The Desulfuration of Thiourea bv Thvroid Cvtoulasmic J J J I

Particulate Fractions*

F. MALOOF AND L. SPECTOR

From the Department of Medicine, Harvard Medical School, The Medical Xervices o,f the Massachusetts General Hospital, and the John Collins Warren Laboratories of the Collis P. Huntington Memorial Hospital of Harvard University

at the Massachusetts General Hospital, Boston, Massachusetts

(Received for publication, November 5, 1958)

Thiourta, a goitrogen (1, 2) and an inhibitor of the iodination of tyrosyl groups in the thyroid (3), is itself metabolized by this organ. The sulfur of thiourea is converted largely to inorganic sulfate (4, 5) and partially to protein-bound sulfur and two un- identified sulfur compounds (5). Recently thiosulfatel has also been found among the products of the metabolism in viva of thiourea by the rat thyroid. This report will describe a cyto- plasmic particulate system in vitro from mammalian thyroid which desulfurates thiourea to sulfur-containing products similar to those observed in vivo.

EXPERIMENTAL

1Materials-S35-thiourea C?4-thiourea were obtained from the iew

and SVhiocyanate England Nuclear Corporation; the

specific activities ranged from 15.0 to 40.0 mc. per mmole. The chromatographically pure compounds were dissolved in triple- distilled water, and the solutions were stored at -20”. S%ul- fide in 0.02 N NaOH, with a specific activity of 4.2 mcuries per mmole, was obtained from the same source.

Dehydroascorbic acid, potassium selenocyanate, and 3,4-di- chlorophcnylserine were kindly furnished by Drs. I’. L. Munson, J. Wolff, and D. Gilbert, respectively. Thyroglobulin (Prepara- tion CH-%9,91921 A) was supplied by Dr. R. L. Kroc of the War- ner-Chilcott Laboratories.

DPNH and TPNH were obtained from the Sigma Chemical Company. Wherever necessary, rompounds were neutralized with potassium hydroxide before use.

Preparation of Thyroid Cytoplasmic Particulate Fractions- Thyroids from freshly killed sheep were obtained at local abat- toirs, chilled on ice, and cleaned of extraneous matter. The tis- sue was minced with a razor blade and disintegrated with 9 volumes of 0.25 M sucrose-O.027 M KHC08 in a chilled Servall Omni-Mixer run at half speed for 15 seconds. The mixture was ground gently in a chilled homogenizer of the Potter-Elvehjem type until a homogeneous suspension resulted. Unbroken cells, nuclei, and tissue debris were removed by two successive centrifu-

* Supported by grants-in-aid (AT-1434) from the National In- stitute of Arthritis and Metabolic Diseases, United States Public Health Service, by Grant No. MET 37-B from the American Can- cer Societv. contracts No. AT (30-11-1207 and No. AT (30-11-609 frdm the “iAtomic Energy Comkssibn, and by an Ins&tutional Cancer Grant (INST 16 K) to the Massachusetts General Hos- pit,al.

1 F. Maloof, unpublished observations.

gations in a refrigerated International Centrifuge at 600 x g for 5 minutes. The supernatant fluid (I) was then centrifuged for 10 minutes at 8,500 X g to collect the “large particles.” The supernatant fluid (II) from the latter centrifugation was centri- fuged for 60 minutes at 105,000 X g in the No. 40 rotor of the Spinco Preparative Ultracentrifuge to collect the “small par- ticles.” The supernatant solution from this centrifugation is designated the “soluble fraction.” For most of the experiments described in this paper, supernatant fluid I was centrifuged di- rectly for 60 minutes at 105,000 x g, thus collecting all the cyto- plasmic particles as one pellet. Pellets were suspcndcd by ho- mogenation in 0.1 M Tris,2 pH 7.4, containing 0.25 M sucrose or 0.15 M KCl. A ml. of this suspension contained the particales from 1.0 gm. of t,hyroid tissue.

Liaer and Kidney Particulate Fractions-lClale rats weighing approximately 150 gm. were decapitated. The liver and kid- neys were excised rapidly, minced, and washed free of red blood cells and urine by suspending the tissue in a chilled solution of 0.25 M sucrose-O.027 M KHC03. Homogenation and differential centrifugation were carried out as above.

