Cysteine thiosulfonate in cystine metabolism

ARCHIVES OF BIOCHEMISTRY Vol.

218, No. 1, October

AND BIOPHYSICS 1, pp. 303-308, 1982

Cysteine Thiosulfonate J. H. FELLMAN’ Department

AND

of Biochemistry, School 3181 S. W. Sam Jackson Received

March

in Cystine Metabolism’ NANCY

of Medicine, Park Road,

A. AVEDOVECH Oregon Health Sciences Portland, Oregon 97201

29, 1982, and in revised

form

June

University,

25, 1982

A sulfur-containing amino acid was observed in mammalian cystine metabolism, in vitro and in uiuo, which we have characterized as 2-amino, 3-(thio-thiosulfonate)propionic acid (cysteine thiosulfonate). Its biosynthetic pathway appears to initiate with the cleavage of cystine by cystathionine y-lyase to form thiocystine, which undergoes sulfinolysis to form cysteine thiosulfonate.

We have been investigating the metabolic precursors of the sulfur-containing anion, isethionate, in mammalian tissue using a dual-ion-exchange analytical procedure (1, 2). When [35S]cystine was examined as a precursor, a major radiolabeled metabolite appeared in an anion-exchange column fraction which was uncharacterized. We wish to report the isolation and properties of this substance which we have characterized as 2-amino, 3-(thio-thiosulfonate)propionic acid, “cysteine thiosulfonate.” We also report evidence for a biosynthetic pathway of this substance from cystine and the appearance of cysteine thiosulfonate in urine from rats maintained on diets in which the cystine content was increased. MATERIALS

AND

method of Segel and Johnson (3). Two ion-exchange resins used throughout were Bio-Rad, AG 5OW-X8, 200-400 mesh, hydrogen form, and Bio-Rad, Bio-Rex 5, 100-200 mesh, chloride form. Radiolabeled L-[U-“C]cystine (290 mCi/mmol) was purchased from New England Nuclear and the L[%S]cystine (0.259-442 Ci/mmol) from Amersham Corporation and New England Nuclear. Radioscanning (with a Packard 7220/21 radiochromatograph scanner) of paper chromatograms (see below) was routinely carried out to determine the purity of labeled amino acids. Occasional batches of cystine required purification by passing through a Bio-Rex 5 column (see below) to remove impurities. The synthesis of L-[?S]cysteine sulfinic acid (220 &i/mmol) was achieved by the method of Emiliozzi and Pichat (4). Pyruvate determinations were carried out using the method of Minakami et al. (5) employing a Gilford 2400 spectrophotometer. Determinations of alanine were carried out using a Beckman Amino Acid Analyzer Model 116 or 120. Sprague-Dawley male rats weighing 150 to 400 g were employed for both in vivo and in vitro studies.

METHODS

Material

Methods

L-Cystine was purchased from Calbiochem-Behring Corporation, N-ethyhnaleimide was obtained from Aldrich Company, and 2-amino-4-pentynoic acid (propargylglycine) was purchased from Sigma Chemical Company. S-Sulfocysteine was prepared by the

a. Enzyme preparation and incubation procedure. Liver homogenates were prepared in 0.05 M potassium phosphate buffer, pH 7.2, one part liver to four parts buffer. After dialysis against the buffer overnight, the homogenate was either used directly or stored for subsequent studies. Liver supematants were prepared by homogenizing the liver with four parts 0.15 M KCl. The homogenate was centrifuged at 25,000g for 15 min and the supernatant obtained was dialyzed overnight against 0.05 M phosphate buffer, pH 7.2, or 0.2

i This investigation was supported by Grant NS01572 from the National Institutes of Health. s To whom correspondence should be addressed. 303

0003-9861/82/110303-06$02.00/O

CopyrightQ 1982by AcademicPregs.Inc. All rights of reproductionin anyformreserved.

