THE JOUBNAL or Bro~oox!~. Cx~xlerar Vol. 237, No. 11. November IQ62

Ptinfd in U.S. A.

Properties of Glutathione Insulin Transhydrogenase

from Beef Liver*

HENRY H. TOMIZAWA

From The Fels Research InAtute, Yellow Springs, Ohio

(Received for publication, April 9, 1962)

The glutathione-insulin transhydrogenase from beef liver haa been found to promote the reductive cleavage of the disulfide bonds of insulin by simple sulfhydryl compounds such as gluta- thione (2). The properties of this enzyme are of interest not only because of its probable involvement in the biological regu- lation of the level of insulin but also because, at present, it is the only isolated mammalian enzyme that can catalyze the re- ductive cleavage of a protein.

The present paper describes some of the properties of the beef liver glutathione-insulin transhydrogenase.

EXPERIMENTAL PROCEDURE AND REBULTS

Chemically reduced TPNH, GSSG, glutathione reductase (6000 Racker units per mg), Tris, and five times crystallized bovine pancreatic ribonuclease were obtained from the Sigma Chemical Company. ATE’ was purchased from Mann Re- search Laboratories, and n-homocystine from General Bio- chemicals, Inc. Crystalline preparations of insulin and glucagon were gifts from Dr. 0. K. Behrens and Dr. W. W. Bromer of the Lilly Research Laboratories, and insulin-Pa1 was obtained from Abbott Laboratories. Purified synthetic oxytocin (450 i.u. per mg) was kindly supplied by Dr. R. Bircher of Sandoz Pharma- ceuticals, and highly purified preparations of ovine prolactin and bovine growth hormone were gifts of the Endocrinology Study Section of the National Institute of Health.

The assay for enzymatic activity employing insulin-Ii51 was developed earlier for use in the purification of the enzyme (3) ; occasional modifications of the assay procedure are appropriately noted in this paper. In a 2-ml incubation system in 0.1 M po- tassium phosphate, pH 7.5, with 5 X 10J M EDTA, the sub- strate consisted of insulin and a trace amount of insulin-Pal; the degradation of insulin was determined by measuring the amount of radioactivity solubilized in 5% trichloroacetic acid. The peptide moiety of the soluble radioactivity was recently shown to be the A chain of insulin (2).

The substrate specificity of the enzyme was investigated by substrate competition studies (4) and by the p-chloromercuri- benzoate titration of the newly formed protein sulfhydryls, ac- cording to the procedure of Narahara and Williams (5).

Requimment for Simple Sdfhydryl Compound-The absolute

* Supported in part by a research grant (A-3854) from the National Institute for Arthritis and Metabolic Diseases. United States Public Health Service. While this manuscript’ was in preparation, Katzen and Stetten (1) published an abstract which confirmed the conclusions drawn concerning the mode of action of the enzyme (2). They also reported several observations which are similar to those described in the present paper.

1 The abbreviation used is: ATE, acetyl-L-tyrosine ethyl ester.

requirement for a simple sulfhydryl compound such as GSH is shown in Table I. It can be seen that the reductive cleavage of insulin can occur chemically with increasing concentrations of GSH; therefore, the optimal concentration of GSH, which is probably in the range of lQd M, will@ be difficult to establish exactly. 2-Mercaptoethanol is also an excellent hydrogen donor and was used as such in the previous studies which involved characterization of A chain aa a product of the enzymatic reac- tion.

Efect of TPNH-Narahara and Williims (5) obtained evi- dence with rat liver preparations that suggested that the reac- tion catalyzed by glutathione reductase (i.e. GSSG + TPNH + H+ + 2GSH + TPN+) is coupled to the reaction catalyzed by an insulin-reducing enzyme between the GSH formed and insulin. The involvement of TPNH was therefore investigated with the isolated enzyme. Table II shows the results of a study in which degradation of insulin-In1 was measured, and Fig. 1 gives the results of an experiment in which utilization of TPNH was followed. These data indicate that TPNH does not directly reduce insulin but functions in the glutathione re- ductase-catalyzed reaction for the generation of GSH, the com- pound which is directly involved in the reduction of insulin.