Incubation-The incubation mixture contained the tissue frac- tion, S35- or Cl*-thiourea (approximately 125,000 c.p.m. in 50 mpmolcs), thiocyanatc, ascorbic acid or dehydroascorbic acid, and Tris buffer, pH 7.4. The flasks were shaken in a nater bath at 37” for 1 hour. The reaction was stopped by placing the flasks on ice.

Analytical Methods-The V5-metabolites were characterized by paper chromatography. A 20.~1. aliquot of the incubation mixture and 0.2 pmole each of thiourea, thiosulfate, and of thio- cyanate carriers were applied along a 1.5.cm. line to Whatman No. 1 chromatography paper. Chromatograms were run in an ascending solvent system of ethanol-ammonium acetate (1 M),

pH 7.5 (7.5:3). The radioactive spots along the paper strips were located by radioautography with the use of No-Screen x-ray film. The carrier spots were identified as follows: thiourea by ultraviolet quenching; and thiosulfate and thiocyanate by spray- ing with 0.1 N iodine and 10 per cent ferric chloride, respectively. A typical chromatogram is shown in Fig. 1A. The percentage of each product formed and of thiourea converted was measured by cutting up the chromatogram and assaying the radioactivity in each area as a percentage of the total number of counts along the strip. Blank values were obtained for each experiment from

2 The abbreviation used is: Tris, tris(hydroxymethyl)amino- methane.

949

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Desulfuration of Thiourea Vol. 234, No. 4

FIG. 1. Radioautogram of paper chromatogram run in an as- cending solvent system of ethanol-l.0 M ammonium acetate, pH 7.5 (7.5:3). A, represents the results obtained after incubation as described in the legend of Table I; B, a control, heated at 100” for 1 minute. This solvent separates a radioactive spot which re- mains at the origin (nrotein-bound S35. PBSs5). an inorganic 535 spot with an RF of 0.25 which is a mixture of thiosulfate-and sul- fate, a thiourea spot with an RF of 0.77, and an unknown spot with an RF of 0.84. The RF of thiocyanate in this solvent is 0.92.

TABLE I Requirements joy desuljuration of thiourea by thyroid

cystoplasmic particles The complete system contained the thyroid cgtoplasmic par-

ticles (1.5 mg. of protein), 10m3 M KSCN, 10-y M ascorbic acid, 5 X 10-S M S3%hiourea, 0.15 M Tris buffer, pH 7.4, in a final vol- ume of 1.0 ml. Incubation, 1 hour at 37”.

system Thiourea desulfurated

Complete system..................................... Complete system minus ascorbic acid.. Complete system minus thiocyanate.. Comp1et.e system minus particulate fraction. Complete syst,em (with heated particulate fraction)

m~moles

12.0 1.0 0.8 0.5 1.0

a heated control (Fig. 1B). The S35 radioactivity was counted by a thin end window Geiger-Mueller tube (window thickness, 0.9 mg. per cm?).

Protein analyses were done by means of the biuret (6) and the micro-Kjeldahl methods (7). Iodine was determined by a modi- fication of Chaney’s method (8).

The amount of thiourea desulfurated varied from one prepara-

tion to another. However, since the particulate preparation was stable at 4’ for 2 weeks, a number of experiments could be carried out with the same material. All experiments were performed at least two or three times. Each table represents a typical run.

RESULTS

In Table I are set out the requirements for the desulfuration of thiourea by cytoplasmic particles from sheep thyroid. It is clear that, in addition to the protein, there is a strict requirement for thiocyanate and ascorbic acid.

Characterization of Thyroid Cytoplasrnic Particles-The “large particles” had succinoxidase activity3 comparable with that of an equal weight of liver mitochondria; the “small particles” showed no measurable succinoxidase activity.

Ultracentrifugal analysis4 of a twice-washed pellet of “small particles” in a medium of 0.25 M sucrose-O.1 M glycylglycine, pH 7.6, revealed the fraction to be mainly heterogeneous except for a small, sharp peak, s?~,~ = 51, probably ribonucleoprotein particles (9, 10). The protein-bound iodine content of this frac- tion was 0.12 per cent. I f this represents thyroglobulin iodine, then thyroglobulin (approximate iodine content, 0.5 to 1.0 per cent) is only 10 to 20 per cent of the total protein of the “small particles.” An alternative explanation is that this fraction con- tains an incompletely iodinated protein, possibly a precursor of thyroglobulin. Electron microscopy5 (RCA model EMU 3B) of a twice-washed pellet of “small particles,” fixed in osmium te- troxide, revealed a heterogeneous mass of components which resembled previously described microsomes (11). Electron mi- croscopy of the “large particles” revealed the presence of mito- chondria plus components similar to those seen in the sections of “small particles.”

