Cysteine oxidase in brain

Brain Research, 97 (1975) 117-126 ~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

I I7

CYSTEIN E O X I D A S E IN B R A I N *

C H A N D R A H. MISRA AND JOHN W. OLNEY**

Washington University School of Medicine, Department of Psychiatry, St. Louis, Mo. 63110 (U.S.A.) (Accepted April 14th, 1975)

SUMMARY

Cysteine oxidase, which catalyzes the oxidation of cysteine to the neuroexcitatory and neurotoxic compounds cysteine sulfinic and sulfonic acids, previously has not been studied succesfully in brain. Here we report optimal conditions for measuring cysteine oxidase activity in rat brain, describe its regional and subcellular distribution and compare activity during several stages of development. Employing N A D + as cofactor, substantial acitivity was measurable in whole brain homogenate, 7 0 ~ of which was localized to the microsomal fraction. Appreciable activity was present throughout brain with a 3-fold difference being detected between p o n s medulla and cerebral cortex, the regions with the highest and lowest levels respectively. Cysteine oxidase activity in adult rat brain was approximately 5 times higher than that measured at birth. For the first 6 days of life there was very little change in activity; thereafter, a steady increase occurred with adult levels being reached between the 25th and 50th days.

1NTRODUCTION

The conversion of cysteine to its oxidation product, cysteine sulfinic or sulfonic acids in mammalian tissues is catalyzed by the enzyme cysteine oxidase. Formation of these acidic sulfur amino acids in brain is of considerable interest as they are known to have both neuroexcitatory 2 and neurotoxic properties s. Further, the conversion of cysteine to these compounds is thought to be an important intermediate step in the formation of taurine, an abundant brain metabolite with neuroinhibitory properties 3. Cysteine itself, when given subcutaneously to immature rodents induces extensive brain damage 9. The conversion within brain of cysteine to one of its neurotoxic

* This research was supported in part by grants NS-09156, MH-07081 and DA-00259. ** Recipient of Research Career Development Award MH-38894.

118 oxidation products has been postulated as the mechanism by which this brain damage O c c u r s .9.

Despite the potential importance of the cysteine oxidase pathway in brain, what little is known about it derives largely from studies of the enzyme in liver: m~d, those who have studied the liver enzyme are not in agreement regarding the cofactors it uses and hence the conditions which may be considered optimal for measuring its activity. Whereas Ewetz and S6rbo 4 and Lombardini and Singe¢ ~ reported that the introduction of N A D H or N A D P H is required for maximal activity, Yamaguchi el al. ~ found higher activity with NAD ~ and N A D P " than with either NADH or NADPH. This study was undertaken to clarify optimal conditions lbr measuring cysteine oxidase activity in rat brain, to explore the regional and subcellular distribution of the enzyme in brain and to compare enzyme activities at various stages in development. MATERIALS AND METHODS

