The Effect of Triton X-100 on the Respiratory Chain Enzymes

Transcription

1 Eur..J. Biochem. 14 (1970) The Effect of Triton X-100 on the Respiratory Chain Enzymes of a Heart -Muscle Preparation Dorota SOCTYSIAK and Zbigniew KANIUGA lnstytut Biochemii Uniwersytetu Warszawskiego, Warszawa (Received December 22, 1969) 1. The effect of non-ionic detergent Triton X-100 on the respiratory chain enzymes of a heartmuscle preparation was compared with that of deoxycholate. 2. The efficiency of the action of the detergents on the activity of membrane-bound enzyme depends on both the detergentlprotein ratio and the concentration of protein in the reaction mixture. 3. No essential differences both in the site and the degree of inhibition or activation of enzymic activities were observed due to the action of Triton X-100 or deoxycholate when a suitable detergentlprotein ratio was applied. 4. The usefulnes of non-ionic detergent Triton X-100 for the studies on several rnembranebound enzymes is discussed. According to Hatefi [l] Triton X-100 belongs to the group of strong detergents which destroy some enzyme activities of the respiratory chain and therefore, in contrast to deoxycholate, Triton X-100 is not used for the fragmentation of mitochondria. However it has been recently succesfully applied to studies on several membrane-bound enzymes. The present report shows that no essential differences were observed either in the site or the degree of inhibition or activation of enzymic activities of heartmuscle preparation due to the action of either nonionic detergent Triton X-100 or deoxycholate when a suitable detergentlprotein ratio was applied. EXPERIMENTAL PROCEDURE The heart-muscle preparation, determinations of protein and enzymic activities were the same as in the previous paper [2], except that the cytochrome c oxidase activity was measured polarographically with a Clark electrode when the ascorbate-cytochrome c system was used as the substrate. All the enzymic activities were determined in 0.1 M phos- -~ Dedicated to Professor Dr. Irena Chmielewska on the occasion of her 40th year of teaching and research work at Warsaw University. Unusual Abbreviutions. Triton X-100, alkylphenoxypolyethoxyethanol with 9 or 10 oxyethylene groups per molecule; TMPI), N,N,N',N'-tetramethyl-p-phenylenediamine. Elzzymes. B-Glucoronidase or p-d-glucoronide glucuronohydrolase (EC ) ; cytochrome oxidase or cytochrome c: 0, oxidoreductase (EC ); NAD(P) transhydrogenase or NADPH,:NAD oxidoreductase (EC ); rhodanase or thiosulphate: cyanide sulfurtransferase (EC ). phate buffer, ph 7.4, in a total volume of the reaction mixture of 1, 1.5 and 3 ml in the manometric, polarographic and spectrophotometric assay, respectively. The manometric and polarographic measurements were conducted at 25" while spectrophotometric were at 18-20'. The preparation was incubated with an aqueous solution of either Triton X-100 or deoxycholate at a given detergentlprotein ratio (w/w) for 1 min at room temperature. The distinct symbols in figures represent experiments carried out with a separate heart-muscle preparation. The enzymic activity of heart-muscle preparations expressed as natoms 0 x min-l x mg protein-l is given in the legends to figures. In all figures the enzymic activity in the absence of detergent was taken as 10Oo/,. RESULTS The efficiency of the action of detergents on the membrane-bound enzymes appeared to be dependent not only on the detergentlprotein ratio but also on the concentration of protein. This relationship is shown in Fig. 1. It is evident that at the same detergent/protein ratio, activity of thenadh oxidase decreases with increase in the concentration of protein in the reaction mixture. The effect of protein concentration during the incubation with detergents appears to be pronounced. Inactivation of NADH as well as succinate oxidase activity by both detergents is less effective (about 7 times) when the protein concentration in incubation mixture is higher ( times) than those used in the experiment shown in

2 Vol. 14, No. 1, 1970 D. SOLTYSIAK and Z. K 4NIITT(lA 71 "00 80 i h fl > ",. 20 A 0 I I I I I I I I 0.02 C Protein (rngirnl) cyt.c Fig. 1. Effect of protein concentration on XADH oxidase activity at constant detergentlprotein ratio during incubation with Triton X-100 (ratio = 0.04 for 0 and 0.06 for e) and deoxycholate (ratio = 1.14 for A and 1.74 for A), NADH oxidase activity of heart-muscle preparations was 500 to 700 natoms 0 x min-l x mg prot,ein-l Triton X-100: protein ratic Fig. 1. These data support some observations on the effect of Triton X-100 on lysvsomal hydroxylases [3] and that of digitonin [2] and cholate [4,5] on the respiratory chain enzymes. As can be seen from Fig. 2, the addition of either Triton X-100 or deoxycholate to the reaction mixture containing heart-muscle preparation results in the same inactivation of the succinate oxidase activity when the Triton/protein ratio is about twice as high as the deoxycholate/protein ratio. Reactivation of the succinate oxidase activity in the presence of exogenous cytochrome c suggests that the cytochrome c region is susceptible to both detergents. The by-pass which is formed round endogenous cytochrome c upon addition of soluble cytochrome c (cf. [S]) is probably responsible for the observed restoration of succinate oxidase activity in preparations inactivated by these detergents. The effect of deoxycholate on succinate and NADH oxidase (see below) is similar to that observed by Ball and Cooper [7]. The effect of both Triton X-100 and deoxycholate on NADH oxidase activity is shown in Fig.3. Two points are interesting. Firstly, the shape of the inhibition curve with both detergents is very similar. However, almost complete inactivation of NADH oxidase activity by both detergents is observed at different detergentlprotein ratios, i. e and 4.5 with Triton X-100 and deoxycholate, respectively. Prom the data of Fig.2 and 3 it can be seen that succinate oxidase activity is about 10 times more sensitive to the action of deoxycholate than NADH C b " Fig.2. Effect of Triton X-100 (A) and deoxycholate (B) on succinate oxidase activity assayed either in the presence (full symbols) or in the absence (empty symbols) of added cytochrome c (10 pm). Succinate oxidase activity of heart-muscle preparations was natoms 0 x min-l X mg protein-'. Protein concentration in incubation mixture 0.10 to 0.12 mg/ml B

