Thyroglobulin iodoamino acids estimation after digestion with pronase and leucylaminopeptidase

ANALYTICAL BIOCHEMISTRY 33, 307-317 (19%)

Thyroglobulin

lodoamino Digestion

with

Acids

Estimation

Pronase

and

after

Leucylaminopeptidase MARCEL

ROLLAND,

Laboratoire Faculte’

ROBERT AQUARON,

de Biochimie de Me’decine,

AND

Me’dicale et Unite’ thyroidienne Bd Jean-MO&n, 1SMarseille

SERGE LISSITZKY de I’INSERM, 6, France

Received July 25, 1969

During the past fifteen years, numerous methods of iodinated amino acid estimation in biological fluids, thyroid extracts, or thyroglobulin have been described. Except for the spectrophotometric method (1)) enzyme digestion is a preliminary step in the quantitation of iodoamino acids after separation by different techniques such as gel filtration (2-7) or chromatography. The latter has been used in different ways, on paper sheets (8-12), thin layer (13)) kieselguhr columns (11, 14)) cellulose powder columns (lo), and anion- (15-18) or cation-exchange resin columns (19122). After separation, iodinated amino acids are estimated by lz51 or 1311 counting when labeled with radioactive iodine or by the sodium arsenite/ ceric sulfate (23) or the ferrichloride/ferricyanide/arsenic acid reactions (24) for their lz71 content. Enzymic digestion of iodinated proteins has been carried out with proteolytic enzymes alone-trypsin (11, 14, 25), pepsin (25-27)) pronase (21, 27-31)) thyroid proteases (27, 32-33)) or in association-pancreatin (6, 8, 11, 22, 25, 28, 34-38)) pancreatin + erepsin (lo), trypsin + pancreatin (11)) pancreatin + pronase (27)) pancreatin or pronase + thyroid proteases (27). Variable degrees of deiodination occurred that depended on the enzyme used and that might reach up to 15% of total iodine. Moreover, incomplete digestion resulted in the persistence of iodinated peptides (up to 20%) which contained mostly 3,5-diiodotyrosine and thyroxine and which lower drastically the quantitative estimation of total iodoamino acids of iodinated proteins. If current methods of separation and estimation of free iodoamino acids are satisfactory (although few methods are concerned with ?I estimation), on the contrary the available enzymic methods of iodopro307

308

ROLLAND,

AQUARON,

AND

LISSITZKY

tein digestion result in incomplete release of iodoamino acids and often in excessive deiodination. This paper shows that the association of pronase and leucylaminopeptidase tends to obviate these difficulties. MATERIALS

1. Chemicals

Ethanol and sulfuric acid (reagent grade) were obtained from Prolabo (Paris, France) ; formic acid, ammonium hydroxide, ceric sulfate, and sodium iodide all puriss.) from Merck (Darmstadt, Germany) ; sodium arsenite (general-purpose reagent) from Hopkin-Williams (Chadwell Heath, England) ; and thiouracil from Serlabo (Paris, France). Pronase (protease type VI) and subtilisin were purchased from Sigma (Saint Louis, MO.) ; pepsin and papain from Worthington (Freehold, N. J.) ; and leucylaminopeptidase (106 U/mg, leucinamide) from Seravac (Maidenhead, England). Iodinated amino acids were obtained from Calbiochem (Lucerne, Switzerland). Bidistilled water was used throughout and obtained from an all-quartz apparatus. 2. Reagents