Heat Stability of Enzyme-Although it is inactivated by heat, the enzyme has some heat stability, and retains about one-third of its activity on exposure for 30 minutes to a temperature of 30” (Table III). Undoubtedly, the moderate heat stability of the enzyme was a major contributing factor in making its puri- fication possible.

pH Optimal Range-The reaction between GSH and insulin probably proceeds in a two-step exchange reaction involving GSH as GS-, a mercaptide ion (6); such chemical reactions usually proceed faster with increased alkalinity of the medium. To keep the nonenzymatic (control) reaction minimal at the higher ranges of pH, the concentration of GSH employed was one-tenth that of the usual (i.e. lo-‘ M instead of lOma M). The pH optimum range is roughly 7.5 to 8.5, as shown in Fig. 2.

Competition Studies-In these experiments, compounds were tested for their effect on the rate of degradation of insulin-1’31 by the GSH-insulin transhydrogenaae system. The assay was performed by the procedure described earlier (4) with only one modification, the use of 0.04 mg of carrier insulin instead of 0.1 mg per tube. When the enzyme concentration is a limiting factor, a sub&rate or inhibitor has the effect of decreasing the rate of degradation of insulin-1”‘.

In Fig. 3 are plotted the results of competition studies with n-cystine, n-homocystine, glucagon, and ATE when their effects are compared with the effect of additional insulin. ATE was

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3394 GSH-Insulin Transhydrogenase Vol. 237, No. 11

TABLE I same type of competition study, with use of 6.65 x 10” pmole of Effect of GSH on activity of GSH-insulin tranahydrogenase each. The order of effectiveness was as follows: added insulin

The assay system with use of insulin-IiS’ was described earlier (3.3) > added ribonuclcasc (5.4) 2 added oxytocin (5.6) > (3); the only modification in this experiment was the variation added prolactin (7.9) > substrate alone (9.6). The percentage in the concentration of GSH.

Final cp&ratioa Control supmatant Net supematant radioactivity radioactivity*t

Y % kkl n3dioactia1y* %

0 0 0 1 x lo-’ 0 0.6 5 x lo-’ 0.3 3.4 1 x 19-a 0.8 4.7 5 x.10-s 3.7 4.1 1 x lo-’ 5.5 1.2

Spxilic activity$

0 428

2428 3357 2928

857

* Corrected for approximately 2% trichloroacetic acid-non- precipitable radioactivity in the insulin-1”’ preparation.

t Net aupernatant radioactivity = y. supernatant radioactiv- ity produced in an enzyn%tic system minus r. supernatant radio- activity produced in the control or nonenzymatic system (the values of Column 2).

$ Specific activity = net supernatant radioactivity per mg of ensyme protein. Specific activity values vary with the insulin- II*1 preparation used; for experiments requiring comparative values, the same batch of insulin-Par was employed for a given series.

TABLE II

E$ect of TPNH, GSSG, and glutathione reductaee on activity of GL’H-insulin ttanahydrogenme

The insulin-Pai assay was performed as previously described (3)) except that substitutions for GSH were made as indicated.

