Toxicology Letters, 23 (1984) 183-188 Elsevier

183

TOXLett. 1304

INFLUENCE OF TRIETHYL LEAD ON THE ACTIVITY OF ENZYMES OF THE ASCITES TUMOR CELL PLASMA MEMBRANE AND ITS MICROVISCOSITY

(Triethyl lead; cytotoxicity; (Na -t K)-ATPase; membrane fluidity, Ehrlich ascites cells)

E.W. HAEFFNER, H.-P. ZIMMERMANN and C.J.K. HOFFMANN

Institut fiir Zell- und Tumorbiologie, Deutsches Krebsforschungszentruum, Im Neuenheimer Fe/d 280, D-6900 Heidelberg (F. R. G.)

(Received November 7th, 1983) (Revision received May 7th, 1984) (Accepted June 8th, 1984)

SUMMARY

The influence of triethyl lead (TriEL) on the activity of plasma membrane-bound enzymes of Ehrlich ascites tumor cells and on membrane fluidity has been investigated. TriEL completely inhibits the (Na+-K+)-ATPase in its membrane-bound and even more pronounced in its solubilized form between 5 and about 20 CM. It also alters the microviscosity of the isolated plasma membrane up to a temperature of about 30°C but it does not have any influence on the fluidity of the membrane-derived liposomes. From these data it is concluded that the inhibitor may interact directly with the catalytic subunit of the (Na+ +K+)-ATPase, and may not exert its influence by interfering with the membrane lipids.

INTRODUCTION

TriEL is a derivative of the antiknock additive TEL, which can be converted metabolically [l-3] or by photolytic decomposition [4,5] to the active, more water- soluble TriEL. This organic lead compound is highly cytotoxic [6,7], especially neurotoxic [8,9] and creates dramatic mental disorders as well as histological and cytological damage in brain and other tissues [9-l 11. It has also been reported that TriEL interferes with chromosome segregation, DNA and protein synthesis, cytokinesis and inhibits cellular growth [7,12-151. Recently, it was shown that

Abbreviations: TEL, tetraethyl lead; TriEL, triethyl lead.

0378-4274/84/g 03.00 0 Elsevier Science Publishers B.V.

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TriEL and other organic lead compounds depolymerize microtubules from mam- malian cells in vitro and in vivo [16]. Very little is known, however, about the biochemistry of TriEL cytotoxicity.

MATERIAL AND METHODS

Plasma membrane preparation The material was obtained from the glycogen-free ascites-cell line and purified by

a combination of discontinuous and continuous sucrose-density gradient centrifuga- tion. Details of this procedure have been described elsewhere [17]. Purified plasma membrane fractions were also prepared by gel filtration of an enriched fraction (12 000 x g pellet) on Sephacryl S-1000 superfine (Pharmacia Fine Chemicals, Upp- sala, Sweden). The membranes were eluted with 0.25 M sucrose, 10 mM Tris-HCl, pH 7.4, at a flow rate of 50 ml/h and the effluent was recorded with a UV-detector at 280 nm. The yield varied between 25 and 35% on a protein basis. The membrane material was collected by ultracentrifugation at 104 000 x g for 1 h, and the pellet was resuspended in Tris-buffered isotonic sucrose solution, pH 7.4, for further experiments.

Enzyme activity measurements The enzyme activities were determined according to standard procedures [17].

Solubilization of (Na+ + K+)-ATPase activity from the purified plasma membrane preparations was performed with saponin (0.005Oro) in the presence of substrate to protect the enzyme against inactivation [ 181. Sodium dodecylsulfate could not be us- ed for this purpose, since it was found to interfere with the procedure for phosphorus analysis. A freshly prepared aqueous solution of TriEL was used for the inhibition studies. The degree of inhibition of (Na+ + K+ )-ATPase activity was calculated as fraction of the total ouabain-sensitive activity. Protein was determined by the method of Lowry et al. [ 191, and Pi by the procedure of Eibl and Lands [20].

Fluorescence polarization Fluorescence polarization was measured essentially as described elsewhere [21],

except that the temperature was automatically controlled with a Haake PG-40 pro- gramming system. The plasma membranes were suspended in 0.25 M sucrose, 2 mM EDTA, 20 mM Tris-HCl, pH 7.4 at a protein concentration of about 0.50 mg/ml in the presence of 2 x 10e6 M 1,6-diphenyl-1,3,5-hexatriene. The plasma mem- brane lipids isolated by the Folch procedure [22] were dispersed in the same buffer at a concentration of about 120 pg/ml based on the content of polar lipids.

RESULTS AND DISCUSSION

The effect of TriEL on the activity of plasma membrane-bound enzymes has been

185

investigated. The data in Table I demonstrate that the (Na+ +K+)-ATPase is strongly inhibited by this compound, whereas the 5’-nucleotidase activity is not af- fected at all, whether in its membrane-bound or its solubilized form (not shown) Fig. 1 shows that the soluble (Na+ + K+)-ATPase is more susceptible to the inhibi- tion by TriEL than the membrane-bound one, which reveals some rest activity at a 2.5 to 5 times higher inhibitor concentration. Table II presents data on both alkaline phosphatase and the Mg2+ -dependent ATPase which are affected only to some extend by the organic lead compound. From these data we conclude that TriEL is mainly an inhibitor of (Na+ + K+)-ATPase.