(1) 0.2 M ammonium formate buffer, pH 3.5, in 30% ethanol (v/v). (2) 0.5 N, 0.8 N, and 1.5 N ammonium hydroxide in 30% ethanol (v/v) (3) 0.3 M ceric sulfate (12 gm/liter) in 3 N sulfuric acid. (4) 2 N sulfuric acid. (5) 0.18 M sodium arsenite (6 gm/liter) in water. (6) Solutions of pronase (5 mg/ml) or leucylaminopeptidase (2 mg/ml) in 0.1 M Tris-acetate, pH 8.6. 3. Solutions

of Reference

Ioodoamino Acids

Iodoamino acids were desiccated for 24 hr on P,Os. A mother liquor containing 0.2 pmole/ml of each of the four iodoamino acids (MIT, DIT, T,, and T,)l was then prepared by dissolving each of them first in a few drops of concentrated ammonium hydroxide and filling up with water. The working solution (10 ,pM in each iodoamino acid) was an adequate dilution of the mother liquor in ammonium formate buffer, pH 3.5. Both ’ Abbreviations: MIT, thyronine ; T,, thyroxine

3-iodotyrosine ; DIT, ; LAP, leucylaminopeptidase.

3,5-diiodotyrosine

; T,,

3,5,3’-triiodo-

THYROGLOBULIN

17. WYNN, J., (1959).

18. 19. 20. 21. 22. 23. 24. 25.

FABRIKANT,

I.,

IODOAMINO

AND DEISS,

W.

ACIDS

ESTIMATION

P., Arch.

B&hem.

317 Biophys.

84,

106

GALTON, V. A., AND PITT-RIVERS, R., Biochem. J. 72,310 (1959). LERNER, S. R., Federation Proc. 19, 171 (1960). REILLY, W. A., SEARL.E, G. L., AND Scorr, K. G., Metabolism 10, 869 (1961). BWCK, R. J., AND MANDL, R. H., Ann. N. Y. Acad. Sci. 102, 87 (1962). LERNER, S. R., Federation Proc. 19, 171 (1960). KOLTHOFF, I. M., AND SANDELL, E. B., J. Am. Chem. Sot. 56, 1426 (1934). GMELIN, R., AND VIRTANEN, A. I., Acta Chem. Stand. 13, 1469 (1959). ROCHE, J., MICHEL, R., LISSITZKY, S., AND YAGI, Y., Bull. Sot. Chim. Biol.

36,

143 (1954). 26. KOUJGLU,

S., SCHWARTZ, H. L., AND CARTER, A. C., Endocrinology 78, 231 (1966). 27. MALAN, P. G., Biochem. J. 109, 787 (1968). 28. TONG, W., RAGHUPATHY, E., AND CHAIKOFF, I. L., Endocrinology 72, 931 (1963). 29. SIMON, C., AND LISSITZKY, S., Biochim. Biophys. Actu 93, 494 (1964). 50. CHEFTEL, C., BOUCHILIAXJX, S., AND LISSITZKY, S., Compt. Rend. Acud. Sci. (Paris)

259, 1458 (1964). 31. MURTHY, P. V. N., RAGUPATHY, E., AND CHAIKOFF, I. L., Biochemistry 4, 611 (1965). 32. MCQUILLAN, M. T., MATHEWS, J. D., AND TRIKOJUS, V. M., Nature 19.2, 333 (1961). 33. PITTMAN, C. S., AND PITTMAN, J. A., Am. J. Med. 40,49 (1966). 34. YAMAZAKI, E., NOGUCHI, A,, AND SLINGIERLAND, D. W., .I. Clin. Endocrinol. 20, 889 (1960). 35. STOLC, V., Endocrinology 71, 564 (1962). 36. AROSENIUS, K. E., &and. J. Clin. Lab. Invest. 16,440 (1964). 37. DIMITRIADOU, A., SUWANIK, R., RUSSEL-FRASER, T., AND PEARSON, J. D., J. Endocrinol. 34, 23 (1966). 38. ERMANS, A. M., KINTHAERT, J., DELCROIX, C., AND COLLARD, J., J. Clin. Endocrinol.

28, 169 (1968). 39. BOUCHILLOUX, S., R,OLL~ND, Biochim. 40. ROLLAND,

M., TORRESANI, J., ROQUES, Biophys. Actu 93, 15 (1964). M., BISMUTH, J., FONDARAI, J., AND LISSIT~KY,

M., AND LISSITZKY,

S., Actn Endocrinol. 286 (1966). 41. AQUARON, R., These de Doctorat en Pharmacie, Marseille, 1967. 42. PIEZ, K. A., AND MORRIS, L., Anat. Biochem. 1, 187 (1960). 43. AQUARON, R., Thke de Doctorat en MCdecine, Marseille, 1969.