Chemica&. L-[:~sS]Cysteine HCI was purchased from Amersham/Searle Co.; Dowex-50 X4-200-R, NAD ~, NADP~; a-NAD% N A D H and N A D P H from Sigma; nicotinic acid, nicotine and nicotinamide from Eastman Organic Co. ; deamino-NAD and 3-pyridine-aldehyde-deamino-NAD from Nutritional Biochemical Corporation and 3-acetylpyridine-NAD and thionicotinamide-NAD from Pabst Laboratories, Animals and tissue preparation. Young adult Wistar albino rats (National Laboratory Animals, St. Louis, Mo.) of either sex weighing 200--250 g were used throughout these experiments except that, for the developmental studies, litters of younger animals of the same strain were used. For the latter, pregnant animals were maintained throughout gestation and delivery in our facility. The animals were killed by decapitation and their brains removed quickly, stripped of blood vessels and cleaned in cold 0.32 M sucrose. They were then homogenized in 0.32 M sucrose (1:9, w/v) using 5 up and down strokes at 1000 rev,/min, in a Potter-EIvehjem homogenizer. For studies of regional enzyme activity brains were dissected"-' betbre homogenization into samples representing cerebral cortex, hippocampus, caudate, hypothalamus-thalamus, cerebellum, pons-medulla and spinal cord. To obtain subcellular fractions a 10°k homogenate of whole brain was centrifuged at 800 x g for 10 rain and the pellet resuspended in 0.32 M sucrose and resedimented. The supernatant and washing fluids were combined and centrifuged at 12,500 < g for 20 min t o sediment a crude mitochondrial fraction. Pellet washings and supernatant were combined and centrifuged at 100,000 x g for 60 rain to obtain microsomal and cytosol fractions. The microsomal pellet was resuspended in 0.32 M sucrose and sedimented again at t00,000 ;-/, g. All fractions were suspended in a known volume of 0.32 M sucrose lbr enzyme assay. The centrifugations were carried out in a refrigerated Sorvall (RC2-B) or Beckman (Model L5-65) ultracentrifuge. E n z y m e assay. The standard assay system was adapted from Yamaguchi et al. ~" with modifications based on findings described in results. The assay mixture consisted

119 of 155 #moles of L-[3zS]cysteine (0.5-0.7 /~Ci), 0.5 #moles Fe(NH4)2(SO4)26H20, 10 #moles hydroxylamine-HC1, 100 #moles phosphate buffer (pH 6.8), 4 #mole. N A D + or other cofactor and enzyme (homogenate) to make 2 ml of total volumes The substrate (L-cysteine) was withheld from the assay mixture in each experiment for a 5-rain preincubation period at room temperature during which N A D ~ or other cofactor and enzyme were present. The reaction was initiated by the addition of substrate and terminated by addition of 15 ~/o trichloroacetic acid. Incubation was carried out for 30 rain at 37 °C with constant shaking. Duplicate samples and one blank were employed in each experiment. Blanks were handled the same as samples except that enzyme (homogenate) was not included until after incubation and T C A was added.The samples and blank were then centrifuged, the precipitates discarded and a 2-ml aliquot of supernatant from each was applied to a Dowex 50 X4-200RH 4 (0.8 cm × 8.0 cm) column. Reaction products were eluted with 10 ml of water, then 1 ml of effluent was mixed with 10 ml Insta-gel (Packard) and counted in a liquid scintillation spectrometer. Protein was measured by the standard method of l o w r y e t a / . 7 using bovine serum albumin as a standard and specific enzyme activity was expressed as #moles cysteine sulfinate formed/h/rag protein. The reaction products in the effluent from the Dowex-50 column were identified as cysteine sulfinic acid and a small amount of cysteic acid by chromatography on Dowex-l-X2 acetate as described by Bergeret and Chatagner t with minor modifications. This was further confirmed by freeze drying the effluent after treating with ether for removing TCA, dissolving it in a small volume of water and subjecting it to thin-layer chromatography with authentic L-cysteine sulfinic acid and L-cysteic acid. Two chromatograms were processed, one sprayed with ninhydrin for identification of the spots and the other used for quantifying the percentage of radioactivity in each spot.

8OOO 7000 I E 6000

5000 i 4000 ~ 3000 u~

2000 1000

2'o- 4'o go 8'o 16o MINUTES

Fig. l, Cysteine oxidase activity in counts/rain with time. Assay was carried out as detailed in Methods, the only variable being the incubation time.

120 . . . . .

r

. . . . . . . . .

