BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL. Xin-Min Li ~, Jia-Zuan Ni 1, Jian-Wen Chen 2 and Fen Hwang 2

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1 Vol. 45, No. 2, June 1998 Pages EFFECTS OF La 3 ON LIPID FLUIDITY AND STRUCTURAL TRANSITIONS IN HUMAN ERYTHROCYTE MEMBRANES Xin-Min Li ~, Jia-Zuan Ni 1, Jian-Wen Chen 2 and Fen Hwang 2 1Laboratory of Rare Earth Chemistry and Physics, Changchun Institute of Applied Chemistry, the Chinese Academy of Sciences, Changchun , China 2National Laboratory of Biomacromolecules, Institute of Biophysics, the Chinese Academy of Sciences, Beijing , China Received January 5, 1998 Received after revision, February 24, 1998 SUMMARY The effects of La 3+ on the structure and function of human erythrocyte membranes were investigated by fluorescence polarization, spin-labeled electron spin resonance (ESR) and differential scanning calorimetry (DSC). The results showed that increasing concentrations of La 3+ inhibited (Na++K+)-ATPase and Mg2+-ATPase activities. La 3+ lowered the lipid fluidity of erythrocyte membranes and induced structural transitions in erythrocyte membranes. Key words: La 3+, Lipid fluidity, Structural transitions, DSC, Erythrocyte membranes INTRODUCTION The lanthanide series of elements comprises the 15 elements between lanthanum and lutetium in the periodic table. Four major motives form the basis of research into the biochemical properties of the lanthanides. In roughly chronological order, these are: research directed into possible medicinal applications(l), toxicological concerns (2,3), intellectual curiosity, and the potential use of these elements as informative probes in biological and biochemical research(4,5). As most of the evidence suggests the primary site of interaction of lanthanide ions with living cells is at the external surface, the biological effects of lanthanides are based on the interactions of lanthanides with biomembranes. Some results were reported concerning the effects of lanthanide ions on Ca z+ movements in erythrocyte membranes. Szasz et al.(6) noted that 0.25 mmol/l La 3+ completely blocked the efflux of 45Ca2+ from preloaded cells. Various other lanthanides ranging from Pr 3+ to Lu 3+ were equally effective inhibitors of these processes (7). La 3+ ions also prevented the efflux of K + produced by either high concentrations of intracellular Ca 2+ or by propranolol treatment of MgZ+-depleted cells /98/080323M) /0 Copyright by Academic Press Australia. All rights of reproduction in any form reserved.

2 However, when cells were preloaded with Ca 2 by A23187, the rate of rapid K transport was unaffected by external La 3+ ions(6). Further work by (8) confirmed that Tb 3+ ions applied externally to resealed erythrocyte ghost membranes inhibited the net effiux of K + via the Ca2+-activated channel. However, internalized Tb 3+ ion could activate this channel as well as Ca 2+. However, the mechanisms of the effects of lanthanides on erythrocyte membranes still remain poorly understood. In this work, the effects of La 3+ on the (Na++K+)-ATPase, Mg2+-ATPase activities, the lipid fluidity and structural transitions of human erythrocyte membranes are investigated. MATERIALS AND METHODS Materials. Fresh human red blood cells were obtained from the Beijing Blood Center of the Red Cross Society. The preparation of erythrocyte membranes is given in (9), protein was measured by the method of (10), using bovine serum albumin as the standard. LaCl3 solution was prepared by dissolving its oxide(>99.99%) in HC1 with elimination of excess acid, and the concentration was determined as described(l 1). 1,6-diphenyl-l,3,5-hexatriene(DPH), 5-doxyl-stearic acid(5-dsa) and 16-doxylstearic acid (16-DSA) were purchased from Sigma Co. St. Louis MO(U.S.A.). All other reagents are of analytical grade. A TPase activity. (Na++K and Mg2 activities of human erythrocyte membranes were measured by the method of (9) and the liberated inorganic phosphate (Pi) was assay as previously described (9). Lipid fluidity. The lipid fluidity was measured by fluorescence polarization (9)~ Fluorescence was measured using an HITACHI F-4010 fluorescence spectrophotometer at 25~ Spin labelling of erythrocyte membranes and the fluidity by ESR were measured as described in (9). The ESR spectra were obtained using a Varian E-109 electron resonance spectrometer. Central magnetic field: 3320G, scanning width: 200 G, modulation range: 2G, microwave power: 200mw, time constant: 0.128s at 25~ Calorimetry. Heat capacity measurements were obtained using a Microcal-2 differential scanning calorimeter(amherst, MA) equipped with 1.0 ml platium cells. membrane samples equilibrated in the desired buffer were loaded into the sample cell, and an equal volume of the identical buffer system was added to the reference cell of the calorimeter. The heating rate in these experiments was 1 ~ RESULTS AND DISCUSSION Effects of La 3 on the (Na++K+)-ATPase and Mg2+-ATPase activities There is a small amount of (Na++K+)-ATPase in erythrocyte membranes. The cation pump utilizes the free energy of hydrolysis of ATP to extrude Na and to accumulate K thereby maintaining the Na and K gradient across the plasma membrane. Nayler et al.(12) studied the effects of La 3+ on a microsomal fraction of pig 324