S., 53,

310

ROLLAND,

HEATING BATH 35’ tl

MIX e I1

AND

AQVARON,

MIX e **

LISSITZKY -

MIX C A. *

1

MI(NOMETER

Q

5

c

0.39 ml / min P.OOml/min

REAGENT

0.00 ml /mm

a/R

k

m

COOLER COLUMh

FIG. 1. Flow diagram of reagent stream for AutoAnalyzer. Reagent A, 0.18 M sodium arsenite. Reagent B, 0.30 M ceric sulfate. Column (0.63 X 30 cm) filled with Dowex 50-X4 (resin P, Technicon) to a height of 23 cm. Equilibration of column with 0.2 M ammonium formate buffer, pH 3.5. Temperature of elution, 40”. Flow rate 0.35 ml/min under pressure of 100 psi. 0.5 ml samples (reference mixture or thyroglobulin digest) are routinely applied on the column. An aliquot of the column effluent volume may be obtained through an “h” tube and fractionated (to fraction collector).

formate, pH 3.5, buffer for 30 min; 20 to 30 analyses can be performed without refilling the column.

4. Enzymic Digestion of Thyroglobulin Thyroglobulin in solution at pH 8.6 is submitted to the action of pronase (10% by weight) and leucylaminopeptidase (20% by weight) consecutively. The standard medium contains: thyroglobulin (6.25 mg/ Composition

of Autograd

TABLE 1 Chambers for Gradient

Elution of Iodoamino

Acids

Chamber number

Ammonium formate buffer, pH 3.5

0.5 N ammonium hydroxide

0.8 N ammonium hydroxide

1 2 3 4 5

40 ml 30 ml 20 ml -

10 ml 20 ml 40 ml

40 ml

All solutions or buffer contained ethanol (30% v/v).

THYROGLOBULIN

IODOAMINO

ACIDS

311

ESTIMATION

ml) 0.4 ml; 0.1 M Tris-Cl, pH 8.6, 0.6 ml; thiouracil (5 mg/ml) 0.025 ml; pronase (5 mg/ml) 0.050 ml; and toluene (1 drop). After standing in a closed tube at 37” for 48 hr, 0.250 ml of leucylaminopeptidase solution (2 mg/ml) is added. After 24 hr at 37”, ammonium formate, pH 3.5, is added to obtain a 1: 2 dilution and an aliquot is submitted to iodoamino acid analysis. Digestion of thyroglobulin for 24 or 48 hr in the presence of both pronase and leucylaminopeptidase is less satisfactory, giving lower yields in DIT and iodothyronines. RESULTS

1. Separation

and Quantitative

Estimation

of Iodoamino

Acids

Figure 2 shows that a good separation of the four iodoamino acids is obtained. The relationship between the surfaces of the peaks obtained by planimetry and the amount of each compound (Fig. 3) is linear up to 5

FIG.

pound

2. Separation of mixture layered on the column.

of reference The mixture

iodoamino contained

acids. 2 nmoles of each no added iodide.

com-

iodinated amino acids (mole j iodide (nmole x IO-’ 1 FIQ. 3. Standard curves for I-, MIT, DIT, T,, and T4. Area vs. nmoles compound. Values in arbitrary units.

measured

by planimeter

312

ROLLAND,

AQUARON,

AND

LISSITZKY

nmoles for MIT and T,, up to 3 nmoles for T,, and up to 2.5 nmoles for DIT. As iodide is not exchanged by the resin it is eluted in front of the eluting buffer as a very sharp peak, the surface of which cannot be estimated. However the height of the peak is directly proportional to its amount up of 0.3 nmole. 2. Enzyme

Digestion

of Thyroglobulin

The records of iodoamino acid separation from enzyme digests of human or ox thyroglobulins (200 to 400 pg) are shown in Figure 4. Quantitative data are summarized in Table 2. The level of deiodination is very low and never exceeds around 3%. As far as iodoamino acids are

FIQ. 4. Recording digests digested

(pronase material

Iodoamino

of ox (upper curve) and human for 48 hr and leucylaminopeptidrtse in ox thyroglobulin.