Activity of Various Cytoplasmic Particles of Thyroid-The com- bined “large plus small particles” of sheep thyroid were used in most experiments, since, as can be seen from Table II, these two fractions possess approximately equal activities. The inability of the whole homogenate to desulfurate thiourea is due to an inhibition by the soluble fraction. This, however, is not a specific property of the thyroid soluble fraction, since the corresponding fraction of rat liver is equally inhibitory. In both instances, coagulation of the soluble proteins destroyed the inhibition.

Although all fractions contain iodine, there is no clearly per- ceptible relationship between iodine content and activity.

Properties of Thyroid Cytoplasmic Particulate Fraction-The system is heat-labile, 1 minute at 100” sufficing to abolish all activity. While it is stable at 4” for at least 2 weeks, there is a 40 per cent loss of activity after 7 days at -20”. Dialysis of the preparation overnight at 5” results in no loss of activity.

Protein Concentration-The desulfuration of thiourea as a func- tion of protein concentration is shown in Fig. 2.

Thiourea-The system is saturated with 40 to 50 mpmoles per ml. (Fig. 3).

Thiocyanate-At a concentration of 1O-3 M, the system is satu- rated (Fig. 4). To date no substitute has been found for thiocyanate. Ions tested were cyanate, selenocyanate, methyl- thiocyanate, perchlorate, iodide, bromide, chloride, fluoride, benzenesulfonate, trichloroacetate, sodium and magnesium Ver-

3 Kindly assayed by Dr. Arnold F. Brodie. * We are indebted to Dr. Karl Schmid and Miss A. Polis for

the ultracentrifugal analyses. 5 Performed by Dr. David Spiro.

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April 1959 F. Maloof and L. Spector 951

TABLE II Desulfuration of thiourea by various cytoplasmic fractions of thyroid

Centrifugal fractionation as described in the “Experimental.” The washing medium was 0.15 M KCI-0.1 M Tris, pH 7.4. Incuba- tion conditions as in Table I.

Tissue fraction

Whole homogenate ......................... Large particles. ...........................

washed once ............................. washed twice. ...........................

Small particles. ........................... washed once ............................. washed twice. .. ........................

Large plus small particles+. ................ Soluble fraction. .......................... Large plus small particles plus soluble frac-

tion$ ..................................

Total iodine*

1.0 0.5 0.3 2.5 2.1 1.2

6.9

d Thiourea

esulfurated

mpmoles/ ng. grotein

0.4 5.6 4.5 5.2 4.5 6.6 8.0 6.1 0.2

0.8

* This iodine is considered to be protein bound, since dialysis removed none of it.

t Washed preparations; on a weight basis, the protein in this incubation was half “large particles” and half “small particles.”

$ Similar to t, plus 1.7 mg. of soluble fraction protein.

senate, 1, lo-o-phenanthroline, urea, cysteine, glutathione, and tyrosine.

L-Ascorbic Acid-The saturation concentration of ascorbic acid is 1O-3 M (Fig. 5). It can be replaced by equimolar con- centrations of dehydroascorbic, cZ-isoascorbic acid, or DPNH. TPNH is inactive. Hydroquinone, dichlorophenolindophenol, and methylene blue are 30 to 50 per cent effective. Cysteine and reduced glutathione are inactive. Attempts were made to replace ascorbic acid with metal ions, Cu+, Fe++, and Mn++, but all of these salts produced a nonenzymatic desulfuration of thio- urea.

Anaerobiosis-The desulfuration of thiourea was inhibited to the extent of 80 per cent by anaerobiosis.

pH Optimum-The pH optimum was found to be 7.2 to 7.4. Kinelks-The desulfuration of thiourea has a maximal rate

during the first 30 minutes (Fig. 6). This rate varied from prep- aration to preparation, ranging from 125 to 420 mpmoles per gm. of thyroid per hour. This approximates the rate in vivo in rats of about 250 mMmoles per gm. of thyroid per hour.1

Liver and Kidney Tissues-The particulate fractions of rat liver and kidney desulfurated only an insignificant amount of thiourea (Table III). Negative results were also obtained when thyroglobulin, P-lactoglobulin, or crystalline bovine albumin served as the protein source.