Part GSH TPNH GSSG chd~lutil Specific activity

rmk 1 2 2 0 3 0 4’ 0 5* 0

pm& ,umok

0 0

i 0 0.1 2 0.1 2 0.1t

UNik

0 0 0 0

2406

2980 0 0 0

1148

* The components listed were preincubated together for 30 minutes at 37” in the usual buffer system to promote conversion of GSSG to GSH.

t A comparatively low concentration of GSSG was used in the presence of excess TPNH and glutathione reductase since con- tinual regeneration of GSH was expected. On reaction of the resultant GSH with insulin, one of the products was probably GSSG which then would have been a source of more GSH. For the sake of uniformity, the same concentration of GSSG was also used in Parts 3 and 4.

found by Williams et crl. (7) to be effective in competition studies with a crude preparation of the enzyme. Cystine and homo- cystine are neither good substrates nor good inhibitors. ATE, an excellent substrate for chymotrypsin, proved to be an in- hibitor, since a competitive &ect was noted, but no hydrolysis of this ester by the GSH-insulin transhydrogenase could be de- tected when the reaction was carried out in a pH-Stat. Gluca- gon must also be an inhibitor of the enzyme since it contains no disulfide linkages.

Ribonuclease, oxytocin, and prolactin, each a disulfide-contain- ing peptide or protein, were also compared with insulin in the

of radioactivity in the trichloroacetic acid supernatant after the 5minute enzymatic incubation, comparable to points plotted on Fig. 3, is indicated in parentheses.

Protein Sdfhydyl Assay-Since the competition method does not distinguish between substrates and inhibitors, the extent of enzymatic conversion of the disulfides to sulfhydryl groups was determined with the three previously mentioned proteins by ti- tration with p-chloromercuribenzoate (5). Unfortunately, oxy- tocin cannot be assayed in this way since the procedure involves trichloroacetic acid precipitation after the reaction with GSH. The protein sulfhydryl value for the reduction products of insulin

INCUBATION TlYl? (min.1

FIG. 1. Oxidation of TPNH. At zero time, the silica cuvette contained 0.2 pmole of chemically reduced TPNH and 0.6 wale of zinc-free insulin (2) in a total volume of 3 ml in the usual phos- phate buffer system. Additions to this solution at the indicated times were as follows: 0, 4 rg of GSH-insulin transhydrogenaae; @,0.5 mole of GSSG; and 0, 126 units of glutathione reductase. Corrections in optical density readings were made for the volume changes resulting from these additions. Temperature within the Beckman model DU spectrophotometer cell compartment was 29”.

TABLE III

Effect of temperature on stability of GSH-insulin transhydrogenase

Before the assay with insulin-I’S’, the enzyme was exposed to the temperatures indicated for 30 minutes.

Temperature Specific activity Yield

Yz x 1G-: %

0 2.9 196 25 2.9 106 37 2.9 196 60 1.9 66 80 1.0 34

106 0 0

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November 1962 H. H. Tomizawa 3395

L 6.50 675 700 725 7.50 7.75 8.00 825 8.50 875 9.00

PH Fxo. 2. The effect of pH on the insulin-Psi-degrading activity

of GSH-insulin transhvdronenase. Since lO+ M GSH was used ” I

instead of 10-a M (see text), the specific activity values are con- siderably lower than usual and have been plotted on an arbitrary scale of 0 to 8.. The buffer used throughout the entire pH range was 0.1 M potassium phosphate with 5 X lea M EDTA.

would be low because of loss of reduced A chain into trichloro- acetic acid. Such loss does not occur with ribonuclease (8) and prolactin (Q), both of which are larger than insulin and are single chains with intrachain disulfides only. Table IV shows results of the sulfhydryl azzay. Although both contain three disulfide bonds, insulin is a much better substrate than pro- la∈ the value for aulfhydryls formed from insulin is about 2.5 times greater than for those formed from prolactin, even without taking into account the loss of reduced A chain of insulin during trichloroacetic acid precipitation. Ribonuclease is not a substrate, so ita effect in the competition study was inhibitory only.

DISCUSSION

The results of the experiments concerned with the general properties of the transhydrogenase were not unexpected in view of the fact that this enzyme promotes a reaction between GSH and insulin. The requirement for a simple sulfhydryl compound as a reactant, however, does not exclude the possibility that the enzyme may only be active in a sulfhydryl form; this possibility has not been investigated. An inhibitory effect of Hg* and Cu+* at concentrations of less than 1P M was observed with the enzyme system. This was not surprising since these cations readily react with sulfhydryl groups.

