CYSTINE AND GLUTATHIONE REDUCTASES IN THE CLOTHES MOTH TINEOLA BISSELLIELLA By R. F. POWNING* and H. IRZYKIEWICZ* , [Manuscript received October 27, 1959] Summary The larva of the clothes moth Tineola bisselliella 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 keratin disulphide bonds may be a necessary prerequisite for digestion of wool by insects. 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 insect 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 <x-glycerophosphate (Light); sodium lactate (B.D.H.); L-cystine (B.D.H.); L-homocystine (C.F.B.R.); dithiodiglycollic acid (Light); dithiodibutyric acid (Light). Oxidized glutathione (GSSG) was prepared by aerating reduced glutathione (GSH) (B.D.H.) in an ammonium carbonate solution until the nitroprusside test was negative and the solution was free of buffer. 62 R. F. POWNING AND H. mZYKIEWICZ 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 <x-Glycerophosphate 35* 0 54* 0 isoCitrate 0 1880 Glucose 6-phosphate 0 2450 Malate 1120 1400 3240 900 . -~-----~----.-- * 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 + <x-glycerophosphate (6 -3) 0-76 0-96 + <x-glycerophosphate (50) 0-64 1-06 + lactate (12-5) 0-70 1-64 + lactate (100) 0-78 2-04 64 R.