I52 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 26044

T H E REACTION OF THIOURACIL W I T H ~-LACTOGLOBULIN

SULFENYL IODIDE

LUDEK JIROUSEK Department of Biochemistry, Vanderbilt University, School of Medicine, Nashville, Tenn. (U.S.A.)

(Received May 29th, I968) (Revised manuscript received August 22nd, I968)

SUMMARY

I. The binding of E14C]thiouracil to fl-lactoglobulin is stimulated by prior t reatment of the protein with 13- or H~O2 plus I- . The highest values are found when all of the thiol groups are converted into sulfenyl iodide groups prior to [14C]thiouracil t reatment and when excess of the oxidant is avoided. The amount of radioactivity bound to the protein corresponds with the stoichiometry of the formation of a mixed disulfide.

2. The product of the reaction of fl-lactoglobulin and thiouracil is decomposed by azide, cyanide, H20~, I- , I~-, sulfite, thiols, thiosulfate and thiourea derivatives.

3. About 60 % of the original thiol groups in /~-lactoglobulin were recovered after t reatment of the mixed disulfide solution with cyanide or mercaptoethanol.

4. H20~ alone does not stimulate subsequent thiouracil binding to fl-lacto- globulin.

5- The stimulation of thiouracil binding by 13- or by H20 ~ plus I - occurs also with ovalbumin but is not observed in proteins which do not form stable sulfenyl iodide derivatives.

6. E14C]Thiouracil can be used as a reagent for the detection of small quantities of sulfenyl iodide groups which have been generated in pure proteins or in tissues.

INTRODUCTION

Reports1, 2 of the preparation of a rather stable sulfenyl iodide derivative of fl-lactoglobulin have made studies of its chemical behavior possible. This derivative reacts at an appreciable rate with non-goitrogenic thiols only when the latter are present in excess; however, with goitrogenic thiourea derivatives it reacts much faster, even in an equimolar ratio 3. These observations and uptake studies of !ssS.I- thiourea and E35Stthiouracil by thyroid in vivo and thyroid slices in vitro have led to the suggestion that the sulfenyl iodide group may be a key intermediate in 13- metabolism 3-5.

The reaction of the fl-lactoglobulin sulfenyl iodide (fl-LGSI) with thiols such as mercaptoethylamine

fl-LGSI + HS-R-+fl-LGS-S-R + H+ + I - (i)

Biochim. Biophys. Acta, 17o (1968) 152-159

THIOURACIL AND SULFENYL IODIDE GROUP 153

has been described and the modified protein isolated s. Upon oxidation by performic acid, the taurine formed accounted for 8o % of the theoretical amount of the mixed disulfide 6. I t was also found that t reatment of fl-lactoglobulin sulfenyl iodide with [14C]thiouracil resulted in formation of a protein derivative containing radioactivity 3, presumably the mixed disulfide, though careful quantitation of this reaction was not attempted.

In the present paper the reaction of fl-lactoglobulin and fl-lactoglobulin sulfenyl iodide with thiouracil is described in more detail and the use of [14C~thiouracil as a reagent for detection of the sulfenyl-iodide group is discussed. Additional information about the chemical behavior of the sulfenyl iodide derivative is also presented.

EXPERIMENTAL

Materials fl-Lactoglobulin was obtained from Pentex Inc. and was stored at --IO °. To

prepare a stock solution a sample was dissolved in o.o5 M NaC1. A small amount of impurity was removed by centrifugation and the supernatant was used. The amount of fl-lactoglobulin (mg/ml) was estimated by reading the absorbance at 280 m/z of a I:iOO (v/v) diluted aliquot and multiplying by IOO and by a factor 1 of I . I I . The stock solution used was freshly prepared or stored overnight on ice.

E14ClThiouracil was obtained from New England Nuclear and contained 1.97 mC per mmole (lot 62-27~3). Sinee~hiouracil solutions are easily oxidized on standing, only freshly prepared solutions were used. A small amount of [14C]thiouracil (about o.I mg) was dissolved in about 5 ml of O 3 free water by the addition of I drop of I M NaOH and used within a few hours. The exact concentration was determined by radioactivity measurements.

Methods All experiments, unless stated otherwise, were performed in an ice bath or in a

cold room at temperatures not exceeding 5 °. Precooled solutions were used. The washing (but not the precipitation) of the samples and radioactivity measurements were performed at room temperature.