304

FELLMAN

AND

phosphate buffer, pH 7. Enzymatic studies always included a boiled enzyme blank and/or zero time control. The enzymatic reactions were terminated by the addition of 20% trichloroacetic acid to bring the reaction mixture to 2.75% trichloroacetic acid final concentration. b. Purification of the new amino acid. After centrifugation of the reaction mixture at 400g for 15 min, the resultant supernatant was applied to a 16 X 0.8cm, AG 50 ion-exchange resin column. The columns were eluted with water and the effluent which emerged from 5 through 28 min, about 4.5 ml, was collected and placed on a 5 X 0.8-cm Bio-Rex 5 ion-exchange resin column. The Bio-Rex 5 column was subsequently eluted with 6 ml of water and the eluate (designated fraction I) was collected. (This fraction could potentially include taurine, thiotaurine, and sulfocysteamine). Three milliliters of water were then applied to the column and the effluent was discarded. The column was next eluted with 10 ml of 0.5 M HCl and the effluent (designated fraction II) was collected. (Potentially included in this fraction were isethionate, sulfite, cysteic acid, sulfocysteine, alanine thiosulfonic acid, and the new amino acid reported here.) Further isolation of the new amino acid was carried out by subsequent descending paper chromatography of fraction II. The fraction was evaporated to dryness, applied to Whatman 3MM paper, and developed with I-butanol:glacial acetic acidwater (12:3:5), (Rf = 0.24) or phenol:water (8~2) (I?, = 0.28). Material developed with the butanol:acetic acidwater solvent system was eluted from the paper with 0.01 M HCl, rotary evaporated to dryness, and used for subsequent analysis. M

RESULTS

Characterization

AND

DISCUSSION

of New Amino

Acid

We undertook these studies on cystine metabolism in search of a biosynthetic pathway for isethionate, a substance which emerges in fraction II from an anion-exchange resin. When [35S]cystine was incubated with dialyzed rat liver homogenates, a radiolabeled anionic metabolite was formed which was characterized as follows. Mixtures consisting of 1.0 ml of rat liver homogenates or supernatants, 1 mM cystine, [35S]cystine (0.08-0.12 Ci), and 0.2 M potassium phosphate buffer, pH 7.0, in a final volume of 1.25 ml were incubated at 37°C for 60 min. The enzymatic reaction was terminated by the addition of 20% trichloroacetic acid and the supernatant was subjected to dual-ion-exchange chromatography. After further separation us-

AVEDOVECH

ing paper chromatography as described under Materials and Methods, this radioactive material behaved like an amino acid yielding a purple color with ninhydrin and also exhibited a thiosulfate-like reaction after treatment with potassium ferrocyanide spray followed by ferric chloride spray (6). The metabolic formation of this new amino acid from cystine was dependent on the presence of oxygen. When incubation mixtures were purged with a stream of nitrogen, a marked decrease in the amount of the new amino acid was observed. However, under nitrogen, no substantial change in the amount of cystine cleavage was noted (indicated by the extent of pyruvate/ alanine formation and amount of residual cystine, Table I) (7, 8). In order to determine this oxygen-dependent step in the metabolic sequence leading from cystine to the new amino acid the following proved informative. When [35S]cysteine sulfinate alone was employed as the substrate in incubation mixtures, the new amino acid product could not be detected. However, when nonradioactive cystine was added to such incubation mixtures, maximum radiolabeled product formation was observed both before or after purging the system with nitrogen. From these observations we concluded that the formation of the new amino acid included, in part, the oxidation of some of the cystine to cysteine sulfinate which was necessary, but not sufficient, for the formation of the new amino acid. The formation of cysteine sulfinate is the likely oxygen-dependent step (9). To determine the number of sulfur atoms in the new amino acid, parallel incubation experiments were conducted using [U-14C]cystine and [35S]cystine substrates of equal specific activity. The ratio of the radioactivity incorporated into the new amino acid derived from [U-14C]cystine and [35S]cystine was determined from the corresponding peaks obtained by radioscanning of the paper chromatograms. The area subsuming the radioactive new amino acid scan was determined by carefully cutting out the peaks and weighing on an analytical balance. The isolated products contained radioactivity in a ratio of 1:3,‘4C:35S. From

CYSTEINE

THIOSULFONATE

IN CYSTINE TABLE

I

ENZYMATICSYNTHESISOFCYSTEINETHIOSULFONATE:EFFECTOFN~ PROPAF~GYLGLYCINE Product

Experiment

Cysteine thiosulfonate

305

METABOLISM

formation

ATMOSPHEREAND

(pmol)