% /

m

36a

i

a,

0.2L

i[

Fig. 2. pH dependence of cysteine oxidase activity. The assay was carried out as described in Methods. All conditions were kept identical except the pH. The following 0.1 M buffers were used: acetate (L --(~), phosphate ( 0 O), and Tris.HCI (-",- ,,L). RESULTS

In pilot experiments we found the time curve for cysteine oxidase activity in whole brain homogenate to be linear up to 30 rain (Fig. 1). Therefore, we employed an incubation period of 30 rain for our assay procedure but doubled the values obtained in order to express results on an hourly basis. The p H optimum for measuring cysteine oxidase activity in brain with phosphate buffer was found to be pH 6.8 (Fig. 2) which does not differ from that established for the liver enzyme by Yamaguchi et al. it. We tested imadazole buffer but rejected it as it inhibited enzyme activity when employed in the same concentration as phosphate. The effect of" L-cysteine concentration on enzyme activity is shown in Fig. 3. The activity increased up to 70 mM. The Lineweaver plot (Fig. 3, insert) gives a

y___.., ~

0.25

~oi

,

l

10

20

.... ~

q0 40 50 60 L-CYSTEINE (raM)

J

70

80

'

i _ _

90

100

Fig. 3. Effect of substrate concentration on cysteine oxidase activity. The insert shows the Lineweaver-Burk plot for the substrate.

121

0I

Lu u

b

mg o~ Proten

Fig. 4. Cysteine oxidase activity v e r s u s enzyme concentration. The assay system was as described in Methods except that enzyme concentration was varied by using different amounts of brain homogenate.

0.25 i(

0.20

0.15 Rf

0

072

010[

o.o5k

0

cysteic acid

28

cysteine

sample

sulfinicacid THIN-LAYERCHROMATOGRAMOF PRODUCTSOF ENZYME OXIDATIQN Fig. 5. Thin-layer chromatogram identifying the products of cysteine oxidation. The chromatograms were developed in n-butanol-acetic acid-water (60:20:20) for 3.25 h in a glass chamber. Spots from the sample which correspond with the Re values for authentic cysteine sulfinic acid and cysteic acid contained 7 2 ~ and 28 % respectively of the recovered counts. The small ninhydrin-positive spot at Rt,, :- 0.20 contained no counts.

122 TABLE 1 E F F E C T S OI- S[-VERAL ( O~?NZYMES O N ( ' Y S T E I N E OXII)ASF, A C ] I V H ",'

Coetlz~vmc *

( vsteine o vidase c,,ctivity* *

None NAD i NADH NADP ~ ct-NAD NADPH

N o activit5 O.744 O.324

0.146 0.055 0.036

* Assay was carried out under standard conditions except tbr the omission of N A D ~. The concentration of each added compound was 2 :, 10 -',~M . * * / ~ m o l e s cysteine sulfinate formed/h/rag protein.

Km for substrate equal to 45.4 mM. Based on these data we employed 155/lmoles of L-cysteine in the assay mixture rather than the lower amount (20 #moles) used by Yamaguchi et al. ~1. The influence of enzyme concentration on the reaction is shown in Fig. 4. The activity of cysteine oxidase increased in a linear fashion up to 5.0 mg of protein in the reaction mixture. A typical thin-layer chromatogram identifying the products of enzyme oxidation as cysteine sulfinic acid and cysteic acid is illustrated in Fig. 5. About 72 % of the counts were recovered from the spot corresponding to cysteine sulfinic acid and 28 !!;; from the cysteic acid spot. To clarify cofactor requirements of the cysteine oxidase pathway in brain, we compared the influence of several potential cofactors on enzyme activity in whole brain homogenate (Table I). With no cofactor added there was no activity (blank values slightly higher than experimental sample). Low to moderate activity was stimulated by NADPH, ~-NAD + and NADP + (0.036, 0.054 and 0.146, respectively), substantially more by NADH (0.324) but this was less than half the activity obtained

TABLE

11

E F F E C T OF N A D ~- A N D N A D

ANALOGUES ON CYSTEINE OXIDASE ACTIVITY

Compound *

Qvsteine o x i d a s e a c t i v i t y * *

NAD +

0.744 0.713 0.418 0.087 0.037

Thionicotinamide-NA D Deamino-NAD

3 -acetyl-pyridine-NA D 3-pyridine-aldehyde-deamino-N A D Nicotine Nicotinic acid

--

* Concentration of each compound : 2 ;< 10 ":3 M . **/tmoles cysteine sulfinate formed/h/mg protein.