In the final step of corresponding experiments the precipitation of proteins was accomplished by addi t ions of 4 ml of an I I % trichloroacetic acid solution to the reaction mixtures. The precipitate was separated by centrifugation at room temper- ature and then washed three times with 2-ml portions of the same solution. The thrice washed precipitates were dissolved in o.i ml of i M NaOH and transferred quantitatively to stainless-steel planchets. The radioactivity was measured in a Nuclear Chicago Scintillation Counter. Corrections were made for self absorption.

Preparation of fl-lactoglobulin sulfenyl iodide solution 0.8 ml of a stock solution of fl-lactoglobulin (38 mg, I #,mole)* was mixed with

1. 9 ml of o.I M sodium phosphate buffer of pH 6.1 and 0. 3 ml of a K I solution of a molarity necessary to make the final I - concentration from o.i /~M to o.i M as required in the various experiments. The final volume was 3 ml. The mixture was

* All calculations are based on a molecular weight for ~-lactoglobulin of 38 ooo and two thiol groups per molecule.

Biochim. Biophys. Aaa, i7o (I968) I52--I59

I 5 4 L. J I R O U S E K

titrated with 0.0I M 12 solution in 0.04 M KI. The titration was performed as described by CUNNINGHAM AND NUENKE 1. From the equivalence point estimated in this titration, the amount of 13- required to convert a /5-1actoglobulin solution to its sulfenyl iodide derivative was calculated. The calculated amount was then added to parallel samples of fl-lactoglobulin solution prepared in the same way as the sample for the titration. In this way fl-lactoglobulin sulfenyl iodide could b6 formed in near maximum yield but an excess of 13 - was avoided. The mixture was allowed to stand for 30 rain to insure completion of the reaction.

Proportionality between sulfenyl iodide formation and []4Cithiouracil binding I ml of fl-lactoglobulin stock solution (41 mg fi-lactoglobulin), 1. 7 ml of o.I M

phosphate buffer of pH 6.I and 0.3 ml of o .o i M KI were mixed and the mixture titrated with o .o i M I2 in 0.04 M KI solution. Absorption at 355 m/~ was measured after successive additions of 13- as described previously 1.

0.5 x

0.3 0.2 / ~ ~ ' ~

OJ • / x x

x

I 2 x 4 5

equlv [21SH

7 ~ 9= .L,=

0 000 ~

8 000 ~ 4C

L 6 0oo =o 3O c 4 000 20

2000 lot 0

o o

/-

5O r60

I00 2OO ~00

O i I

200

400 500 300 nmole$,atGSt 600 nequlv-SI

Fig. I. Titration of f l- lactoglobulin by 13- and parallel b inding of [*4C~thiouracil to formed fl-lacto- globulin sulfenyl iodide. O - - Q , t i trat ion of 1.o8 /~moles of /~-lactoglobulin by 13- fo l lowed by absorbance readings at 355 mp against water; × - - - × , a l i q u o t s ( I / IO) from 13- t i trat ion treated with [14C]thiouracil and the bound radioact iv i ty measured. Detai ls g iven under EXPERIMENTAL.

Fig. 2. E14C]Thiouracil binding to f l- lactoglobulin and fl- lactoglobulin sulfenyl iodide (fl-LGSI) as a function of the quant i ty of protein. Curve a: indicated amount of f l- lactoglobulin in 3 ml of o .I M phosphate buffer of p H 6.1 was treated wi th o . i ml (98.2) m/~moles) I14C]thiouracil for 3 ° min at o °. Curve b : indicated amount of f l- lactoglobulin was converted to f l- lactoglobulin sulfenyl iodide by calculated amount of o .oo i M 13 in 0 .004 M KI in 3 ml of phosphate buffer of p H 6 . i and treated wi th o . i ml (98.2 m/~moles) [14C]thiouracil. Curve c : theoret ical t i tration curve. - S I s tands for sulfenyl iodide group.

In parallel with this titration a sample of the same solution of fl-lactoglobulin was divided into IO parts (o.3 ml each) and increasing amounts of o .ooi M I2 in o.oo 4 M KI added to each aliquot. In the first aliquot I3- was omitted. After standing for 30 min o.I ml of the [14CJthiouracil solution (70 m/~moles, about 5o00o counts/ min) were added and the mixture allowed to stand for another 3o min. The protein was precipitated, washed and the radioactivity measured (Fig. i) .

