CYSTINE AND GLUTATHIONE REDUCTASES IN THE CLOTHES BISSELLIELLA By R. F. POWNING* and H. IRZYKIEWICZ*

, [Manuscript received October 27, 1959]

Summary The of the possesses an enzyme which catalyses the reduction of L-cystine by reduced triphospyridine nucleotide (TPNH). Extracts of whole larvae reduce up to 14 pmoles of cystine per g larvae per hr at pH 7· 3. Reduced diphosphopyridine nucleotide (DPNH)-linked cystine reductase and DPNH- and TPNH-linked glutathione reductase of somewhat lower activity are also present in the clothes moth extracts.

I. INTRODUCTION The reduction of the disulphide bonds of wool has been shown to increase its susceptibility to digestion by proteolytic enzymes (Geiger et al. 1941) and it was suggested by Linderstr0m-Lang and Duspiva (1936) that a reductive break of disulphide bonds may be a necessary prerequisite for digestion of wool by . Since thiol compounds are efficient reducing agents of disulphide bonds in wool (Geiger, Kobayashi, and Harris 1942) clothes moth larvae were examined for enzymic activity which would produce thiols by reduction of disulphide com­ pounds. Enzymic reduction of disulphide bonds in biological materials is now known. Glutathione reductase was described in peas (Mapson and Goddard 1951) and in wheat (Conn and Vennesland 1951), and cystine reductase was found in peas and yeasts (Nickerson and Romano 1952). There is also a protein disulphide reductase known (Nickerson and Falcone 1956). A preliminary note on a reduced triphosphopyridine nucleotide (TPNH)­ linked cystine reductase in clothes moth larvae has already appeared (Powning and Irzykiewicz 1959) and further details of cystine and glutathione reductases in this are presented in this paper.

II. METHODS AND MATERIALS The insects used in this work were Tineola bisselliella (Humm.) larvae about 4 weeks old, bred on a diet of casein containing 3 per cent. yeast. Batches of larvae were homogenized in a "Virtis 45" homogenizer in 4 volumes of water or 0·05M tris (tris(hydroxymethyl)aminomethane) buffer adjusted with HCI to pH 7 '3, and centrifuged at 30,000 g for 30 min before use. Dialyses were carried out against 25 volumes of the same buffer for 20 hr with one or two changes of buffer. All

* Division of Entomology, C.S.I.R.O., Canberra. 60 R. F. POWNING AND H. IRZYKIEWICZ operations were carried out in the cold and the extracts were used as soon as possible after preparation. The activity of disulphide bOhd reductases was measured in three ways: (1) the disappearance of reduced diphosphopyridine nucleotide (DPNH) and TPNH was measured at 340 mIL, in special cuvettes for anaerobic conditions, in a Beckman model DU spectrophotometer. The increase in -SH groups was estimated either (2) by a modification of the Grunert and Phillips (1951) colorimetric nitro­ prusside method, or (3) by a titrimetric method similar to that of Katchalski, Benjamin, and Gross (1957), using phenyl mercuric nitrate. Reactions were carried out at 25°0 under anaerobic conditions.

TABLE 1 CYSTINE REDUCTASE IN NON-DIALYSED TINEOLA EXTRACT Reaction mixture contained 1 ml Tineola extract, 1 ml 0 ·125M tris-HCI pH 7· 3, plus additions as indicated; final volume 2· 5 ml. Incubated for 2 hr

--"'------~~"------.~ ------.. --"-.~----- fLMoles Cysteine

Colorimetric Method Titrimetric Method Additions (fLmoles)

Total Increment Total Increment .------

Boiled extract Nil 0·91

L-Cystine (4·2) 1·25 0·34 I

Fresh extract I

DPN (2·9) 1·14 o 1·75 o DPNH (2·2) 1·14 o 1·64 o L-Cystine (4, 2) 3·52 2·38 4·16 2·41 L-Cystine + DPN (2·9) 5·04 3·90 5·90 4·15 L·Cystine + DPNH (2, 2) 2·13 0·99 2·63 0·88