Pyruvate

Alanine

Cystine remaining bmol)

1.391 1.151

0.330 0.382

0.116 0.191

0.983

0.219

0.009

0.054

I [%I Cystine” [36S]Cystine

+ nitrogen

0.110 0.020

+ 0.04* + 0.02*

II [s’S]Cystine [s’S]Cystine

+ propargylglycine’

0.204 0.211 0.023 0.025

Note. Incubation mixtures consisted of 1 ml rat liver supernatant prepared as described under Materials and Methods and containing 25 mg/ml of protein. Other components in the 1.25-ml volume were 1 mM cystine, [??l]cystine (0.06-0.12 pCi), 0.1 mM propargylglycine, and 0.2 M phosphate buffer, pH 7. After incubation at 37°C for 60 min, 0.2 ml 20% trichloroacetic acid was added and the products and unreacted cystine contained in the supematants were analyzed as described under Materials and Methods. ’ Zero time control contained no detectable cysteine thiosulfonate, pyruvate, or alanine. *n=4,mean+SD. ’ Propargylglycine was preincubated with liver supematant for 60 min at 37’C before addition of substrate.

this we hypothesized that the new amino acid possessed an alanine side chain, appended to three sulfur atoms, the terminal sulfur in its maximal oxidized state: cysteine thiosulfonate (Fig. 1). Suitable reducing agents should cleave this substance to cysteine, H&S, and sulfite. When the 35Slabeled new amino acid, (isolated by dualion-exchange chromatography followed by paper chromatography as outlined above) was reduced with metallic zinc dust and 3 M H2S04 the products included [35S]cysteine isolated as its N-ethylmaleimide derivative. The latter, which cochromatographed with a sample of 3[(l-ethyl - 2,5 - dioxo - 3 - pyrrolidinyl)thiolalanine (prepared from cysteine and iV-ethylmaleimide), was rendered visible with ninhydrin and by radioscanning of the paper strip. Also observed in the reduction products was radiolabeled HPS which was trapped in a cadmium chloride solution (10). Another volatile radiolabeled material was also detected which was trapped with hyamine base which we tentatively ascribed to [35S]sulfite. When 35Slabeled new amino acid was treated with

hydrogen peroxide, [“5S]cysteic acid was obtained. The latter was characterized by its behavior, compared to authentic cysteic acid, on the dual-ion-exchange chromatography system and also by paper chromatography as outlined under Materials and Methods. Sufficient new amino acid for mass spectral studies was prepared from incubation of rat liver supernatant with cystine and isolated by dual-ion-exchange chromatography followed by paper chromatography and elution of the appropriate area in the chromatograms. Mass spectrometry was carried out by direct probe in a Finnigan 9000 Mass Spectrometer employing ammonia gas chemical ionization (11). A base peak fragment at 108 m/e was assigned to NH&Hz-CHz-S-S. A major fragment at 152 m/e was assigned to HOOC-CH(NH.JCHz-S-S. No parent ion was observed. Chemical synthesis of the amino acid was undertaken by the reaction of cystine with thiosulfate. Cysteine thiosulfonate was prepared using a modification of a reported method (12) by heating 240 mg (1 mmol) of cystine dissolved in 5.0 ml 15 M NHIOH

FELLMAN AND AVEDOVECH

306 PYRUVATE 0

+

Nli;

NH;

-o-E-CH-CH*-S

0 NH; ILL

-o-gyi-c”2-s 0 Ml;

b-&CH2-S-W

THIOCYSTEINE

\

CYSTINE

0

NH;

-0-&-Cl+,-S

0

CISTEINE NH;

4

; CYSTEINE

-0-&CH,-S

SULFINATE I

Y _ so;

THIOCYSTINE

CYSTEINE w

“CYSTEINE

THIOSULFONATE

”

FIG. 1. Proposed biosynthetic pathway thiosulfonate.