Acid

TABLE 2 of Thyroglobulin

Composition

y0 by weight

Species Man

ox Horse

Dog Sheep Rat Rabbit

Iodine (est’d.)a

Iodine (ca1cd.P

0.26 0.37 0.38 0.68 0.46

0.26 0.38 0.33 0.66 0.48 0.46 0.65

0.49 0.61

Residues

MIT

4.9 6.5 5.4 6.8 6.3 4.6 7.0

(lower curve) thyroglobulin for 24 hr). Arrow shows un-

of Several

Animal

Species

per mole

DIT

Tq

T,

Undigestedc

1.9 3.5 2.0 4.2 3.4 4.4 4.6

1.0

2.3 2.1 3.7 2.2 2.5 3.6

0.2 0.7 0.7 0.5 0.5 0.3 0.5

0 6.0 0 6.5 4.5 3.0 3.4

Deiodination rated

1.5 1.8

1.5 3.2 1.8

2.8 2.5

o Total iodine estimated after chloric acid digestion. b Sum of iodine contained in iodoamino acids, iodide, and, if present, undigested iodinated material. c Area of undigested iodinated material X lOO/area of iodoamino acids + area of undigested iodinated material. d Estimated iodide X loo/total iodine.

THYROGLOBULIN

IODOAMINO

ACIDS

ESTIMATION

313

concerned, the digestion is complete for human and horse thyroglobulins. Undigested iodinated material amounts, respectively, to 3 and 4.5% for rat and sheep and about 6% for ox and dog thyroglobulins. Table 2 also shows a good agreement between the value of total thyroglobulin iodine calculated from iodoamino acid analysis after enzyme digestion and the value obtained by direct total iodine estimation. The method may be applied to extracts of total soluble thyroid proteins or to crude thyroglobulin preparations obtained by salting-out. In such cases, calculation of enzyme to protein ratios are done by taking into account the total protein content of the extract. DISCUSSION

1. Separation and Quantitative

Estimation of Iodoami?zo Acids

Quantitative separation of iodoamino acids in a reference mixture or in thyroglobulin digests is achieved by the method described. Total recovery of the iodoamino acids is asserted by the following observations: (1) the discoloration of the ceric reagent is linear in a satisfactory zone of iodine concentration ; (2) no iodine is present in the solution (1.5 or 2.0 N ammonium hydroxide) used to wash the resin after analysis; (3) a good agreement is obtained between the iodine calculated from recovered iodoamino acids after separation and the total iodine estimated directly; (4) when a digest of radioactive iodine labeled thyroglobulin (human or rat) is separated on the column all the radioactivity is recovered after fractionation of the effluent in the peaks corresponding to iodide, iodoamino acids, and eventually undigested iodinated material. Variations in the estimation of each of the iodo compounds separated on the resin column do not exceed &2.5% whether iodoamino acid reference solution or thyroglobulin digests are used. Since the iodine content of normal thyroglobulins ranges between 0.3 and 0.6% by weight, the sensitivity of estimation of the sodium arsenite/ ceric sulfate reaction has been voluntarily limited by performing the absorbance measurement at 440 nm and allowing the reaction to proceed at 35”. Under these conditions 0.2 to 0.4 mg thyroglobulin is sufficient for a precise estimation of iodoamino acid content. A fivefold increase of sensitivity can be obtained by raising the temperature to 50” and reading fading of the colored solution at 420 nm, but the “noise” level is increased. However, these conditions are useful for analysis of thyroglobulins of low iodine contents to avoid overloading of the column with excessive amounts of thyroglobulin digest. If the analysis is carried out on the digest of a radioactive iodine labeled iodoprotein, an aliquot (1: 10) of the effluent volume can be