The values in Table 1 illustrate slight differences between the results from the two methods of -SH estimation. The titrimetric method measures total -SH groups in the reaction mixture, and therefore in the presence of boiled enzyme there was not any increase of -SH groups on addition of cystine. The colorimetric method measures only -SH soluble in the metaphosphoric acid reagent, and the increase of cysteine in the boiled enzyme mixture is explained by the non-enzymic reduction of a little cystine by protein-bound -SH groups. Although the values for the basic reaction mixture from the titrimetric method are higher than those from the colorimetric method (due to protein-bound -SH groups) the net increase of -SH due to reductase action is about the same in both methods. There is a small amount of sulphide produced in the reaction mixtures, probably from cysteine (Powning 1954), and this is not estimated by the colorimetric method. In most -S-S- REDUCTASES IN THE CLOTHES MOTH 61 experiments the colorimetric method was used; however, many confirmatory tests were done with the titrimetric method. The dehydrogenase substrates glucose 6-phosphate and malate were provided in these experiments only in amounts sufficient for the hydrogen transfer reaction, as higher amounts were found to inhibit the cystine reductase.

"2

,'0

0'8 ..z iii lii >- ..U 0'6 "'oJ o :IE ;\.

0'4

0·2

0 1 I 5 6 7 8 9 pH

Fig. I.-Effect of pH on cystine reductase in non-dialysed Tineola extract. Reaction mixtures: 1 ml Tineola extract, 4·2 pmoles L-cystine; final volume 2·5 ml. Incubated 2 hr. -SH estimated by the colorimetric method and the results corrected for -SH produced in controls without cystine. Buffers (final concn.): • O'125M Na2HP04-HCI; 0 O'125M Na4P207-HCI; • O·04M barbitone-HCI; X O'125M tris-HCI.

Chemical reagents used in this work included diphosphopyridine nucleotide (DPN). (Sigma "90"); triphosphopyridine nucleotide (TPN), 84 per cent. (Sigma); DPNH, 53 per cent. (Sigma); TPNH, 50 per cent. (California Foundation for Biochemical Research); malic acid (B.D.H.); trisodium DL-( +allo)isocitrate (C.F.B.R.); glucose 6-phosphate, disodium salt (Sigma); sodium

III. RESULTS (a) Non-dialysed Extracts (i) Effect of pH on Cystine Reduction.-The enzymic reduction of cystine by non-dialysed extracts of Tineola larvae was found to have an optimum pH of about 7·3 (Fig. 1). Phosphate and pyrophosphate caused considerable inhibition of reductase activity and their further use was avoided. Tris buffer at pH 7·3 was used for all experiments.

'·0 .,

} ~., ~ ., " '" 0'6 I-« z '(...... , ...... CONTROL o ;:: ~ 0-4 ----.--.-.-.-. o Inm « ~ 0·2 "- CYSTINE ...... ~ ...... J o I 60I eoI 100I 120 I 2'0 40 TIME (MIN)

Fig. 2.-Cystine reductase in non-dialysed Tineola extract. Reaction mixtures: 1· 4 ml Tineola extract, 0·54 /Lmole DPNH, 2 ml 0 . 125M tris-HOI pH 7·3; final volume 3·5 ml. 2·1 /Lmoles L·cystine added where indicated. The reaction was carried out in evacuated Beckman cuvettes.

(ii) Effect of DPNH.-The cystine reductase of pea seeds and yeasts is DPNH-specific (Nickerson and Romano 1952), and insect tissues were examined for similar activity. A non-dialysed extract from Tineola larvae reduced added cystine; however, the addition of DPNH to this reaction mixture resulted in a strong inhibition of reduction and DPN addition caused an activation. The addition of DPN and DPNH to the enzyme without cystine had no significant effect on -SH production. Boiled enzyme was inactive (Table 1). Spectrophotometric tests of the oxidation of DPNH by Tineola extract revealed an activation of this reaction on addition of cystine (Fig. 2). Calculations from the net decrease of absorption at 340 mfL of the mixture containing cystine show that 0·16 fLmole DPNH was used per ml of enzyme and this is equivalent to 0·32 fLmole cysteine produced. This is only a fraction of the amount of cysteine which is actually formed with the same enzyme preparation under similar con­ ditions (Table 1). Studies with dialysed preparations indicated that endogenous activity of TPNH-linked cystine reductase accounted for this discrepancy. -S-S- REDUCTASES IN THE CLOTHES MOTH 63