123 TABLE 111 SUBCELLULAR

DISTRIBUTION

OF C Y S T E I N E O X I D A S F IN R A T B R A I N

Two rat brains, weighing 1.8068 and 1.7703 g were homogenized separately in Potter-Elvehjem tissue grinder with 5 strokes up and down in 0.32 M sucrose and subfractionated. Results from each brain are indicated individually in the Table. Fractions

Cysteine oxidase activity

Homogenate Nuclear M itochondrial M icrosomal Cytosol

SpcciJlc *

Total* *

Per cent

0.692 0.697 0.468 0.411 0.361 0.267 1.446

104.00 109.00 12.30 10.30 13.60 10.70 72.20 80.70 5.19 6.17

100

0.200 0.183

Recovery from fractions

100 11.0 9.5 13.0 9.5 69.5 72.2 5.0 97.1

5.7 98.9

*/~moles of cysteine sulfinate formed/h/mg of protein. ** Total activity in ,amoles of cysteine sulfinate formed/h. with N A D ~ (0.744). Except where otherwise stipulated N A D + was the cofactor employed in these experiments. A c o m p a r i s o n of N A D + with 7 of its analogues (Table I1) revealed only one, t h i o n i c o t i n a m i d e - N A D ~ which stimulated activity at a rate (0.713) c o m p a r a b l e to N A D ~ (0.744). Appreciable activity was supported by d e a m i n o - N A D (0.418) but relatively little or n o n e by the other 4 analogues tested. Assaying the activity of cysteine oxidase in subcellular fractions (Table III) revealed the majority of it (70-74 ~o) to be associated with the microsomal fraction. This is in contrast to rat liver where cysteine oxidase activity is reportedly4,10, asTABLE lV RF(.HONAL DISTRIBUTION

OF C Y S T E I N E O X I D A S E A C T I V I T Y 1N R A T C E N T R A L N E R V O U S S Y S T E M

Number of experiments given in parentheses. The values are given as means 3_ S.E.M. Region

Cysteine oxidase activity (lmtole of eysteine sMfinate formed/h/mg of protein)

Cortex Cerebellum Hippocampus Caudate Hypothalamus and thalamus Pons-medulla Spinal cord

0.421 ~ 0.051 (5) 0.690 _L 0.072 (5) 0.675 ~_ 0.127 (4) 0.732 ~ 0.113 (4) 0.999 _-+_0.079 (3) 1.321 ~_ 0.106 (4) 0.855 :t- 0.080 (4)

124 TABLE V C H A N G E S IN C Y S T E I N E O X U ) A S E AC'I [VITY I ) U R I N G D E V E L O P M E N T OF RAI" B R A I N

Age of rat* (days)

(),steine oxidase activity (/~moles of cysteine sMfinateJbrmed /h/mg of protein)

Birth 1 2 5 6 9 11 16 25 50

0.143 0.166 0.159 0.147 0.152 0.236 0.239 0.369 0.562 0.742

Adult

0.704!

!: 0.024(4) 0.020 (4) ~ 0.008(51 i 0.021(5) 0.016(5) : 0.010(4) 0.010(4) 10.058(5) 0.039(4) 0.034(5) 0.039(6)

* A d u l t s were n o t of the s a m e group. The values are given as m e a n J: S.E.M. with the n u m b e r o f observations in parentheses.