314

ROLLAND,

AQUARON,

AND

LISSITZKY

collected through the “h” tube (Fig. 1). Under standard conditions, fractions are collected each 10 min. By measuring the total radioactivity of each iodoamino acid peak, the specific radioactivity of the latter is easily calculated. 2. Enzymic

Digestion

of Thyroglobulin

Digestion of thyroglobalin by pancreatin or pronase is most commonly used. Even with pronase (10% by weight), which allows a more complete liberation of iodoamino acids, the uncompletely digested iodinated material (iodinated peptides) reaches about 10 to 15% of total thyroglobulin iodine, and under the better conditions about 5% deiodination is observed. Increasing the enzyme to iodoprotein ratio (up to 20%) and the digestion time (up to 48 hr) reduces to a slight extent the amount of iodinated peptides but the amount of iodide formed by deiodination becomes prohibitive. In an attempt to improve the digestion method, several systems including the consecutive action of trypsin, chymotrypsin, and pronase, pancreatin or papain or subtilisin and pronase, were tested at different ratios of enzymes to iodoproteins and for different digestion times. No improvement over the use of pronase alone was observed. The association of pepsin and pronase recently described (27) was less satisfactory than the association of pronase and leucylaminopeptidase studied in this paper. For the latter, addition of ethanol (lo%, v/v) (21) or bivalent cations does not modify the final result. Whereas in the last period of digestion the two enzymes coexist in the medium and digest themselves to some extent (Table 3)) it is shown that heat inactivation of pronase (100” for 0.5 to 2 min) before the addition of leucylaminopeptidase does not improve the extent of digestion but increases deiodination. Besides the total or subtotal liberation of iodoamino acids (Table 2)) the extent of over-all digestion of thyroglobulin by the system pronase/leucylaminopeptidase is shown in Table 3, which compares thyroglobulin digestion by pronase (column 6)) by pronase plus leucylaminopeptidase (column 4)) and by 6.0N HCl. Except for serine and threonine (which are not separated from asparagine and glutamine and therefore cannot be estimated), for half-cystine, and to a lesser degree for glycine and proline, hydrolysis is almost complete and anyhow far better than that obtained with pronase alone. Whatever the origin of thyroglobulin or the nature of the enzyme(s) used for digestion, two “negative” peaks are always observed between iodide and MIT and between MIT and DIT. These peaks correspond to an increase in the reagent color. They are absent when reference iodo

THYROGLOBULIN

(pg amino

Amino acid

Asp Thr Ser Glu Pro GlY Ala Val cys-1 Met Ile Leu Tyr Phe LYS His Arg

/2

acid

IODOAMINO

ACIDS

TABLE 3 Digestion of Sheep Thyroglobulin residues liberated during digestion

Autolysis pronase 48 hr + LAP 24 bra 1

Pronase 48 hr + LAP 24 hr 2

Pronase 48 hr + LAP 24 hr deducted valuesb 2-l

1.95 d d

14.38 d d

12.43 d d

2.58 1.07 2.11 2.27 2.47 0.20 0.52 1.58 3.28 2.93 1.76 2.82 1.23 2.81

25.56 20.29 14.93 21.32 21.99 9.79 6.27 11.36 40.15 16.19 27.33 14.23 6.71 34.98

22.98 19.22 12.82 19.05 19.52 9.59 5.75 9.78 36.87 13.26 25.57 11.41 5.48 32.17

315

ESTIMATION

of 400 pg protein)

Digestion by 6.0 N HClc 3 26.93 14.25 26.13 55.12 21.75 14.44 18.69 18.93 13.97 5.51 9.05 35.77 14.03 25.17 10.64 5.34 33.42

0 Complete medium but thyroglobulin omit,ted. Amount of material umn corresponded to 40 rg pronase and 80 pg LAP. b Figures of column 2 minus figures of column 1. c Digestion times: 20 and 70 hr. d Not measurable due to overlaping with asparagine and glutamine.