In related experiments on [14C~thiouracil binding the amounts of fl-lactoglobulin or fl-lactoglobulin sulfenyl iodide and [14CJthiouracil were varied (Fig. 2.) The proteins

Biochim. Biophys. Acta, I7 o (1968) I 5 2 - I 5 9

THIOURACIL AND SULFENYL IODIDE GROUP 155

with bound [x4CTthiouracil were separated from free Ex4Clthiouracil either by precipi- tation with trichloroacetic acid as described above or by fractionation on Sephadex G-25 column (o.8 cm × 40 cm), using o.I M phosphate buffer of pH 6.I for the elution.

Regeneration of the thiol group by treatment of the mixed disulfide of ~-lactoglobulin and thiouracil with KCN or mercaptoethanol

To o.8 ml of a stock fl-lactoglobulin solution containing 76 mg (2 /*moles) of /3-1actoglobulin, i.e., 4/*equiv of -SH, 0.8 ml of o.i M phosphate buffer of pH 6.1 was added and the solution converted into fl-lactoglobulin sulfenyl iodide by addition of 0.4 ml of o.oi M 12 in o.04 M KI (4/,moles of Is). To this solution 0. 4 ml of o.o2 M thiouracil (8/,moles) were added to form the fl-lactoglobulin-thiouracil mixed di- sulfide at a ratio of 2 moles of thiouracil per thiol group. In the control experiment thiouracil was replaced by the same volume of water.

The formed mixed disulfide was separated from excess of thiouracil by column chromatography performed either after the reaction mixture was left standing on ice for IO min or after it was standing on ice overnight.

An aliquot (o.6 ml) of the reaction mixture containing the mixed disulfide was transferred to a Sephadex G-25 column (0.8 cm × 4o cm) and eluted with o.I M phosphate buffer of pH 6.1. The protein peak was pooled and, after addition of KI (2 ml of the pooled fraction + I ml of o.I M KI in o.I M phosphate buffer of pH 6.1) t i trated with 13- as described earlier.

Another aliquot of the same reaction mixture (0.6 ml) was treated by the addition of 25 mg of solid KCN (or 0.05 ml of mercaptoethanol). The mixtures with KCN or mercaptoethanol were left standing in an ice bath overnight and then transferred to the column. After fractionation the regenerated thiol groups in the pooled protein fraction were ti trated by 13- as described above.

I00

"~ 80

,.~ so

g 4.0

"6 zo

o i 6 5 4 3 2 I

-io~ [x-}

Fig. 3. Spl i t t ing off of r ad ioac t iv i ty f rom ~-lactoglobulin-~14C]thiouracil mixed disulfide. A p repa ra t i on of /~-lactoglobul in su l fenyl iodide was t r ea t ed wi th [14C]thiouracil to m a k e t he m i x e d disulfide. To a l iquots of t he resu l t ing solut ion a r eagen t was added to m a k e t h e concen t r a t i on indica ted . P ro te in was p rec ip i t a ted and rad ioac t iv i ty measured . For deta i ls see EXPERIMENTAL. Curve I, th iourea ; cu rve 2, I -me thy l -2 -mercap to imidazo le (Tapazole); curve 3, t h io su lpha t e ; curve 4, azide; curve 5, t h i o c y a n a t e ; curve 6, sulfite; curve 7, cyan ide ; curve 8, iodide; curve 9, H 2 0 ~ ; curve IO, p h o s p h a t e buffer (pH 6.i) ; cu rve I I, Na~SO 4. U n t r e a t e d sample ( reagent replaced by water) = ioo~/o of b o u n d rad ioac t iv i ty .

Biochim. Biophys. Aaa, x7o (x968) I52--I59

156 L. JIROUSEK

Cleavage of the fl-lactoglobulin-[14CIthiouracil mixed disulfide To 0. 4 ml of a stock fl-lactoglobulin solution containing 38 mg of fl-lactoglobulin

(I /,mole) 2.3 ml of o.I M phosphate buffer of pH 6.1 and 0.2 ml of o.oi M 12 in 0.04 M KI was added and the fl-lactoglobulin sulfenyl iodide solution prepared. To this solution o.I ml of I14Clthiouracil (70 m/~moles) was added and allowed to react for 30 rain. The resulting solution containing the mixed disulfide was then divided into IO equal parts (0.3 ml each) and to every part o. 7 ml of phosphate buffer (pH 6.i) (or 7.4) added. Next, I ml of water or I ml of a solution of a reagent having the neces- sary molarity was added to individual parts to make the final concentrations of the reagent as given in Fig. 3- After standing for 30 rain the protein was precipitated with trichloroacetic acid and radioactivity measured as described. No significant difference was observed between the experiments at both pH values.