(b) Dialysed Extracts Attempts to purify the enzyme by acetone and ammonium sulphate precipi­ tation led to greatly decreased activity_ However, quite consistent results were obtained with fresh extracts dialysed and used without further treatment or storage_

TABLE 2

DPN- AND TPN-LINKED DEHYDROGENASES IN DIALYSED TINEOLA EXTRACT Reaction mixture contained 0-05 ml dialysed extract, 0-05M (final concn_) tris-HCl pH7-3 or 9-1, 0-2pmole DPN or TPN, 24JLmoies substrate; final volume 1 -2 mI_ 1 unit of activity = change of absorption of 0 -001 per min at 340 mJL

Activity (units/ml enzyme)

pH7-3 pH9-1 Substrate

DPN TPN DPN TPN

Lactate 720 44* 360 0 Glutamate 0 0 9* 0

. -~-----~----.-- * 0 -5 ml dialysed extract_

TABLE 3

CYSTINE REDUOTASE IN DIALYSED TINEOLA EXTRAOT Reaction mixture contained 1 ml dialysed extract, 1 ml 0 -125M tris-HCI pH 7 -3, and 0-1 pmole DPN or TPN where required plus additions as indicated; final volume 2 -5 m!. Incubated for 2 hr _ -SH estimated by the colorimetric method

fLMoles Cysteine

Additions (fLmoles) Without DPN TPN Coenzyme

Nil 0-16 L-Cystine (4 -2) 0-70 0-80 0-82 + malate (12-5) 5-96 6-30 6-38 + malate (50) 5-28 5-42 + glucose 6-phosphate (3 -I) 5-22 6-42 + isocitrate (20) 3-34 3-76 +

(i) Dehydrogenases.-Dialysed clothes moth larval preparations were examined for the presence of dehydrogenases which could reduce DPN or TPN. Appreciable malate-TPN, malate-DPN, isocitrate-TPN, and glucose 6-phosphate-TPN activities

TABLE 4

CYSTINE REDUCTASE IN DIALYSED TINEOLA EXTRACT Reaction mixture contained 1 ml dialysed extract, 1 ml 0 ·125M tris-HCI pH 7'3, plus additions as indicated; final volume 2·5 ml. Incubated for 1 hr. -SH estimated by the colorimetric method

~------~------~~- /LMoles Cysteine Additions (/Lmoles) Total Increment

Nil 0·06 L-Cystine (4,2) 0·36 0·30 + DPN (0·07) 0·48 0·42 + TPN (0·06) 0·48 0·42 + DPNH (1'0) 0·75 0·69 + TPNH (1·0) 1·73 1·67 + glucose 6-phosphate (3·2) 2·76 2·70 + glucose 6-phosphate (3'2) + TPN (0·06) 4·09 4·03

TABLE 5

REDUCTION OF VARIOUS DISULPHIDE COMPOUNDS Reaction mixture contained 1 ml dialysed extract, 1 ml o ·125M tris-HCI pH 7· 3, 4·2 I'ffioles disulphide com­ pounds as indicated, 0·06 /Lmole TPN, 3· 1 /Lmoles glucose 6-phosphate; final volume 2·5 ml. Incubated for 1 hr. -SH estimated by titrimetric method and the results corrected for control without disulphide com­ pound. Different batches of enzyme were used in experiments A and B

Experiment I Disulphide Compound /LMoles -SH

A L-Cystine 5·09 GSSG 1·63 L-Homocystine 0·76 Dithiodiglycollate 0·05 Dithiodibutyrate 0·17