sociated primarily with a soluble fraction. Our findings suggest that the brain enzyme is predominantly membrane bound with only negligible amounts (5-6~) being demonstrable in the soluble fraction. Regional variations in cysteine oxidase activity were demonstrated (Table IV) with pons-medulla (1.321) and cerebral cortex (0.421) having the highest and lowest activities respectively. Although, dissections were not performed so as to reflect differences on a white v e r s u s grey matter basis, the low activities in cerebral cortex and cerebellum suggest lower activity might be associated with grey matter. On the other hand, relatively high activity in the predominantly grey hypothalamus-thalamus (0.999) renders this observation invalid as a generalization. Measurement of cysteine oxidase activity at different ages (Table V) revealed a relatively low level at birth, about 1/5 that of the adult with no appreciable increase until after the 6th day. Each measurement thereafter (days 9, 11, 16, 25 and 50) provided evidence for a steady rise in activity until the 50th day when adult activity levels were present. DISCUSSION

Although prior studies of cysteine oxidase have focused primarily upon liver, Ewetz and SSrbo a and Yamaguchi e t al. i l in testing their liver assays on other tissues did detect activity in brain. Ewetz and Sfrbo a using a homogenate prepared in 0.15 M KCI and reaction mixture containing Fe 2 v, NADPH and hydroxylamine reported negligible activities (/tmoles cysteine sutfinate/h/100 mg t i s s u e ) in brain (0.25) compared to liver (3.7). Yamaguchi e t al. 1~ employing a supernatant of brain prepared in 0.01 M phosphate buffered 0.25 M sucrose (pH 7.3) and a reaction mixture containing Fe 2+, hydroxylamine, phosphate buffer (pH 6.8) with NAD + as a cofactor found

125 0.154 #moles cysteine sulfinate/h/mg protein, or about 1/5 the activity in our whole brain homogenate. Possible reasons for the low activity obtained by Yamaguchi et al. H in brain despite use of NAD ~ as cofactor would be that they performed their measurements on a supernatant fraction rather than whole homogenate; also, only 20/~moles of substrate (L-cysteine) was included in their assay mixture compa red to 155 #moles contained in ours. In any event, the conditions described here appear to be more suitable for assaying cysteine oxidase activity in brain than any described elsewhere; moreover, the activity we demonstrate in brain is higher than has been reported thus far in any mammalian tissue. Of course, if cysteine oxidase activity in other tissues is primarily localized to the microsomal fraction as we found it to be in brain, but for some reason the activity of the microsomal fraction has remained masked in prior experiments, this would readily explain low readings. A reexamination of the microsomal fraction of liver under various assay conditions to rule out a previously unidentified reservoir of cysteine oxidase activity would seem warranted. The fact that cysteine oxidase in brain seems to be definitely NAD ~-dependent also should be taken into consideration in further attempts to elicit activity of the enzyme in other tissues. Curtis and Watkins z have demonstrated that certain acidic sulfur amino acids, including sulfinic acid and cysteic acid, excite neurons as glutamate does when introduced by microelectrophoresis into mammalian brain. Olney et al. 8 have shown that subcutaneous administration of the same compounds to experimental animals destroys neurons in a particular region of the hypothalamus (arcuate nucleus) as does glutamate. A different and more widespread pattern of brain damage was described more recently by Olney et al. 9 in infant or fetal rodents following subcutaneous administration of L-cysteine. It is unclear whether L-cysteine has a direct toxic action on brain cells or induces widespread brain damage by converting intracerebrally to a toxic metabolite such as cysteine sulfinic acid. In the latter case, a competent cysteine oxidase pathway in immature brain would be necessary. Our findings indicate that cysteine oxidase activity is present from birth on, although at only I/5 adult levels for approximately the first week of neonatal life. It is of interest that it is in this early period that certain doses of cysteine readily produce brain damage. In the ensuing weeks as cysteine oxidase activity in brain is steadily increasing it is difficult to induce the brain damage syndrome because the same or even lower doses of cysteine are rapidly lethal 9. Our data on cysteine oxidase are consistent with the interpretation that conversion of cysteine to its oxidation products plays a role in the perinatal brain damage it induces. However, it will be necessary to obtain additional information regarding the metabolic fate in brain of subcutaneously administered cysteine and changes in other relevant enzyme systems during development before a more definitive interpretation can be made. Errors of sulfur amino acid metabolism are relatively common metabolic disorders and serious neuropsychiatric disturbances frequently accompany such disorders. A genetic defect has been identified in many such cases without the pathogenesis of the CNS disturbance being elucidated. Attempts to trace the metabolic fate and enzyme pathways of various sulfur amino acids in brain, of which this study