3 VOI. 45, No. 2, 1998 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL heart muscle and showed that 10 to 100 I_tmol/L La 3+ had an uncompetitive inhibitory effect on the (Na +jr_ K + )-ATPase activity. The effects of La 3 on the (Na++K*)-ATPase and Mg2*-ATPase activities of +q_ + human erythrocyte membranes is shown in Tablel. La 3+ inhibits the (Na K )-ATPase and Mg2+-ATPase activities, the higher the concentrations of La 3+, the stronger the inhibition of the ATPase. When the concentration of La 3 is 5 gmol/l, (Na++K+)- ATPase and Mg2+-ATPase activities were decreased to 47% and 74.3%, respectively. But the inhibitory effect of La 3* on the Mg2+-ATPase was weaker than that on the (Na++K Effects of La 3+ on the lipid fluidity The fluorescence spectra of DPH labeled erythrocyte membranes were investigated (9) and the results showed that DPH entered the hydrophobic region of the membranes. The effects of La 3 on DPH fluorescence polarization in erythrocyte membranes is shown in Table lamol/l La 3+ increases the fluorescence polarization, which indicates that La 3+ enhances the order of the membrane lipid and decreases the fluidity. In order to further investigate the conclusion that La 3 may lower the fluidity of erythrocyte membranes, 5- and 16-doxyl stearic acid (5-DSA and 16-DSA) spin probes were used to label the polar surface and the terminus of the hydrocarbon chains, Table 1 Effects of La 3+ on the (Na++K+)-ATPase and Mga+-ATPase activities of erythrocyte membranes CLa 3+ (Na++K+)-ATPase Mg2+-ATPase (~tmol/l) Specific activity* Relative activity(%) Specific activity* Relative activity(%) _ _ _ *The enzyme activity is expressed as Bmol Pi/mg/hour, values are the mean + S.E., n=

4 Table 2 Effects of La 3+ on the fluorescence polarization (P), the order parameter (S) and rotational correlation time (zc) of erythrocyte membranes CL. 3+ Polarization Order parameter Rotational correlation time (gmol/l) (P) (S) ('cox 109 s) _ _ Excitation: 360 nm, Emission: 430 nm, Slit: 5 nm. Value are the mean_+ S.E, n=4-6 and spin-labeled ESR technique were used to study the effects of La 3+ on the dynamic characterization of the membrane lipids. ESR spectra of erythrocyte membranes labeled with 5-DSA and 16-DSA at 25~ were performed as in (9). Table 2 shows that La 3+ enhanced the order parameter S, namely, by increasing the order of the membrane and lowering the fluidity of the 5-DSA labeled erythrocyte membranes. 5 ~tmol/l La 3~ increases the rotational correlation time "to of 16-DSA labeled membrane mobility which indicates that it slows the terminal mobility, increases the order, and mainly lowers the fluidity of the membranes. These results correspond with the data gained by fluorescence polarization. La 3+ induces structural transitions in erythrocyte membranes As the interaction of lanthanides with erythrocyte membranes influences the structure and function of the membranes, the membrane proteins are probably involved in the reactions. The advent of highly sensitive differential scanning calorimetry (DSC) has introduced a tool for use in characterizing the structure and behavior of biological membranes. A typical heat capacity profile for human erythrocyte membranes in isotonic sodium phosphate ( 20 mmol/l NaH2POa-Na2HPO4, 125 mmol/l NaCI, ph7.4) was shown in Fig. 1. Of the four transitions labeled in the diagram, the A transition (49~ has been shown to involve the denaturation of spectrin, a component of the red cell membrane cytosketon(13). This transition is sensitive to adenine nucleotides and other small ligands which perturb the morphology of the cell. The B1 326