B L-Cystine 2·51 D-Cystine 1·26 were observed at pH 7· 3. Reduction of DPN by lactate was moderate at pH 7·3 and 9 ·1, and very rapid reduction of DPN by malate was observed at pH 9·1 (Table 2). -S-S- REDUCTASES IN THE CLOTHES MOTH 65

(ii) Effect of Dehydrogenase Substrates and Coenzymes on Cystine Reductase.-In the experiment shown in Table 3 all of the dehydrogenase substrates added increased the activity of the cystine reductase system. Glucose 6-phosphate and isocitrate yield considerable amounts of cysteine, and since these substrates are coupled only to TPN in the insect extracts (Table 2), it appears that TPN is mostly responsible for this reduction. The effect of malate addition supports this, although malate is known to be coupled to both DPN and TPN in clothes moth extracts (Table 2).

0·6 DPNH ".'. TPNH \\' ......

::!.. CONT~OL o:; 0'5~~ --....:.--.-. CONTROL 0·5 \ 0,,- ~ o~o ~ --0 G55G z \\ '.~. 0'4 o.~ "\ "G55G I ...... CY5TINE

\ • 0'3 0·3

o 5 10 15 20 o 5 10 20 TIME (MIN) TIME (MIN)

Fig. 3.-DPNH- and TPNH-linked reductases. Reaction mixtures: 1 ml dialysed Tineola extract, 0·27 pmole DPNH or 0·32 ,.mole TPNH. 2·1 ,.moles GSSG or L-cystine added where indicated, diluted to 3·2 ml with o ·125M tris-HOI, pH 7· 3. The reaction was carried out in evacuated Beckman cuvettes.

Further evidence for moderate DPNH-linked and also for considerably higher TPNH-linked reductase activity is presented in Table 4. A slight increase in cysteine formation occurred when cystine or cystine and DPN or TPN were added to the dialysed enzyme; however, DPNH, TPNH, glucose 6-phosphate, and glucose 6-phosphate plus TPN added together with cystine yielded cysteine in increasingly larger amounts. Figure 3 also shows that the cystine-TPNH reductase is much more active than the cystine-DPNH reductase. (iii) Course of the Reductase Reaction.-Figures 4 and 5 show that the TPNH­ linked reductase using glucose 6-phosphate as hydrogen donor takes a linear course within certain limits of time and cystine concentration. No evidence was obtained for a reversal of the cystine reductase reaction by attempts to reduce TPN by 66 R. F. POWNING .AND H. IRZYKIEWIOZ

cysteine, or to inhibit the oxidation of TPNH by cystine on addition of cysteine. The maximum amount of cysteine observed in our experiments using Tineola preparations was about 27 fLmoles per g larvae per hr. (iv) Other Disulphide Compounds.-Table 5 (expt. A) shows that, compared with L-cystine, GSSG and L-homocystine produce considerably less -SH, and that negligible amounts are obtained from dithiodiglycollate and dithiodibutyrate. The reduction of D-cystine takes place at about half the rate of L-cystine (Table 5, '[ /x 6

5

zOJ iii 4 Iii >- U

2

2 3 TIME (HR)

Fig. 4.-Relationship of TPNH-cystine reductase activity of Tineola with time. Reaction mixture: 1 ml dialysed extract, 1 ml 0 ·125M tris-HOI pH 7· 3, 4·2 JLmoles L-cystine, 3·1 JLmoles glucose 6-phosphate; final volume 2·5 m!. -SH estimated by the colorimetric method and the values corrected for controls without cystine.

expt. B). A spectrophotometric test confirms that the reduction of GSSG by TPNH and DPNH is slower than the reduction of cystine. At the same time GSSG is reduced at a higher rate by TPNH than by DPNH (Fig. 3).