Reaction Products-S35-thiourea is converted chiefly to protein- bound $Y5 (about 54 per cent of the products), thiosulfate (27 per cent), sulfate (9 per cent), and an unidentified compound (10 per cent) (Fig. I).

Protein-bound S3j-The reasonable conclusion that the 535 is bound to protein stems from the following observations: (a) the radioactivity precipitates along with the protein on denaturation with trichloroacetic acid; (6) the radioactivity is nondialyzable; and (c) the radioactivity does not separate from the protein under a variety of conditions of paper chromatography and paper elec- trophoresis.

PROTEIN CONCENTRATION (mg/ml) FIG. 2. The amount of thiourea desulfurated as a function of

the protein concentration. The preparation was washed once in a medium of 0.15 M KCl-0.1 M Tris, pH 7.4. Incubation conditions as in Table I.

17.5

3 l5.0- 0

z 12.5-

5 lO.O-

2

g 7.5- a 0

z 3 5.0- 0

z 2.5 --

40 80 160 3;t

TH IOUREA (m)c moles /ml)

FIG. 3. The amount of thiourea desulfurated as a function of the substrate concentration. Incubation conditions as in Table I.

Attempts were made to identify this protein by paper electro- phoresis with the use of Whatman No. 1 paper, barbital buffer (0.12 M), ionic strength 0.1, pH 8.6, and 125-150 volts for 18 hours. The paper was dried in an oven for 30 minutes at 100” and then stained with Amido Schwartz (Bender & Hobein, Munich, Germany). The protein-bound S35 does not move in this electrophoretic system, in contrast to thyroglobulin which travels between serum ocl-globulin and albumin. Solubilizing the protein-bound V5 with sodium desoxycholate (1 or 4 per cent) at 37” for 2 hours led to the movement of only a small portion of the protein along with its radioactivity. The mobility

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1.0 2.0 5.0

TH IOCYANATE OJ moles/ml)

FIG. 4. The amount of thiourea desulfurated as a function of the concentration of thiocyanate. Incubation conditions as in Table I.

m I W

0

E 12.5 c

2 I l I

- m-m-

lb 5.0

0 I I : ! ---

2.5 -

1.0 2.0 5.0 10.0

L-ASCORBIC ACID OJ moles/ml)

FIG. 5. The amount of thiourea desulfurated as a function of the ascorbic acid concentration. Incubation conditions were the same as in Table I.

of this small fraction varied, but was not that of thyroglobulin similarly treated with sodium desoxycholate. Unlike thyro- globulin, only a very small fraction of the protein-bound S35 is extracted by isotonic saline.

Attempts were made to determine the nature of the sulfur bound to protein. It was possible to demonstrate that the in- tact thiourea molecule does not become attached to protein. When V-labeled thiourea was incubated in place of the S35- thiourea, no protein-bound Cl4 was found after incubations rang- ing from zero to 60 minutes. Hence, only the sulfur portion of thiourea becomes attached to protein during the course of the reaction. C14-urea was identified as one of three C14-labeled products formed during this incubation; cyanamide was not one of the products.

Dialysis of the protein-bound S35 for 24 hours at 4” against water, Tris buffer, or sodium chloride results in the loss of less than 5 per cent of the radioactivity. The S35 is not displaced by reincubation of the protein-bound S36 with nonradioactive thio-

0 IO 20 30 60

TIME (minutes) FIG. 6. The amount of thiourea desulfurated as a function of

time. Incubation conditions as in Table I, except that dehydro- ascorbic acid (10-a M) was used in place of ascorbic acid.

TABLE III Desulfuration of thiourea by cytoplasmic particulate fractions

of sheep thyroid and rat liver and kidney

Centrifugal fractionation as described in the “Experimental.” Incubation conditions as in Table I.

Tissue particles Thiourea desulfurated

Thyroid, large plus small particles .................. 5.9 Liver, large plus small particles*. .................. 0.6 Kidney, large plus small particles*. ................ 0.5

* Results were similar whether these fractions were tested

separately or together.

urea (1O-2 M), sulfide (5 X 1O-3 M), or sulfate (10-Z M) at 37” for 30 minutes. After 2 weeks in solution at 4” 80 per cent of the radioactivity remains attached to the protein. The protein- bound S35 also resists the action of 1 N HCl at room temperature for 2 hours.