5 C " B! B2 I., I I 32, T(~ 72.5 Figure 1 Heat capacity as a function of temperature of human erythrocyte membranes suspended in 20 mmol/l sodium phosphate/125 mmol/l NaCI, ph7.4. The heating rate was l~ the sample cell of the calorimeter contained 6-7 mg/ml protein. transition (59~ may relate to band 2.1, 4.1 and 4.2, but the nature of the endotherm is unclear. The Bz transition (63~ has been identified with the thermal unfolding of the cytoplasmic domain of band 3. The C transition (69~ was shown to derive from the denaturation of the membrane-spanning domain of the anion transport protein, band 3. These results are coincident with those reported in (13). As lanthanide phosphates are insoluble, phosphate buffers are to be avoided in order to assign possible functional roles in La 3+ to any of the structural regions giving rise to these thermal transitions. One technique for determining the site of interaction of a particular solute molecule on a structurally heterogeneous membrane is to observe the effects of that solute on the various calorimetric transitions of the membrane. Thus, any solute induced change in the size (enthalpy) or temperature of a transition would indicate a direct or indirect interaction of that solute with the membrane components associated with that transition. The calorimetric transitions change markedly as the concentration of buffer is increased (14). Curve a shown in Fig. 2 is the heat capacity profile for erythrocyte membranes suspended at middle salt solution (10 mmol/l Tris- HC1, 28.5 mmol/l NaC1, ph 7.4). Erythrocyte membranes in different La 3+ concentration (0, 0.1, 0.5, 0.8 mmol/l) have been examined by DSC. Two specific 327

6 0.05 T Cal'gLdeg-~._ / ~ I I I ,0 T(~ 72.5 Figure 2 Effects of La 3~ on the calorimeteric transitions of erythrocyte membranes. The buffer was 10 mmol/l Tris-HCl, 28.5 mmol/l NaCI, ph7.4, and the membrane concentrations were 6-7 mg/ml protein. Three transition are seen, designated as the A (Tm=51~ B(T,,=59~ C(Tm=63~ a: control, b: 0.1 mmol/l La p~+, c: 0.5 mmol/l La 3+, d: 0.8 mmol/l La 3+. changes are apparent as the concentration of La 3~ is increased. First, there is a gradually significant increase in the width of the A and C transitions. The effect at La 3+ concentration is greater on A than C. The second effect of La 3+ involves a change in the B transition. Although the transition is quite prominent in the absence of La 3~, most of the transition remains at 0.1 mmol/l La 3', and the transition disappears by 0.5 mmol/l La 3+. The change in the transition temperature for the A and C transitions is very small over this range of La 3+ concentration. The scans in Fig.3 show results obtained for sample at high salt solution (20 mmol/l Tris-HCl, 135 mmol/l NaCI, ph7.4). Serveral prominent changes can be seen in going from solutions of low to high buffer concentrations. To begin with, both the A and C transitions become more prominent. Of more interest is the process which occurrs in the B transition. As the buffer concentration is increased to high salt, it can be seen that the B transition is split into smaller transitions, one of these is ultimately localized on tbe lower temperature side of the B transition and one on the high 328