IV. DISOUSSION Cystine reductase in pea seeds and yeasts is DPNH-specific (Nickerson and Romano 1952), but it is evident from the present work that clothes moth larvae possess not only DPNH-linked reductase activity but a more active TPNH-linked reductase. The yeast reductase has quite a wide range of optimum pH in phosphate buffer: about pH 6·2-7· 3 (Proskuryakov and Buachidze 1956), whereas the clothes moth preparations in tris or barbitone buffers reduce cystine optimally at pH 7·3, -S-S- REDUCTASES IN THE CLOTHES MOTH 67 and phosphate and pyrophosphate inhibit the reaction strongly. The equilibrium of the reaction catalysed by the clothes moth cystine reductase appears to strongly favour reduction of cystine, and in this respect it is similar to the reduction of GSSG by tissue extracts (Rail and Lehninger 1952). The insect -8-S- reductases may be coupled in vivo to DPN with lactate or malic dehydrogenases, or to TPN with glucose 6-phosphate or isocitrate dehydro­ genases or with "malic enzyme", all of which have been demonstrated in clothes

• • 2·01- / IIIz iii

Iii •

•

I I I 0 2 3 4 5 6 7 I"MOLES L-CYSTINE

Fig. 5.-Effect of cystine concentration on TPNH-cystine reductase of Tineola. Reaction mixture: 1 ml dialysed extract, 1 ml O· 125M tria-HCI pH 7·3, L.cystine as shown, 3·1 p.Illoles glucose 6-phosphate and 0·06 p.mole TPN; final volume 2·5 ml. Incubated for 1 hr. -SH estimated by the colorimetric method and the values corrected for controls without cystine. moth preparations. It is interesting to note that Proskuryakov and Buachidze (1956) observed a slight stimulation of cystine reductase on addition of malate and DPN to plant extracts, but they did not study the effect of TPN. The substrate specificity of the dialysed insect preparations is fairly broad; L-homocystine, GSSG, and D-cystine are ail reduced at appreciable rates but L-cystine reductase was the most active. Reduction of GSSG by insect preparations is more rapid with TPNH than DPNH (Fig. 3) and this is in accordance with the properties of yeast and liver glutathione reductase (Racker 1955). Since the extracts used in this work were from whole larvae it is difficult to assign a metabolic role for the cystine reductases. However, any enzyme capable of producing thiol compounds, which could be secreted into the gut of these insects, could play an important part in the process of wool digestion.

v. REFERENCES CONN, E. E., and VENNESLAND, B. (1951).-Nature 167: 976-7. GEIGER, W. B., KOBAYASHI, F. F., and HARRIS, M. (1942).-J. Res. Nat. Bur. Stand. 29: 381-9. 68 R. F. POWNING AND H. IRZYKIEWICZ

GEIGER, W. B., PATTERSON, W. S., MIZELL, L. R., and HARRIS, M. (1941).-J. Re.y. Nat. Bur. Stand. 27: 459-68. GRUNERT, R. R., and PHILLIPS, P. H. (1951).-Arch. Biochem. Biophys. 30: 217-25. KATCHALSKI, E., BENJAMIN, G. S., and GROSS, V. (1957).-J. Amer. Chem. Soc. 79: 4096-9. LINDERSTROM-LANG, K., and DUSPIVA, F. (1936).-C.R. Lab. Carlsberg. (Ser. Chim.) 21: 53-83. MAPSON, L. W., and GODDARD, D. (1951).-Nature 167: 975-6. NICKERSON, W . .T., and FALCONE, G. (1956).-Science 124: 318-9. NICKERSON, W . .T., and ROMANO, A. H. (1952).-Science 115: 676-8. POWNING, R. F. (1954).-Aust. J. Biol. Sci. 7: 308-18. POWNING, R. F., and IRZYKIEWICZ, H. (1959).-Nature 184: 1230-l. PROSKURYAKOV, N. 1., and BUACHIDZE, 1. D. (1956).-Biochimiya 21: 799-805. RACKER, E. (1955).-J. Biol. Chem. 217: 855-65. RALL, T. W., and LEHNINGER, A. L. (1952).-J. Biol. Chem. 194: 119-30.