126 r e p r e s e n t s a b e g i n n i n g , s h o u l d be helpful in c l a r i f y i n g the brain d a m a g e s y n d r o m e ~ w h i c h can be i n d u c e d in e x p e r i m e n t a l a n i m a l s w i t h sulfur a m i n o acids. H o p e i u l b it will s h e d light s i m u l t a n e o u s l y on the C N S d e g e n e r a t i v e c o n d i t i o n s a c c o m p a n y i n g h u m a n e r r o r s o f sulfur a m i n o acid m e t a b o l i s m .

REFERENCES 1 BERGERET, B., ET CHATAGNER, E., Utilisation d'6changeurs d'ions pour la separation de quelques acides amin6 form6s au cours de la d6gradation enzymatique de racide cyst6inesulfinique. Application it l'isolement de I'hypotaurine (acide 2-amino6thanesulfinique), Biochim. biophys. Acla (Amst.), 14 0954) 543-550. 2 CURTIS, D. R., AND WATKINS, J. C., Acidic amino acids with strong excitatory actions on mammalian neurons, J. Physiol. (Lond.), (1963) 166 1-14. 3 CURTIS, D. R., AND WA'rKINS,J. C., The pharmacology of amino acids related to gamma aminobutyric acid, Pharmacol. Rev., 17 (1965) 347-391. 4 EwE/z, L., AND Si3RBO, B., Characteristics of the cysteine sulfinate forming enzyme system in rat liver, Biochim. biophys. Acta (Amst.), 128 (1966) 296-305. 5 GLOWINSKI,J., ANt) IVERSEN, L. L., Regional studies of catecholamines in the rat brain. 1. The disposition of [3H]norepinephrine, [3H]dopamine and [aH]DOPA in various regions of the brain, J. Neurochem., 13 (1966)655-669. 6 LOMBARDINI,J. B., AND SINGER, T. P., Cysteine oxygenase. II. Studies on the mechanisms of the reaction with oxygen, Jr. biol. Chem., 244 (1969) 1172-1175. 7 LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., Protein measurement with folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 80LN~Y, J. W., Ho, O. L., AND RHEE, V., Cytotoxic effects of acidic and sulphur containing amino acids on the infant mouse central nervous system, Exp. Brain Res., 14 (1971) 61-76. 90LNEY, J. W., Ho, O. L., RHEE, V., AND SCHAINKER,B., Cysteine-induced brain damage in infant and fetal rodents, Brain Research, 45 (1972) 309-313. l0 SORBO, B,, AND EWIETZ, L. The enzymatic oxidation of cysteine to cysteine sulfinate in rat liver, Biochem. biophys. Res. Commun., 18 (1965) 359-363. 11 YAMAGUCm, K., SAKAKmARA,S., KYOICmRO, K., AND UEDA, 1., Induction and activation of cysteine oxidase and tyrosine transaminase activities of intact and adrenalectomized rats, Biochhn. biophys. Acta (Amst.), 237 (1971) 502-512, 12 YAMAGUCHI,K., SAKAKIBARA,S., ASAMIZU,J., AND UEDA, 1., Induction and activation of cysteine oxidase of rat liver. 11. The measurement of cysteine metabolism in vivo and the activation of hi vivo activity of cysteine oxidase, Biochhn. biophys. Acta (Artist.), 297 (1973) 48-59.