death .

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The drawing of retina and the separat i on of t he pri ma r y and secon dary subcel lular fractions were carried out as previ ousl y described by us (15) . The enzyme activity was assayed by the method of OJa et al .(19). The homogenate and the primary and secondary subcel I ul ar f ract i ons In 0. 32 M-sucrose were buffered wl t h 1 M-Tris-HC 1, pH 7.4 to make t he final concentration equal to 0 .1 N, . The reacts on mixture contal ned I n a f I nal vol ume of 250 jil 0.4 Nmoles of hypotaurine, 2 moles of oxidl zed NAD+, 2 rmoles of BAL and the enzyme . The samples were incubated for 120 min at 37°C and the enzymic reaction was stopped by adding 250 pi of 20% TCA ( w/v) .After centrifugation the basal and extra formed taurine of supernatants was quantified with an amino acid analyzer C . Erba Mod. G. P. on a 55 x 0.9 cm 3 AR/2/A/55 resin column washed with 0 .35 N-1I thi um hydroxide. Elution was started at 55°C with lithium citrat e buffer pH 4 .15 (90 ml/h flow rate) ; ninhydrin flow rate 45 ml/h .

Protein contents of total homogenate and subcel I ul ar f ractlons were determined according to Lowry et al . (26) . Results are expressed I n moles of taurine/g wet wt/120 min . Results The distribution of hypotaurine oxidase In the primary subcel lular fractions Is shown in Fig. 1 . Approximately 78% of enzyme activity was recovered in soluble fraction . In the mlcrosomal fraction hypotau ri ne oxldase Is very I ow wh ile In crude mltochondrial fraction the amount of enzymic activity was assayable. Further local ization of hypotaurl ne oxi dase activl ty i n the P2 A, P2 B and P2 C fractions, obtaine d from t he crude mi t ochondri a of t er centrifugation on a discontinuous sucrose grads ent, has shown that about 55°ßr of the recovered enzyme act[ v sty is present 1 n synaptosomal fraction (P2B), IF Ig .2).

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The enzymic activity was linear as a function of time, up to 90 min, In the supernatant and crude mitoc.hondrial fractions (FIg .3) and as a function of protein up to 2 .5 mg, in the synaptosomal and crude ml tochondrial fractions (Fig . 4) .

10

FI G . 1

Distribution of hypotaurine oxidase activity in primary subcelluIar fractions of ox retina . Hom ogenates prepared i n 0. 32 M-sucrose were centrifuged In order to obtain the respective subfractlons which were then assayed for hypotaurine oxidase under condl tlons described I n Methods. Distribution calculated as % of tot al recovered act I vi ty . DI s cussion Mammals seem to biosynthesize most of t hell r t surf ne f rom cyst ell ne via cysteinesulphlnic acid and hypotau rine (21) . In our previous papers, synthesis of cyst einesulphlnl c acl d from cystei ne was demonstrated In rat and ox ret Ina ( 16, 22) . In ox reti na about 36!iÄ of cystelne oxidase recovered from crude mi t ochondri a was found In synaptosomal fraction . The enzyme carrying out the conversion of cystelnesulphinl c acid to hypotaurine, cystelnesul phi nic acid decarboxylase, was found primarl ly associated with the particulate components of the primary subcel I ulcer fract tons of ox retina ; about 46% of this enzyme activity, recovered from crude mitochondria was present in synaptosomes ( 15) . I t Is wel I known that taurine I eve I s decrease in r at brain d uri ng development (23) but this has not been observed 1 n ret I na ( 7) .l n the r at

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an high Increase In retinal taurine concentration was observed between

40

R: A F IG . 2

Distribution of hypotaurlne oxidase in secondary subcellular fractions obtained from crude mitochondria of ox retina . Distribution calculated as % of total recovered activity .

160

é

80

P

60

10 70

60

90

110

100

1g0

min

FIG . 3

Activity of hypotaurlne oxidase i n crude ml tochondri a and supernatant of ox retina . The subcellular fractions ( 1 mg protein) were Incubated at 37°C and enzymic activity was measured as described In the text . crude mitochondria (O-O); supernatant ( ~-

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the 10 th and 30 th day of postnatal I i fe (7). In t hi s specs es the inc rease In taurine content Is coincident with the Increase of cystel ne sul phi nate decarboxylase activity (24) . In retina as In brat n, the processes that regulate taurine concentration In vivo are not clear . Young (25) demonstrated the passage of taurine from plasma to retina . The resul is of Pasantes-Morales (24), together with ours on the cystei ne oxidise act l vi ty In retina (22) Indicate that both synthesis in si to as well as uptake from exogenous sources may be operating. 280