On the other hand, it was possible to remove the sulfur label from the protein-bound F5 by simple incubation of the latter mix- ture at room temperature for 2 hours with certain alkaline re- agents, such as cyanide (0.01 M), bisulfite (0.01 M), or 0.1 N sodium hydroxide. The products were identified by paper chromatog- raphy and by the system of paper electrophoresis described below. These alkaline reagents displaced 60 to 80 per cent of the radio- activity from the protein. Thiocyanate was the major product resulting from the reaction with cyanide, while sulfite gave a good yield of thiosulfate. Sulfite was found as a product of the incubation with NaOH. These data suggest that the S35 exists on the protein at more than one level of oxidation. About 20 per cent of the radioactivity was not removed by these alkaline reagents.

Inorganic P5 Spot-The heterogeneity of the inorganic F5 was established by paper electrophoresis with the use of thin What- man No. 31 paper, Tris-citrate buffer (0.06-0.03 M), ionic strength 0.18, pH 7.0, 1000 volts for 35 minutes (12). This method sep-

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April 1959 F. Maloof and L. Spector 953

arates sulfite with a mobility of 15.0 cm., sulfate with a mobility of 19.0 cm., and thiosulfate with a mobility of 21.0 cm. under a potential gradient of 16.7 volts per cm. Sulfite and thiosulfate were identified by spraying the paper with 0.1 N alcoholic iodine; sulfate by using W-labeled sulfate as a marker. The inorganic S35 spot was found on electrophoresis to consist of thiosulfate and sulfate in the proportion of 3: 1. Thiocyanate has the same mobility as sulfate in this system, but is easily separated from it by paper chromatography (Fig. 1).

Experiments with S35 &u&de-The possibility was considered that the desulfuration of thiourea might result from a preliminary hydrolysis to inorganic sulfide followed by oxidation of the latter in the manner described by several investigators (13-19). To test this point, labeled sulfide (50 mpmoles) was incubated in the usual way with tissue preparations from sheep thyroid and rat liver and kidney. It was found that sulfide was transformed to approximately the same extent in all these systems to yield thio- sulfate and protein-bound S35. Since oxidation of sulfide, in these small amounts, occurred as well in the absence of protein, the process is clearly nonenzymatic.

These experiments, however, revealed certain important dif- ferences between the reactions of thiourea and sulfide. The lat- ter was found to give rise to no detectable amount of sulfate, whereas thiourea formed small, but reproducible, quantities of this ion. Furthermore, the presence of mersalyl (1O-3 M) had a profound effect on the reaction of sulfide, completely inhibiting the formation of protein-bound W, while the conversion of thio- urea-sulfur to protein-bound S35 was barely affected (25 per cent inhibition). For these reasons, the desulfuration of thiourea is thought to proceed by way of direct transfer of sulfur to protein and not via preliminary hydrolysis to inorganic sulfide.

Inhibitors-The desulfuration of thiourea was found to be in- hibited by iodide, cyanide, azide, sulfide, a number of thiol com- pounds, and several aromatic antithyroid substances. These and a number of other common enzyme inhibitors are listed in Table IV. Of considerable interest is the fact that, of the halides, only iodide is inhibitory, and that this inhibition is partially re- versed by increasing the thiocyanate concentration. Thiosul- fate is inhibitory but not at the low concentration produced in the desulfuration reaction. There is no inhibition by 3,4-di- chlorophenylserine which is reported to be a specific copper- enzyme inhibitor (20).

DISCUSSION

This study describes a cytoplasmic particulate system derived from thyroid tissue which is capable of desulfurating thiourea and oxidizing the sulfur to inorganic sulfate. This system is heat-labile, nondialyzable, and pH dependent. Particulate frac- tions from liver and kidney, prepared and incubated in a similar manner, are much less active. This difference is also apparent in vivo; the thyroid can metabolize a much greater quantity of thiourea than an equal weight of liver (5).

Several of the sulfur products formed in the experiments in vitro are the same as those found in vivo. The major product of the metabolism in vivo of thiourea in the thyroid is inorganic sulfate (4, 5), but protein-bound sulfur (5) and thiosulfater are also found. The system in vitro, however, produces mainly pro- tein-bound sulfur, along with some thiosulfate and sulfate.

From the experiments with labeled sulfide, it is concluded that the desulfuration of thiourea does not result from the hydrolysis to inorganic sulfide. On the contrary, it appears that sulfur is

TABLE IV Inhibition of desulfuration of thiourea

Incubation conditions as in Table I.