The results of studies with TPNH indicate that the isolated

proposal of Narahara and William that the “insulin-reductase”- catalyzed reaction is coupled to that promoted by glutathione reductase (5).

The finding that the simple disulfides cystine and homocystine are poor either as substrates or as inhibitors for the GSH-in&ii

IO

9U69lRATE+pfCMOOSTlNC

9 6wrrrATE+&x3TlNE

I- 6

E

9usSTRATE+*TE

g7

2 WBSlRAlE+AlE -LX

l 6 :! SU66lRAT2 + OLUCA6ON z

ii 5

2 9

%’

2

s 3 6MSTNAT(: + AKilWNAL

2 INSULIN

t 0 2

0 3 s

INOUMTION TM? th)

Fro. 3. In the GSH-insulin transhydrogenase system, the com- petitive effects of L-cystine, L-homocystine, ATE, glucagon, and additional unlabeled insulin on the rate of degradation of a sub- strate which consisted of 0.04 mg of insulin with a trace of insulin- Par; 0.65 X 10-2 mole of each of these substances were used. ATE-$X indicates the use of twice the usual amount or 1.3 X 10-l pmole of this ester; L-cystine and L-homocystine at this higher concentration still did not exhibit competitive effects. Some 3-minute values were plotted to indicate the linearity of the rate of reaction over the b-minute period. Each point indicates the average value of a determination performed in triplicate.

TABLE IV

p-Chloromercuribenzoate assay for protein sulfhydryla formed from disuljide-containing proteins in GSH-insulin

trarwhydrogenase system

Procedure was ae described by Narahara and Williams (5), except that the phosphate buffer system was used instead of glycylglycine buffer. In the absence of and in the presence of the enzyme, 0.3 pmole of each protein was incubated anaerobically for 15 minutes with 8 rmoles of GSH.

System EllZyllX

PS

0 80 0

80 0

80

-

_. pmole

Ribonuclease 0 Ribonuclease 0 0 Prolactin 0.01 Prolactin 0.06 0.05 Insulin 0.01 Insulin 0.13 0.12

l Net trichloroacetic acid-precipitable sulfhydryl formed - protein sulfhydryls formed in the enzymatic system minus those produced in the nonenzymatic systems (values in horizontal

enzyme is not a glutathione reductase. These data support the Rows a, c, and e).

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3396 GSIi-Insulin Transhydrogenme Vol. 237, No. 11

transhydrogenase suggests that a substrate must contain peptide linkages. Also, the lack of effect of homocystine distinguishes the GSH-insulin transhydrogenase from GSH-homocystine transhydrogenase, a beef liver activity described by Racker (10). The inhibition of the enzyme by ATE, a synthetic substrate for rhymotrypsin, also suggests that certain peptide bonds of a sub- strate are involved in the binding of substrate to the enzyme.

The inhibitory effect of glucagon may have physiological sig- nificance. At a time of relatively high concentration of estra- pancreatic glucagon with resultant hyperglycemia, some sparing of insulin from degradation because of glucagon inhibition of GSH-insulin transhydrogenase might possibly occur. Such an inhibition would assist in the maintenance of a higher conccn- tration of insulin to produce the beneficial effect of utilization of the increased available glucose. The reverse effect has been noted with liver slices in which insulin effectively inhibited the degradation of glucagon so that a more pronounced glycogeno- lytic effect was produced by a given amount of glucagon (11, 12).