for cysteine

with 1.3 g (5 mmol) of sodium thiosulfate dissolved in 5 ml of Hz0 and heating the mixture on a steam bath for 4 h. The solution was cooled, rotary evaporated to dryness, and the residue triturated with 10 ml water. The material was filtered to remove the unreacted cystine and chromatographed on paper using the butanol: acetic acidwater solvent system as described above. Two ninhydrin reaction spots were observed, one of which cochromatographed with sulfocysteine (minor) and the other (major) was cysteine thiosulfonate. The amino acid thus synthesized was identical to the new amino acid by exhibiting identical (a) mass spectra, (b) elution characteristics on dual-ion-exchange resin chromatography, and (c) cochromatography with paper partition using two different solvent systems and with high-voltage paper electrophoresis. Savant Instruments High Voltage Electrophoresis Apparatus in formic:acetic acid (pH 1.9) buffer at 4500 V for 120 min was employed. We undertook next to explore the biosynthetic pathway and formulated the following proposal (Fig. 1). This hypothesis is predicated, as shown,

on the initiation of the sequence by the activity of the cytosol enzyme, cystathionine y-lyase. The metabolism of cystine by cystathionine y-lyase (EC 4.4.1.1) has been demonstrated in uitro. The cleavage of cystine by this enzyme occurs by ,B-elimination leading to thiocysteine, ammonia, and pyruvate (4-7). Evidence for thiocysteine and thiocystine formation in this process has been substantial (10, 13, 14). Since propargylglycine is a powerful enzyme-activated irreversible inhibitor of this enzyme (15, 16), we examined the effect of this inhibitor on the rat liver metabolism of cystine. The results presented in Table I indicated that a marked decrease in cysteine thiosulfonate was observed and congruent with this finding, the formation of pyruvate and alanine was profoundly inhibited when rat liver extracts were preincubated with propargylglycine. The hypothesis proposes a nonenzymatic nucleophilic attack of cystine by the thiocysteine produced by cystathionine y-lyase action on cystine to form thiocystine and cysteine. Evidence for such a sequence has been reported (13). We propose that cysteine generated in this sequence is converted into cysteine sulfinate by the O,-dependent enzyme, cysteine dioxygenase (EC 1.13.11.20) (9, 17); cysteine sulfinate is subsequently transaminated with pyruvate as cosubstrate,3 and thus desulfinated or desulfinated directly via cysteine sulfinate desulfinase to SOZ and alanine. Further evidence for this proposal was obtained by conducting experiments in which cystine was incubated with rat liver extracts under nitrogen. A marked decrease in cysteine thiosulfonate was observed. However, when these anaerobic experiments were done in the presence of added sulfite or cysteine sulfinate, cysteine thiosulfonate formation was observed. Our hypothesis proposes that a nucleophilic attack of the thiocystine by sulfite occurs nonenzymatically. Evidence for this was obtained when we added bisulfite to thio3 Unpublished ’ Manuscript

observation. in preparation.

CYSTEINE

THIOSULFONATE

cystine prepared synthetically (18). Rapid conversion to cysteine thiosulfonate was observed. This chemically synthesized compound behaved like the new amino acid when characterized by the criteria cited under Materials and Methods. In Vivo Formation Thiosulfonate

and Cysteine

The question of the significance of the cysteine thiosulfonate formation in the in vivo metabolism of cystine was examined. When rats were fed a diet containing cystine an outpouring of sulfur metabolites including cysteine thiosulfonate could be observed in the urine. Groups of rats were individually caged and maintained on rat chow for 3 days during which time the urine was collected in 2 ml 20% trichloroacetic acid. The urine was found to be free of cysteine thiosulfonate. Food in the cages was next changed to biscuits made from ground rat chow mixed with 1, 5, 7, and 10% cystine. The urine was collected over a period of 4 days. Cystine as well as other metabolites including cysteine thiosulfonate were detected in the urine. Cysteine thiosulfonate was characterized by passing the sample of urine through the dual-ion-exchange resin columns and separating the effluent on paper chromatography as stated under Materials and Methods. In frac:tion II only one major amino acid was detected with ninhydrin and this spot cochromatographed with authentic cysteine thiosulfonate using the phenol: water and I.-butanol:acetic acidwater solvent systems as described under Materials and Methods. While no attempt was made to quantitatively determine the urinary output of cysteine thiosulfonate, it was observed that rats on normal chow diet did not excrete detectable amounts of cysteine thiosulfonate; rats on diets supplemented with 1% cystine were observed to excrete trace but detectable amounts of cysteine thiosulfonate; rats fed 5, 7, or 10% cystinesupplemented chow diets excreted much greater amounts of cysteine thiosulfonate