7 I I I T(~ 72.5 Figure 3 Effects of La 3 on the calorimeteric transitions of erythrocyte membranes. The buffer was 20 mmol/l Tris-HCl, 135 mmol/l NaC1, ph7.4, and the membrane concentrations were 6-7 mg/ml protein. Four transition are seen, designated as the A (Tm=50~ Bl(T,~=57~ Bz(T,,=62~ C(T.,=68~ a: control, b: 0.1 mmol/l La 3~, c: 0.5 mmol/l La 3+, d: 0.8 mmol/l La 3+, temperature side. These two transitions are called the B1 and the Bz transitions, respectively. In the presence of 0.1 mmol/l La 3., there is no significant change of the heat capacity profile for erythrocyte membranes. With increasing La 3+ concentration, these three transitions, particularly BL and B2, exhibit a high sensitivity to La 3.. La 3+ causes B~ and B2 to decrease in size, and the transitions appear to be absent in 0.5 mmol/l La 3+. There is a gradual increase in the width of the C transition, the effect at increasing La 3+ concentration is greater on C than A. This paper describes a method for locating the sites of interaction of lanthanide ions with structurally heterogeneous biological membranes. Major perturbations are observed in the A, B~, B2 and C transitions upon addition of La 3+ at different concentrations, and these perturbations suggest that L~ + may interact with the membrane domains associated with these transitions. Previous work demonstrated that 329

8 the A transition is associated with the spectrin protein complex (13). B~, B2 transitions have been identified with a protein-phospholipid domain which contains the membrane transport band 3, and the C transition was shown to derive from the denaturation of band 3. It is, therefore, suggested that La 3+ preferentially interacts with phospholipids, band 2.1, band 4.1 and spectrin at low salt concentrations, and the effect of La 3+ on spectrin is weak at high salt concentrations. To summarize: the results show that La 3~ inhibits the (Na++K+)-ATPase and Mg2+-ATPase activities, lowers lipid fluidity and induces structural transitions of band 3, band 2.1, band 4.1, spectrin and phospholipids in erythrocyte membranes. Acknowledgements: We are grateful to the National Natural Sciences Foundation of China and the President Foundation of the Chinese Academy of Sciences for financial support. We also thank Prof Niu Chunji for helpful discussion. REFERENCES 1. Evans, C.H.(1990) Biochemistry of the Lanthanides, Academic Press, New York and London. 2. Haley, T.L(1979)Toxicity, in Handbook on,the Physics and Chemistry of Rare Earths (K.A.Gschneidner and L.R. Eyring, eds.) Vol.4, North-Holland, Amsterdam, Ni, J.Z.(1995) Bioinorganic Chemistry of the Rare Earth Elements, Science Press, Beijing. 4. Zha, X.H. and Morrison, G.J.(1995) Am. J, Physiol., 269, C923-C Komiyama, M. (1995) J. Biochem., 118, Szasz, I., Sarkadi, B., Schubert, A. and Gardos, G.(1978) Biochim. Biophys. Acta, 512, Sarkadi, B., Szasz, I., Gerloczy, A. and Grardos, G. (1977) Biochim. Biophys. Acta, 464, Wood, P.G. and Mueller, H.(1985) Eur. J. Biochem., 146, Li X.M., Ni J.Z, Chen Y., Chen, J.W. and Hwang F. (1994) Acta Biophysica Sinica, 10, Lowry, O.H, Rosebrough, N.J., Far, A.L. and Randall, R.J.(1951) J. Biol. Chem., 193, Li, X. M., Zhang, Y.F., Ni, J.Z., Chen, J.W. and Hwang F. (1994) J. Inorg. Biochem., 53, , Nayler, W.G. and Harris, J.P. (1976)J. Mol.Cell Cardiol., 8, Davio, S.R. and Low, P.S.(1982) Biochemistry, 21, Brandts, J.F., Taverna, R.D., Sadasivan, E. and Lysko, KA.(1978)Biochim. Biophys. Acta, 512,