Iodide

All other halides Cyanide

Aaide Sulfide

Cysteine Diethyldithiocarbamate Glutathione (reduced) Thiosulfate

Bisulfite

Urea Sulfate Sulfanilamide Sulfadiazine (Na salt) p-Aminobenzoic acid Dinitrophenol Arsenite Dichlorophenylserine Hydroxylamine Antimycin A Mersalyl Perchlorate

Concentration Inhibition

M %

1 x 19-a 78

1 x 10-b 22 4 x lo-3 5 1 x lo-4 91 1 x lo-6 30 1 x lo-3 84 5 x 19-4 82 1 x lo-6 4 1 x 1w 94 1x lo-4 93 5 x lo-4 80 1x l&3 88 4x lo-6 0 1x lo-4 92 1x lo-6 6 1x lo-3 0 1x lo-2 14 4 x lo-3 46 4 X1&3 38 4 x IO-3 52 1x10-3 4 1x lo-3 0 1x l&3 0 1 x lo-3 0 1x10-4 0 1x lo-4 0 1x lo-3 1

transferred directly from thiourea to thyroid protein; and only thyroid, of the tissues tested (Table III), is capable of dissociat- ing the sulfur atom from thiourea and converting it to the in- organic state. The desulfuration reaction is inhibited by in- organic iodide and the aromatic antithyroid compounds, but not by perchlorate. This is the same pattern of inhibition observed in the iodination of tyrosyl groups by thyroid cytoplasmic par- ticulates (21).

Astwood (22) has already demonstrated that the effective sul- fur-containing goitrogens invariably have a thiocarbamide group as a feature of their molecular constitution. From the present study, the possibility emerges that it is the cleavage of the car- bon-sulfur bond, by thyroid tissue, that underlies the antithy- roidal activity of these compounds.

Thiocyanate is a specific requirement for the desulfuration in vitro of thiourea. Noteworthy is the fact that perchlorate can- not replace thiocyanate even though it is 10 times as active as an inhibitor of the concentration of iodide by the thyroid (23). Moreover, it is significant that, of the halide ions, only iodide is inhibitory; the partial reversibility of this inhibition by increasing the concentration of thiocyanate is in accord with the well estab- lished competition between these two anions in thyroid phys- iology (22). These facts raise again the question as to a possible role of thiocyanate in the normal metabolism of the thyroid (24).

Although it has been reported that thiocyanate is oxidized to sulfate in the rat thyroid (25)) no such oxidation in vitro, with S35-

labeled thiocyanate added, was demonstrable under the present experimental conditions.

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954 Desulfuration of Thiourea Vol. 234, No. 4

The function of ascorbic acid in the desulfuration of thiourea appears to be less specific, since it can be replaced by dehydro- ascorbic acid, d-isoascorbic acid, and DPNH. TPNH, an es- sential cofactor in the deiodination in vitro of monoiodotyrosine and diiodotyrosine by thyroidal microsomes, does not replace ascorbic acid (26, 27). The requirement for a reducing agent is reminiscent of similar requirements in the enzymatic oxidation of tyrosine (28, 29) and various drugs (30).

A considerable effort was made to identify the protein in the thyroid cytoplasmic particles to which the S35 becomes attached; more specifically, it seemed important to know if this protein might be thyroglobulin. From its electrophoretic and ultra- centrifugal behavior, its iodine content, and its very limited extractability into isotonic saline, it appears that the major part of the protein-bound S35 is not sulfur-labeled thyroglobulin.

SUMMARY

A cytoplasmic particulate system, derived from sheep thyroid, has been described which desulfurates thiourea in the presence of thiocyanate and a reducing agent, such as ascorbic acid. The system is heat-labile, nondialyzable, and pH dependent. Partic- ulate fractions from rat liver and kidney are inactive.

The sulfur products of this system in vitro are protein-bound sulfur, thiosulfate, and sulfate. The protein-bound sulfur is ap- parently not sulfur-labeled thyroglobulin.

Acknowledgments-The authors wish to thank Drs. J. B. Stan- bury, 0. Cope, and P. Zamecnik for their encouragement and Dr. M. Soodak for helpful discussions. We are especially in- debted to Dr. Elizabeth B. Keller for invaluable technical ad- vice. Miss Lena Neri provided technical assistance.

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F. Maloof and L. SpectorThe Desulfuration of Thiourea by Thyroid Cytoplasmic Particulate Fractions

1959, 234:949-954.J. Biol. Chem. 

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