IN CYSTINE

307

METABOLISM

but roughly similar to one another as judged by the area and intensity of the ninhydrin spot obtained as outlined above. The rats on diets supplemented with cystine exhibited cysteine thiosulfonate in the first 24-h urine collection and throughout the period they were maintained on these diets. Since the K,,, value for cystathionine y-lyase is at least an order of magnitude lower for cystine than that for cystathionine (19), the metabolism of cystine by cystathionine y-lyase may represent a substantial byway for the metabolism of this sulfur amino acid. Earlier it was reported that the products of this pathway were thiocysteine, thiocystine, pyruvate, ammonia, and H2S (7, 13, 14) but these conclusions were based upon experiments conducted with purified cystathionine y-lyase. The evidence supports the view that the metabolism of cystine in the liver can proceed by cystathionine y-lyase cleavage to cysteine thiosulfonate, with pyruvate and alanine as coproducts. ACKNOWLEDGMENT We gratefully acknowledge Doyle Daves and Mr. Robert the mass spectral studies.

the contributions of Dr. Rodgers in carrying out

REFERENCES 1. FELLMAN, J. H., ROTH, E. S., AVEDOVECH, N. A., AND MCCARTHY, K. D. (1980) Arch. Biochem. Biophys. 204, 560-567. 2. FELLMAN, J. H., ROTH, E. S., AVEDOVECH, N. A., AND MCCARTHY, K. D. (1980) Life Sci. 27, 1999-2004. 3. SEGEL, I. H., AND JOHNSON, B&hem. 5, 339-337. 4. EMILIOZZI, R., AND PICHAT, Chim. Fr., 1887-1888. 5. MINAKAMI, IKAWA, 550. 6. WALDI, phy, New

J. J. (1963) L. (1959)

Bull.

Anal. SOC.

S., SUZUKI, C., SAITO, T., AND YOSHH. (1965) J. Biochem. (Tokyo) 58,543-

D. (1965) in Thin Layer Chromatogra(Stohl, E., ed.), p. 493, Academic Press, York.

FELLMAN

308

AND AVEDOVECH

7. CAVALLINI, D., DEMARCO, C., MONDOVI, B., AND MORI, B. G. (1961) Enzymolagia 22, 161-173. 8. CAVALLINI, D., MONDOVI, B., DEMARCO, C., AND SCIOSCIA-SANTORO, A. (1962) Enzyrrwlogia 24,

253-266. 9. SAKAIBARA, S., YAMAGUCHI, K., HOSOKAMA, Y., KOHASHI, N., UEDA, I., AND SAKAMOTO, Y. (1976) Bzixhim. Biophys. Acta 422, 273-279. 10. FLAVIN, M. (1962) J. Biol. Chem. 237,768-777. 11. CARROLL, D. I., DZIDIC, I., HORNING, M. G., MONTGOMERY, F. E., NOWLIN, J. G., STILLWELL, R. N., THENOT, J. P., AND HORNING, E. C. (1979) And. Chem. 51, 1858-1860. 12. SZCZEPKOWSKI, T. W. (1958) Nature (London)

182,934-935.

13. SZCZEPKOWSKI, T. W., AND WOOD, J. L. (1967) B&him. Biophys. Acta 139, 469-476. 14. LOISELET, J., AND CHATAGNER, F. (1966) Bull. Sot. Chim. Biol. 48, 595-606. 15. ABELES, R., AND WALSH, C. (1973) J. Amer. Chem. sot. 95, 6124-6125. 16. UREN, J. R., RAGIN, R., AND CHAYKOVSKY, M. (1978) Biockem. Pkarrnncol. 27, 2807-2814. 17. YAMAGUCHI, K., HOSOKAWA, Y., KOHASHI, N., KORI, Y., SAKAKIBARA, S., AND UEDA, I. (1978) J. Biockem. (Tokyo) 83, 479-491. 18. FLETCHER, J. C. AND ROBSON, A. (1963) Biothem. J. 87. 553-559. 19. YAO, K., KINUTA, M., AND AKAGI, R. (1979) Physiol. Ckem. Phys. 11, 257-260.