3 72 Effect of Triton X-100 on the Respiratory Chain Enzymes Riir. J. Biochern. I I I I I I I B Secondly, in the presence of added cytochrome c only a small reactivation of NADH oxidase activity is observed. It suggests that both detergents act on the respiratory chain in the site localized before cytochrome c1 and therefore exogenous cytochrome c is unable to establish a by-pass of the detergentsensitive site. The effect of detergents on cytochrorne c oxidase was studiedin two systems : ascorbate-tmpd (Fig. 4A and B) and ascorbate-cytochrome c (Fig.4C and D). The main difference between these systems concerns the site at which electrons are transfered to the respiratory chain (cf. [a]). As can be seen, a similar and pronounced inactivation of cytochrome c oxidase by Triton X-100 and deoxycholate (Fig.4A and B, respectively) was only observed when the system ascorbate-tmpd was used. On the other hand, when the ascorbate-cytochrome c system was used as the substrate, cytochrome c oxidase activity was stimulated in the presence of Triton X-100 (Fig.4C). This observation is in agreement with the effect of other non-ionic detergents such as digitonin [Z] and Emasol 4130 [S]. The stimulatory effect of deoxycholate was observed only at a low detergentlprotein ratio (Fig. 4 D). Fig.3. Effect of Triton X-100 (A) and deoxycholate (B) on XADH oxidase activity assayed either in the presence (full symbols) or in the absence (empty symbols) of added cytochrome c (10 pm). NADH oxidase activity of heart-muscle preparations was natoms 0 x min-l x mg protein-l. Protein concentration in incubation mixture 0.06 to 0.07 mg/ml oxidase (Eig.2B and 3s). This resembles the differential effects of digitonin 04 these two enzymic systems described by Kaniuga, Gardas and Jakubiak [Z]. On the other hand, NADH oxidase activity is more sensitive to the action of Triton X-100 than succinate oxidase, similarly as to the action of solvents, phospholipase, urea and freezing (cf. [2]). DISCUSSlON Action of the detergents on the membrane-bound enzymic systems is manifold. It can result in : (a) activation or inactivation of several enzymic activities, (b) fragmentation of subcellular particles and (c) solubilization of membrane-bound enzymes. It seems that the usefulness of a detergent for one of these purposes cannot be predicted only on the basis of its ionic nature and/or whether it is a strong or weak detergent. This suggestion appears to be supported by following findings : (a) activation of several enzyme activities e. g. cytochrome c oxidase [9] and latent rhodanase [lo] was observed independently of the ionic nature of the detergents. (b) Inactivation of the enzymic systems of the respiratory chain by Triton X-I00 and deoxycholate, shown in the present paper, is similar to that induced by digitonin [Z, 111. (c) Ionic as well as non-ionic detergents are successfully applied for fragmentation of both mitochondria [I, 12, 131 and chloroplast [14,15]. For solubilization of membrane-bound enzymes both determined properties of the detergent and some critical conditions under which the process must be carried out seem to be necessary. This is evident from the application of Triton X-100 for solubilization of 8-glucoronidase [16], cytochrome c oxidase [17] NADH dehydrogenase of the respiratory chain [18,19], NADPH transhydrogenase [20] and a-glycerophosphate dehydrogenase [21]. Triton X-100 appeared to be more useful for the isolation of all these enzymes than the ionic detergents.

4 Vol. 14, No. 1, 1970 D. SOLTYSIAK and Z. KANIUGA 73 A c.: I 01 ' I I I C D 6o t I I I I I I I I I I Fig.4. EIfect of Triton X-100 (A and Cj and deoxycholate (B and 0) on cytochrome c oxidase activity. In A and B ascorbate- TMPD and in C and D ascorbate-cytochrome c were the substrates. Soluble cytochrome c which could interfere in the reaction when TMPD was used was removed from the particles as described previously [2]. Cytochrome c oxidase activity of heart-muscle preparations was and natoms 0 x min-l x mg protein-l with ascorbate-tmpd and ascorbatecytochrome c used as substrates, respectively. Protein concentration in the incubation mixture was mg/ml The authors wish to express their appreciation to Dr. Darrell W. Haas for the gift of Triton X-100. This work was partially supported by a grant from the Polish Committee of Science and Technique. REFERENCES 1. Hatefi, Y., In Comprehensive Biochemistry (edited by M. Florkin and E. H. Stotz), Elsevier, Amsterdam 1966, Vol. 14, p Kaniuga, Z., Gardas, A,, and Jakubiak, M., Biochim. Biophys. Acta, 118 (1966) Wattiaux, R., and de Duve, C., Biochem. J. 63 (1956) Takemori, S., and King, T. E., J. Biol. Chem. 239 (1964) Bryxa, J., Kaniuga, Z., and Slater, E. C., Biochim. Biophys. Acta, 189 (1969) Smith, L., and Camerino, P. W., Biochemistry, 2 (1963) Ball, E. G., and Cooper, O., J. Biol. Chem. 226 (1956) Yonetani, T., J. Biochem. (Tokyo), 46 (1959) Wainio, W. W., and Aronoff, M., Arch. Biochem. Biophys. 57 (1955) 11.5.

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