Influence of the pH on the stability of the mixed disulfide The mixed disulfide, prepared as in the previous experiment, was divided into

equal parts, 0.3 ml each and to each I. 7 ml of a o.I M phosphate buffer in the range of pH 4 to 9 was added. After standing for 30 rain, tile proteins were precipitated and radioactivity measured. The pH did not influence the amount of bound radioactivity in the range of 4 to 8; a slight decrease was found at pH 9.

Stimulation of I14C] thiouracil binding to fl-lactoglobulin by H20 ~ and the e~ect of I 0.3 ml of a stock solution containing 3.8 mg of fi-lactoglobulin were mixed with

0. 7 ml of o.I M phosphate buffer of pH 6.I (or 7.4), I ml of KI solution and I ml of H~O~ solution. In a series of experiments the molarities of both KI and H 2 0 2 solutions were varied in order to make the final concentrations of iodide or peroxide in the reaction mixtures as given in Table I. Peroxide was added as the last component and the reaction was allowed to proceed for 30 rain. In controls, either KI or H~02 or both were replaced by the same volume of water. Then, 7 ° re#moles of !14Clthiouracil in o.I-ml volume were added and allowed to react for another 30 rain. Precipitation and radioactivity measurements were performed as above.

TABLE I

STIMULATION OF ~I4C~THIOURAGIL BINDING TO fl-LACTOGLOBULIN AS A FUNCTION OF H202 AND

I-- CONCENTRATIONS AT p H 6.1 AND 7-4

Samples conta ined o,I /~mole of fl-lactoglobulin in phospha te buffer and final concentra t ions of KI and H~O 2 as indicated, and 5 ° ooo counts /min [14C]thiouracil (0.07 /~moles) in total volume 3.1 ml. Values in phospha te buffer pH 6.1 or (in parentheses) phospha te buffer p H 7.4- Details see under EXPERIMEI,,'TAL.

Final cohen, of H202 (raM)

Final conch.

Counts/min incorporated into fl-lactoglobulin-[14C]thiouracil mixed disulfide at p H 6.I (7.4)

of K I (raM) o o.oi o.Y I Io 500

o 61 (65) 95 (97)

o.oooi 62 (63) o.ooi 78 (99) o.oi 76 (81) o.i 72 (189)

129 (352) IO 136 (3Io)

76 (56) 156 (33) 289 (182) 99 (i2o) 51 (90)

lO3 (91) 155 (174) 3Ol (258) 60 (93) 41 (64) 79 (lO6) 142 (137) 253 (3Ol) 112 (124) 38 (67) 85 (155) 115 (282) 418 (296) 80 (334) 47 (99)

115 (235) 168 (263) 117o (688) 881 (1256 ) 71 (299) 134 (4o5) 579 (513) 3859 (1668) 621 (lO92) 311 (89) 383 (5 ol ) 1437 (iOlO) 1139 (676) 329 (425) 458 (112)

Biochim. Biophys. Acta, 17o (I968) 152-159

THIOURACIL AND SULFENYL IODIDE GROUP 157

Stimulation of [14C~thiouracil binding to various proteins by I a- or a peroxide generating system with I -

Procedure A. 2 mg of a protein were dissolved in o.I M phosphate buffer of pH 7.4- 0.2 ml of o.ooi M I~ in o.oo 4 M KI were added and the volume made to be 2 ml. After 3o rain o.i ml of [x4C]thiouracil (7o m/tmoles) was added for another period of 3 ° rain. Then, precipitation and counting of the samples were performed. In controls, 18 - was replaced with water.

Procedure B. 2 mg of a protein were dissolved in o.I M phosphate buffer of pH 7.4- o.i ml of o.i M glucose, o.I mg of glucose oxidase in o.I ml of water, o.i ml of o.oI M KI and 7 ° m/tmoles of [~4C~thiouracil in o.I ml were added to make the final volume of 2 ml. The mixture was incubated for I h at 37 ° and then the protein precipitated and counted as described above.

RESULTS AND DISCUSSION

After reaction of [x4C]thiouracil with native fl-lactoglobulin a negligible amount of radioactivity (about 0.5% of the theoretical) can be found in the protein. Pre- treatment of fl-lactoglobulin with 18- increases the amount of bound radioactivity. The increase is in proportion to the amount of sulfenyl iodide formed and the highest values of bound thiouracil were found at the equivalence point of the reaction of fl-lactogiobulin with I8-. At that point all the thiol groups are converted into sulfenyl iodide groups and the reaction mixture does not contain an excess of Ia-; the latter apparently leads to oxidative cleavage of the radioactive product (Fig. I).