The results in Fig. 1 indicate that the possible mechanism of this inhibition may be envisaged to occur by a direct interaction of TriEL with the active centre of the enzyme rather than indirectly by altering the physical state of the membrane. To fur-

TABLE I

EFFECT OF TRIETHYL LEAD ON THE ACTIVITY OF BOTH MEMBRANE-BOUND AND SOLUBILIZED (Na+ +K+)-ATPase AND 5’-NUCLEOTIDASE

Enzyme TriEL Spec. activity Inhibition

PM nmol/min/mg protein in %

(NA+ + K +)-ATPase Membrane-bound

Solubilized

0 248.8 * 36.0 2 187.6 + 42.5 5 103.9 * 40.7

15 19.0 + 4.4 0 309.3 + 38.3 5 21.8 f 5.8

10 0

5 ’ -Nucleotidase 0 10 20

37.0 i 6.4 38.1 * 3.6 37.9 + 6.7

24.6 58.2 92.4

93 100

0 0

The data represent the means f S.D. of 3 to 5 experiments with duplicate analyses.

TABLE II

EFFECT OF TRIETHYL LEAD ON MEMBRANE-BOUND ALKALINE PHOSPHATASE AND Mg’ + -DEPENDENT ATPase ACTIVITY

Enzyme TriEL Spec. activity Inhibition

fiM nmol/min/mg protein in %

Alkaline phosphatase 0 3.18 + 0.03 2 3.11 f 0.15 2.2

10 2.90 * 0 8.8

Mg’ + -ATPase 0 163.9 t 32.2 2 162.3 f 16.2 1.0

10 121.0 f 11 26.2

The data represent the means f S.D. of 3 experiments with duplicate analyses.

TRIETHYL LEAD (KM)

Fig. 1. Kinetics of the (Na’ + K+)-ATPase inhibition by triethyl lead for the membrane-bound (X-X), and the solubilized enzyme (O--O). The degree of inhibition is expressed in percent of the untreated control.

ther investigate this possibility we have analyzed the effect of TriEL on the microviscosity of both isolated plasma membrane vesicles and liposomes prepared from lipid extracts of these membranes. Fig. 2a shows a significant difference in fluorescence depolarization between the untreated and the inhibitor-treated mem- branes in the temperature range between 8 and 29”C, i.e. 3.60 and 3.30 x 10m3 “K. On the other hand, no difference was found when the liposomes were measured with respect to fluidity changes (Fig. 2b). These results confirm the above data that the (Na+ + K+)-ATPase activity appears to be inhibited by a direct interaction of TriEL with the enzyme, and this interaction may in turn impose a certain degree of steric hindrance upon the mobility of lipid molecules within the bilayer causing an increase in fluorescence polarization. This steric hindrance, however, vanishes at higher temperatures where the lipids apparently possess a higher degree of motional freedom. The complete absence of any effect of TriEL on the microviscosity of membrane lipids in aqueous suspension further shows that no lipid peroxidation [23], which is known to alter lipid fluidity, could have occurred in our preparation. This observation is substantiated by the gas chromatographic analysis of the fatty acid profile of the TriEL-treated material, which did not differ from the untreated sample (not shown).

The interaction of triethyl lead with the (Na+ + K+)-ATPase could be one of the main reasons for the cytotoxicity of this organic lead compound, and may also ex- plain its highly neurotoxic effects with respect to the direct influence of (Na+ + K+ )- ATPase activity on the potential value of the plasma membrane in neuronal cells.

I87

000 ! x

, / / / 3b

I I I 3.2 34 36

1 / T ( K ) X ?@

b

oww 30 32 54 36

I/~(K)~Io -5

Fig. 2. Temperature dependence of fluorescence polarization P, (a) Plasma membranes without (C--Q) and in the presence of 80-100 FM triethyl lead (X -x), and (b) extracted plasma membrane lipids without (O--O) and in the presence of 80-100 bM triethyl lead (x-x). Suspensions of about 0.60 mg membrane material or 4.4 hg lipid phosphorus per ml were labelled with 2 x low6 M I,&diphenyl- 1,3&hexatriene for 1 h at 37°C and then analyzed for fluorescence polarization. Evaluation of the data from 2 ~~d~v~du~ experiments was made by regression analysis, yielding the following equations: y = Q.245~10-3~ -0.7223 (x-x), andy = 0.371~~O-3x - 1.136 (w> with a high degree of correla-

tion (r = 0.95-0.97). Since the limits of the 95Vo confidence intervals are not overlapping between 8 and 29”C, the two curves are significantly different in this range.

ACKNQWLEDGEMENTS

The technical-assistance by A. Ho11 in the preparation of the plasma membranes and in performing the enzyme assays is gratefully acknowledged. We thank Dr. G, Rtiderer for providing us with highly purified triethyl lead. Thanks are also due to Professor Dr. E. Weber from the Institut fiir Dokumentation und Statistik of this Research Centre for making the statistical analysis of the fluorescence polarization measurements.

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