Pronase 48 hr + ethanol 10% (v/v) 4 1.49 d d 0.90 0 2.05 10.24 11.49 1.63 4.46 7.47 25.69 8.32 20.16 5.25 3.84 14.52 layered

on col-

compounds or other iodoprotein digests (iodo serum albumin, iodinated neurotoxins from scorpion venom) are analyzed. On the contrary, a fetuin digest supplemented before analysis with free iodoamino acids discloses the presence of the “negative” peak between MIT and DIT. Although other experiments tend to correlate the presence of these peaks with the carbohydrate moiety of thyroglobulin, a definitive conclusion on the nature of the compounds increasing the color of the sodium arsenite/ceric sulfate reagent must await further experiments. Figure 4 and Table 2 show that, using the same mixture of pronase and leucylaminopeptidase operated under the same conditions: (1) hydrolysis of iodoamino acids from man and horse thyroglobulins is complete, whereas undigested peptides persist in ox, dog, rabbit, rat, or sheep digests (2) undigested iodine-containing peptidcs have elution volumes which differ according to the origin of the thyroglobulins.

316

ROLLAND,

AQUARON,

AND

LISSITZKY

Despite a very similar amino acid composition (40) of the thyroglobulins studied in this paper these differences toward an identical system of proteolysis lend support to the idea that primary structure of their constitutive polypeptide chains should differ to some extent. SUMMARY

A method for the quantitative estimation of thyroglobulin iodoamino acid residues is described. It combines (1) digestion of thyroglobulin (or iodinated proteins) with pronase (10% by weight for 48 hr) and leucylaminopeptidase (20% by weight for 24 hr), (2) separation of the free iodoamino acids liberated by chromatography on a cation-exchange resin column (Dowex 50-X4) and their quantitative estimation by the sodium arsenite/ceric sulfate reaction operated automatically according to a modification of the procedure of Block and Mandl. The complete release of iodotyrosines and iodothyronines is obtained in the case of man and horse thyroglobulins. For other thyroglobulins (rat, sheep, ox, dog) undigested iodinated material represents between 3 and 67% of total iodine. Deiodination is low and never exceeds around 3% of total protein iodine. The procedure described in this paper definitely improves procedures that are already known. ACKNOWLEDGMENT The skillful

technical

assistance of Mrs. S. Lasry is gratefully

acknowledged.

REFERENCES 1. EDELHOCH, H., J. Biol. Chem. 237, 2778 (1962). 2. LISSITZKY, S., BISMUTH, J., AND ROLLAND, M., C&n. Chim. Acta 7, 183 (1962). 3. LISSITZKY, S., AND BISMUTH, J., Clin. Chim. Acta 8, 269 (1963). 4. MOUGEY, E. H., AND MASON, J. W., Anal. Biochem. 6,223 (1963). 5. MAKOWETZ, E., MULLER, K., AND SPITZY, H., Microchem. J. 14 194 (1966). 6. JONES, J. E., AND SHULTZ, J. S., J. Clin. Endocrinol. 26, 975 (1966). 7. OSBORN, R. H., AND SIMPSON, T. H., J. Chromatog. 34, 110 (1968). 8. TONG, W., AND CHAIROFF, I. L., J. Biol. Chem. 232, 939 (1953). 9. TONG, W., AND CHAIKOFF, I. L., Endocrinology 72,931 (1963). 10. MANDL, R. H., AND BLOCK, R. J., Arch. Biochem. Bbphys. 81, 25 (1959). 11. KLEIN, E., AND REINWEIN, D., Acta Endocriwl. 41,170,584(1962). 12. POSTMES, T., Acta Endocrinol. 42, 153 (1963). 13. SOFIANIDES, D., MEL~NI, C. R., ALCAR, E., AND CANARI, J. J., Proc. Sot. Eqtl. Biol. Med. 1~23, 646(1966). 14. BRAASCH, J. W., ALBERT, A., KEATING, F. R., AND BLACK, B. M., J. Clin. Endo-

crinol. 15, 732 (19.55). 15. BLANQUET, P., MEYNIEL, G., MOUNIER, J., STOLL, R., DUNN, C. A., Arch. Biochem. Biophys. 58, 502 (1955). 16. LI~SITZKY,

S., AND L.~sRY, S., Bull.