In experiments in which I /~mole of fl-lactoglobulin (2 ~uequiv of -SH) was converted to sulfenyl iodide derivative and the latter reacted with o.4 /umoles of [14C]thiouracil, and then the reaction mixture fractionated over a Sephadex G-25 column, 92 % of the added [14CJthiouracil was found in the protein peak and no free thiouracil has been recovered. In similar experiments 0.5 /~mole of fl-lactoglobulin was changed to sulfenyl iodide derivative and then reacted with 1.25 ~umoles of E14C] - thiouracil; after fractionation on Sephadex G-25 column the radioactivity bound to the protein fraction amounted to 87-114% of the theoretical. Excess of thiouracil formed a separate peak but only a part of it was recovered. Considering experimental error in radioactivity measurements and in recovering a substance on a Sephadex column the results can be interpreted as quantitatively corresponding to the stoichio- metry of Eqn. 2

fl-LGSI + HS-uracil-+fl-LGS-S-uracil + H + + I- (2)

where ~-LGSI stands for fl-lactogiobulin sulfenyl iodide. After isolation by ocolumn chromatography of the protein from the reaction

mixture of fl-lactoglobulin sulfenyl iodide and thiouracil (I sulfenyl iodide group: 2 thiouracil), the protein was t i trated with Ia-. The consumption of 13- was found to be only about 8 % of that of native fl-lactogiobulin. When the reaction mixture was treated with KCN prior to column chromatography the consumption of 13- due to recovery of thiol group from the disulfide group increased to 56 (45-66)%. Using mercaptoethanol in place of cyanide resulted in 6o% recovery of the thiol group. Although optimal conditions for the cyanide or mercaptoethanol treatment were not studied in more detail it is apparent that the conversion to fl-lactoglobulin sulfenyl

Biochim. Biophys. Acta, 17o (1968) 152-159

I58 L. JIROUSEK

iodide and subsequent binding of thiouracil modify the thiol group of fl-lactoglobulin and protect it from reacting with low concentrations of I s . Cyanide and mercapto- ethanol regenerate the protein thiol group. Cyanide, mercaptoethanol and other reagents such as sulfite, thiosulfate, thiols and thiourea derivatives, known to be able to split disulfide bonds, also released the radioactivity from the product of reaction of fi-lactoglobulin sulfenyl iodide and [14C]thiouracil. The results of these cleavage experiments (Fig. 3) indicate that the product is a mixed disulfide. Other anions, such as thiocyanate, azide and I - were also effective in splitting off the radioactivity from the mixed disulfide. Cleavage of a sensitive disulfide bound by iodide has been suggested by MALOOV AND SOODAK in a thyroidal enzyme metabolizing thiourea 4.

Our s tudy of the reaction of fl-lactoglobulin sulfenyl iodide with thiouracil was intended to see if [a~C]thiouracil can be used as a reagent for detection of sulfenyl iodide groups in tissues and biological preparations. However, such preparations as the thyroidal subcellular fraction containing the iodinating system cannot be fraction- ated on gel columns. Therefore, after t reatment of such a fraction with [14C]thiouracil, the excess of radioactivity cannot be removed by gel fractionation. Neither can gel fractionation be conveniently used in a large series as for example in cleavage experi- ments (Fig. 3). I t was found convenient to precipitate the labeled proteins and remove the free [14C 1 thiouracil by washing with trichloroacetic acid solution. However, this is not without problems. As evident from experiments with the disulfide of fi-lactoglobulin and [14Clthiouracil, trichloroacetic acid precipitation removes a considerable part of the bound radioactivity. The yields of the labeled protein in various experiments were only 5-15 % of the theoretical.

However, the decrease in the yield is proportional to the calculated amount of the mixed disulfide formed in the reaction mixture, as can be seen in Fig. 2. A con- s tant amount of [14C]thiouracil was treated with increasing amounts of /3-1acto- globulin in various experiments and maximal values of bound radioactivity were :reached at the equivalence point of Reaction 2. Similarly, when a constant amount of fl-lactoglobulin sulfenyl iodide was treated with increasing quantities of [14C~- thiouracil, the maximum of bound radioactivity was reached at a ratio of i mole of thiouracil per I sulfenyl iodide group.