Inhibition of GSH-insulin transhgdrogenase by pancreatic ribonucleaae is of doubtful physiological significance. It was of considerable interest, however, that ribonuclease, which contains four disulfide bonds, is not a substrate. This may be because ribonuclease is a tightly coiled molecule with none of its four disulfide linkages readily accessible for reductive cleavage (8). Demonstration that prolactin is a substrate shov.s that insulin, at least in vitro, is not the only substrate, although it is a much better one, comparatively. The results of experiments with bovine growth hormone, which xas not extensively investigated because of its poor solubility, suggest that growth hormone is comparable to prolactin as a substrate. The competitive effect of oxytocin and the known ease of chcmi- cal reduction with resultant inactivation of this peptide (13) sug- gest that oxytocin, and probably vasoprcssin, may be physio- logical substrates for the enzyme. If this is true, insulin still is probably a more important substrate for the hepatic GSH- insulin transhydrogenase since, unlike the posterior pituitary hormones, newly secreted insulin is transported directly to the liver via the pancreatic to portal venous route.

As proposed by Katzen and Stetten (l), GSH-insulin trans- hydrogenase is most appropriate as a name for this enzyme. The mode of action of the enzyme is similar to that of the en- zyme which Racker (10) designated as GSH-homocystine trans- hydrogenase. Also, demonstration that the “insulin-reductase”

and the glutathione reductase make up a coupled system makes inclusion of GSH in the name quite fitting.

SUMNARY

Some of the properties of the beef liver glutathione-insulin transhydrogenase have been described. Evidence is presented which indicates that the reaction catalyzed by this enzyme is coupled to the reaction promoted by glutathione reductase. Included are data that indicate that the transhydrogenase is not a nonspecific disulfide-reducing enzyme but one with some degree of specificity for insulin.

~lcknorxledgments-The early phase of this investigation was performed in the laboratories of Dr. Robert H. Williams at the University of Washington. I wish to thank him for his con- tinued encouragement and to acknowledge the technical assist- ance of Mrs. Ann Harmon, Mrs. Adele Hopkins, and Mrs. Pearl Forth. Thanks are due to Mrs. Riaryan Stanfield and Mr. Matthew Lake for technical assistance at the Fels Research Institute.

REFEREENCES

1. KATZES, H. RI., ASD STETTER, D., JR., Federation Proc., 21, 201 (1962).

2. TO~IZAWA, H. H., J. Biol. Chem., 237,428 (1962). 3. TONIZAWA, H. H., AXD H.~LSEY, Y. D., J. Biol. Chem., 234,

307 (1959). 4. TOMIZAWA, H. H., ASD WILLIAYS, R. H., J. Biol. Chem., 217,

685 (1955). 5. NARAHARA, H. T., .4SD WILI.I?L~~S, R. H., J. Biol. Chem., 234,

71 (1959). 6. CECIL, R., AKD XICPIIEE. J. R., in C. F3. AXFINSEW, JR., M. L.

Axs&N,‘K. BAILEY, a&~ J. T. EDSALL (Editors), Aduances in protein chemistry, Vol. 14, Academic Press, Inc., New York, 1959, p. 255.

7. WILLIAIIS, R. H., M?LRTIN, F. B., HENLEY, E. D., AND SWAS- sm. H. E.. Metabolism. 3. 99 (19593.

8. SI~A&NAS, 6. H., STEI;, k. G., AND MOORE, S., 3. Biol. Chem., 236, 648 (1960).

9. LI, C. H., J. Biol. Chem., 229, 157 (1967). 10. RACKER, E., J. Biol. Chem., 217, 867 (1955). 11. VUYLSTEKE, C. A., AND DE DUVE, C., J. physiol. (Paris), 47,

308 (1955). 12. TYBERGHEIN, J. M., TOYIZAWA, H. H., AND WILLIAMS, R. H.,

J. Biol. Chem., 222. 945 (1956). 13. AUDRIAN, L., AND CLAUSER, H., Biochim. et Biophys. Acta, 30,

191 (1958).

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Henry H. TomizawaProperties of Glutathione Insulin Transhydrogenase from Beef Liver

1962, 237:3393-3396.J. Biol. Chem. 

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