Sot,

Chim.

Biol.

40,609

R. W., AND TOBIAS,

(1958).

THYROGLOBULIN

17. WYNN, J., (1959).

18. 19. 20. 21. 22. 23. 24. 25.

FABRIKANT,

I.,

IODOAMINO

AND DEISS,

W.

ACIDS

ESTIMATION

P., Arch.

B&hem.

317 Biophys.

84,

106

GALTON, V. A., AND PITT-RIVERS, R., Biochem. J. 72,310 (1959). LERNER, S. R., Federation Proc. 19, 171 (1960). REILLY, W. A., SEARL.E, G. L., AND SCOTT, K. G., Metabolism 10, 869 (1961). BWCK, R. J., AND MANDL, R. H., Ann. N. Y. Acad. Sci. 102, 87 (1962). LERNER, S. R., Federation Proc. 19, 171 (1960). KOLTHOFF, I. M., AND SANDELL, E. B., J. Am. Chem. Sot. 56, 1426 (1934). GMELIN, R., AND VIRTANEN, A. I., Acta Chem. Stand. 13, 1469 (1959). ROCHE, J., MICHEL, R., LISSITZKY, S., AND YAGI, Y., Bull. Sot. Chim. Biol.

36,

143 (1954). 26. KOUJGLU,

S., SCHWARTZ, H. L., AND CARTER, A. C., Endocrinology 78, 231 (1966). 27. MALAN, P. G., Biochem. J. 109, 787 (1968). 28. TONG, W., RAGHUPATHY, E., AND CHAIKOFF, I. L., Endocrinology 72, 931 (1963). 29. SIMON, C., AND LISSITZKY, S., Biochim. Biophys. Actu 93, 494 (1964). 50. CHEFTEL, C., BOUCHILIAXJX, S., AND LISSITZKY, S., Compt. Rend. Acud. Sci. (Paris)

259, 1458 (1964). 31. MURTHY, P. V. N., RAGUPATHY, E., AND CHAIKOFF, I. L., Biochemistry 4, 611 (1965). 32. MCQUILLAN, M. T., MATHEWS, J. D., AND TRIKOJUS, V. M., Nature 19.2, 333 (1961). 33. PITTMAN, C. S., AND PITTMAN, J. A., Am. J. Med. 40,49 (1966). 34. YAMAZAKI, E., NOGUCHI, A,, AND SLINGIERLAND, D. W., .I. Clin. Endocrinol. 20, 889 (1960). 35. STOLC, V., Endocrinology 71, 564 (1962). 36. AROSENIUS, K. E., &and. J. Clin. Lab. Invest. 16,440 (1964). 37. DIMITRIADOU, A., SUWANIK, R., RUSSEL-FRASER, T., AND PEARSON, J. D., J. Endocrinol. 34, 23 (1966). 38. ERMANS, A. M., KINTHAERT, J., DELCROIX, C., AND COLLARD, J., J. Clin. Endocrinol.

28, 169 (1968). 39. BOUCHILLOUX, S., R,OLL~ND, Biochim. 40. ROLLAND,

M., TORRESANI, J., ROQUES, Biophys. Actu 93, 15 (1964). M., BISMUTH, J., FONDARAI, J., AND LISSIT~KY,

M., AND LISSITZKY,

S., Actn Endocrinol. 286 (1966). 41. AQUARON, R., These de Doctorat en Pharmacie, Marseille, 1967. 42. PIEZ, K. A., AND MORRIS, L., Anat. Biochem. 1, 187 (1960). 43. AQUARON, R., Thke de Doctorat en MCdecine, Marseille, 1969.

S., 53,