Precipitation of the protein with trichloroacetic acid to remove the unreacted !14CIthiouracil diminishes the sensitivity of an assay for sulfenyl iodide groups but in many experiments where quantitation is not of pr imary importance it can still be used for qualitative detection, offering the advantages of time-saving and less com- plex experiments.

As suggested earlier by CUNNINGHA.'a and co-worker 3,7 and by MALOOV AND SOODAK a, the sulfenyl iodide group may be involved in enzymatic iodination. I t is known (see refs. 4 and 8 for review of the literature) that the iodinating activity of an active preparation can be detected in the presence of small quantities of I - and HzO2. If sulfenyl iodide groups can be generated by the action of H202 and I - under similar conditions, [14C]thiouracil binding to fl-lactoglobulin should be stimulated by small quantities of peroxide and I- .

In corresponding experiments the binding of [14CJthiouracil to/5-1actoglobulin was stimulated by H202 and I7 (Table I), and also by a peroxide generating system (glucose and glucose oxidase) and I , as well as by Is pretreatment. In the absence of I- , H~O2 alone did not significantly stimulate the binding of [14C!thiouracil to

Biochim. Biophys. Acta, 17o (1968) ~52-159

THIOURACIL AND SULFENYL IODIDE GROUP I59

fl-lactoglobulin. Increasing concentrations of I-, in absence of peroxide, had also no significant influence on E14Clthiouracil binding. Maximal values were obtained when the concentrations of I - and peroxide were both I raM.

In a reaction of the thiol group with either iodine or peroxide, intermediary oxidation forms such as thiol radical, a sulfenyl cation or a sulfenic acid might be formed by the oxidant and then react with I- to form the sulfenyl iodide. They might react also, directly, with the thiouracil to form the mixed disulfide. These reactions, although not completely excluded in our study, are quantitatively not significant as compared with the binding of thiouracil by the fl-lactoglobulin sulfenyl iodide (Table I). The indispensability of I- is a further evidence for the significance of the sulfenyl iodide group in the thiouracil binding. Finally, for the detection of a sulfenyl iodide group in general, a test based on the different reaction rates of thiols and thioureylenes with the sulfenyl iodide group, as reported previously with fl-lacto- globulin sulfenyl iodide 3, can be used.

Of the proteins examined (fl-lactoglobulin, ovalbumin, bovine 7-globulin, yeast-alcohol dehydrogenase, human serum albumin, trypsin and chymotrypsin) only with fl-lactoglobulin and ovalbumin was the stimulation of [14C]thiouracil by Ia-, or by enzymically generated H20 2 plus I-, observed. The other mentioned proteins are known to be unable to form the sulfenyl iodide derivatives.

Summarizing our data we conclude that the [l*C]thiouracil can be used as a reagent for the detection of small quantities of sulfenyl iodide groups in proteins and tissues. These can be generated in a suitable protein and presumably in tissues, in- cluding the thyroid, by I 3- pretreatment, as described previously 1, by H20 * and iodide or by a peroxide generating system, consisting of glucose and glucose oxidase~ and I-.

ACKNOWLEDGEMENTS

The technical assistance of Miss PATRICIA ESTES is gratefully acknowledged. The work has been supported by a grant from the American Cancer Society, No. P-39 o. The author is indebted to Dr. L. W. CUNNINGHAM for his advice and help during the performance of this work and the preparation of this manuscript.

R E F E R E N C E S

I L. W. CUNNINGHAM AND B. J. NUENKE, J. Biol. Chem., 234 (1959) 1447. 2 L. W. CUNNINGHAM AND B. J. NUENKE, J. Biol. Chem., 236 (1961) 1716. 3 L. W. CUNNINGHAM, Biochemistry, 3 (1964) 1629. 4 F. MALOOF AND M. SOODAK, Pharmacol. Rev., 15 (1963) 43. 5 F. MALOOF AND M. SOODAK, Endocrinology, 61 (1957) 555. 6 L. W. CUNNINGHAM AND S. J . NUENKE, J. Biol. Chem., 235 (196o) 1711. 7 L. W. CUNNINGHAM AND B. J . NUENKE, Biochim. Biophys. Acta, 39 (196o) 565. 8 A. TAUROG AND E. 1V[. HOWELLS, J. Biol. Chem., 24I (1966) 1329.

Biochim. Biophys. Acta, z7o (1968) 152-159