_C

240

E 200

Ç T

160 120

0 40

FIG. 4

Effect of protein concentration on hypotaurlne oxi dase of crude mitochondrial (0 and synaptosomal fractions (H) of ox ret i na . Enzyme activity was determined as describe d In t he text . The resul is are the means of duplicate determinations from 2 separate experiments . In brain samples from adult rats Oja et al .(19) found no detectable activity of hypotauri no oxidase while thl s activity was present In the newborn rats . These authors postuI ated th at oxi dat i on of hypot aurine to taurine might be the limiting step In the blosynthesis of taurl ne In adult brain. The present results show, on the contrary, that hypotaurlne oxi dase is present in ox retina and the subcellular dIstribut Ion of enzym le activity appears to confirm a compartmentatlon between the soluble fraction and the particulate fractions (see F1 g . 1 ) . The results reported In FI g.2 also suggest that the hypotaurlne oxidase activity Is significant in nerve endings; In fact about 55% of enzymic activity recovered from crude mitochondri a i s I ocated within Isolated synaptosom es .The presence of cystelne oxidase, cystelnesulphinic decarboxylase, taurine and hypotaur I no oxidase In thl s f ract I on suggests that synaptosomal taurine can be synthesi zed in situ, and this may further on confirm the role of taurine as neurotransmitter .

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Re fer ences 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11 . 12. 13. 14. 15. 16. 17. 18. 19. 20. 21 . 22. 23. 24. 25.

D. R .CLIRTIS and J . C . WATK1NS, J.Neurochem ., 6, 117-141 (1960). A. N . DAVISON and L . K . K AC ZMAREK, Na t ure Lond., 234 107-108 ( 1971) . R. KUBICEK and A. DOLEN EK , J . Chrornat . , l . 266-268 (1958) . H. PASANTES-MORALES, J . KL ETHI, P. F . URBAN and P .MANDEL, Physiol .Chem .Physics . ,4, 339-348 (1972). A. J .COHEN,M .McDANIEL and H.TORR, 1nvest .Ophthal ., 12, 686-693 (1973) . M .S.STARR and M .J.VOADEN, Vision Res ., 12. 1261-1269 (1972). S .MACAIONE, P .R000ERI , F . DE LUCA and G . TUCCI, J .Neurochem ., 22. 887-891 (1974) . H. PASANTES- MORALES, J .KL ETHI, P . F. URBAN and P .MANDEL, Brain Res . , 51 . 375-378 (1973) . H.PASANTES - v10RALES,J .KLETHI,P .F .URBAN and P .MANDEL, Expl .Brain .Res ., 19, 131-141 (1974) . A.N. DAVISON, Biochim .Biophys .Acta. _19, 66-73 (1956) . E. PECK and J . AWAPARA, Biochi m . Blophys . Acta, 141, 499-506 (1967) . J.AWAPARA and W. J. WINGO, J . BI ol .Chem . , 203. 189-194 (1953) . D.CAVALLINI and B.MONDOVI, Glornale dl Blochi mica 3, 170-183. (1951) . D . CAVALLIN1, B. MO ND OV 1 and C . DE MARCO, J. Blol . Chem . , 216, 577-582 (1955) . S.MACAIONE, G. TUCCI, G. DE LU CA and R. M . DI GIORGIO, J.Neuro chem .,27, 1411-1415 (1976) . S .MACAIONE and R .M . DI GIORG 10, Life Sci . , 20 . 617 (1977) . K .SUMIZU, B1ochIm .Biophys .Acta., 63, 210-212 (1962) . A.FIORI andM .COSTA, Acta Vitamin . , 23. 204-207 (1969) . S . S OJA, M . L . KARVON EN and P . LAHDESMAK 1, Br al n Res ., 55, 173-178 (1973) . O. H . LOWRY, N. J . ROS ESROU GH, A. L. FAR R and R . J . RANDALL, J. Biol . Chem . , 193, 265-275 (1951 ) . 48 424-511 (1968) . J . B.JACOBSEN and L .H .SMITH Jr ., Physiol .Rev., ~ R.M .DI GIORGIO, G. TUCCI and S.MACAIONE, LI fe Sci . , 16, 429-436 (1975). H. C . AGRAWAL, A. N. DAV ISON and L . K . KACZMAREK , Biochem . J. , 122, 759-763 (1971). H. PASANTES- MORALES, A. M . LOPEZ-COLOME, R. SALC ED A and P .MANDEL, J .Neurochem . , 27, 1103-1106 (1976) . R . W . YOUNG, in The Retl na: Mo rphology, Function and Clinical Characteristics . (Straa tsm a et al ., eds.) p . 181, University of Cal 1forni a Press, Los Angeles .

Subcellular distribution of hypotaurine oxidase activity in ox retina.

Life Sciences Vol . 20, pp . 1657-1662, 1977 . Printed in the U .S .A . Pergamon Press SUBCELLULÀR DISTRIBUTION OF HYPOTAURINE OXI DASS ACTIVITY IN...
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