Interaction of Erythrocyte Spectrin with Some Nonbilayer Phospholipids

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1 Gen. Physil. Biphys. (1994), 13, Shrt cmmunicatin Interactin f Erythrcyte Spectrin with Sme Nnbilayer Phsphlipids K. MICHALAK 1, M. BOBROWSKA 1, K. BIALKOWSKA 2, J. SZOPA 2 and A. F. SIKORSKI 2 1 Department f Biphysics, Academy f Medicine, Wrclaw, ul. Chahibmskieg 10, Wrclaw 2 Institute f Bichemistry, University f Wrclaw, ul. Przybyszewskieg 63/77 PL Wrclaw Pland Abstract. Bvine erythrcyte spectrin was fund t interact with lysphsphatidylchline and lysphspatidylserine what was detected by small changes f the intrinsic flurescence f spectrin. Lysphsphatidylethanlamine in cntrast t its diacyl, natural cunterpart did nt affect the intrinsic flurescence f spectrin at all. Dileylphsphatidylethanlamine induced distinct changes in the intrinsic flurescence frm these induced by natural phsphatidylethanlamine suspensins. Our data may indicate an imprtance f the presence f bth fatty acyl chains in phsphatidylethanlamine mlecule and perhaps, its bilayer structure fr the interactin f this phsphlipid aggregates with spectrin. Key wrds: Erythrcyte spectrin Lysphsphlipids Dileylphsphatidylethanlamine Intrinsic flurescence quenching Erythrcyte membrane Intrductin Spectrin, which accunts fr 75% f the mass f the erythrcyte membrane skeletn is cmpsed f tw nnidentical subunits a and j3 f M r ~ and respectively (Sahr et al. 1990; Winkelman et al. 1990). Physilgically relevant unit f spectrin is (a/3) 2 tetramer (cntur length 200 nm) that is frmed by headt-head assciatin f tw a(3 heterdimers. Spectrin binds t erythrcyte membrane hydrphbic dmain (membrane bilayer frmed by lipids and intrinsic prteins) by interactins with ankyrin-anin transprter prtein cmplex (fr a review see e.g. Gdman et al and Bennett 1990). The ends f the tetramers are engaged in s called junctinal cmplexes Crrespndnce t: A. F. Sikrski, Institute f Bichemistry, University f Wrclaw, ul. Przybyszewskieg 63/77 PL Wrclaw, Pland.

2 58 Michalak et al. cmpsed f shrt actin filament, prtein 4.1, adducin and prtein 4.9 (Bennett 1990; Derick et al. 1992). Interactin with membrane lipid is a well knwn prperty f red bld cell spectrin that was demnstrated in mdel systems (Julian et al. 1971; Mmbers et al. 1980; Bitbl et al. 1989) as well as in the natural membrane (Haest et al. 1978; Sikrski and Kuczek 1985). Spectrin was als fund t bind many amphipatic cmpunds as fatty acids and detergents (Isenberg et al. 1981; Sikrski et al. 1987b). Suggested in early studies specificity f this interactin twards phsphatidylserine was nt cnfirmed in ur and ther's studies (Sikrski et al. 1987a; Bitbl et al. 1989). It seems that spectrin exhibits higher affinity and lwer capacity twards PS, while lwer affinity and much higher capacity fr PE suspensins (Sikrski et al. 1987a; Michalak et al. 1993). Amng several amphipatic cmpunds whse interactin with spectrin were studied (Isenberg et al. 1981; Sikrski et al. 1987a; Kahana et al. 1992) lysphsphlipids have nt been included. The fact that lys-pe did nt affect the intrinsic flurescence f spectrin indicated the imprtance f the presence f bth fatty acyl chains and perhaps, bilayer structure in the interactin f ethanlamine phsphlipids with spectrin. The results f the experiments f the effect f DOPE n the intrinsic flurescence f spectrin seem t cnfirm this suggestin. Abbreviatins used: DOPE - dileyl-phsphatidylethanlamine, lys-pc - lysphsphatidylchline, lys-pe - lysphsphatidylethanlamine, lys-ps - lysphsphatidylserine, PC - phsphatidylchline, PE - phsphatidylethanlamine, PS - phsphatidylserine. Materials and Methds Bvine erythrcyte spectrin dimer was islated as described previusly (Michalak et al. 1993). Sepharse CL 4B clumn (2 x 60 cm) was equilibrated with the buffer: 20 mml/1 Na 2 HP0 4, 50 mml/1 NaCl, 0.1 mml/1 EDTA, 1 mml/1 NaN 3, 0.1 mml/1 2-mercaptethanl, ph 7.4. Lipids: phsphatidylethanlamine (bvine brain) was frm Kch Light, Clnbrk, England, dileylphsphatidylethanlamine, lysphsphatidylchline (egg ylk), lysphsphatidylethanlamine (egg ylk) and lysphsphatidylserine (bvine brain) were purchased frm Sigma Chem. C. St. Luis, MO. USA. Lysphsphlipids were disslved in the abve buffer. Phsphatidylethanlamine suspensins were prepared as described previusly (Sikrski et al. 1987a and Michalak et al. 1993). Prtein was determined accrding t Bradfrd (1976) and phsphlipid phsphrus accrding t Bartlett (1959). Flurescence measurements were perfrmed at 20 C with the use f a Perkin-Elmer MPF-3L spectrflurimeter as described previusly (Sikrski et al. 1987a; Michalak et al. 1993). Fr each experimental pint a cntrl measurements fr the light scattering were perfrmed. The cntrl sample cntained an apprpriate vlume f the suspensin f phsphlipid in the sample buffer. Samples at and belw lipid cncentratins indicated in Results were clear and displayed very lw light scattering effect (n mre than 10% f maximal flurescence). The average values f triplicate measurements differing by n mre 10% are presented.

3 Spectrin Interactin with Nnbilayer Phsphlipids 59 Results and Discussin The effect f lys-pc n the intrinsic flurescence f islated spectrin dimer is shwn in Fig. 1A. The quenching f the flurescence was in the range f 10% what is similar t the values btained fr phsphatidylchline vesicles at the same lipid t prtein ratis (Sikrski et al. 1987a). It shuld be nted that the maximal lipid:prtein rati in Fig. \A was 900; at higher lipid cncentratins the slutin became turbid. Lys-PS caused ~ 10% increase f flurescence at lwer and ~ 10% decrease at higher cncentratins f this phsphlipid (Fig. IB). Described abve data might suggest that the effect f these tw lysphsphlipids, particularly lys- PC mre r less resembled the effect f their diacyl cunterparts n islated spectrin (see: Sikrski et al. 1987a, Fig. 1). On the ther hand, the effect f lys-pe did nt resemble the effect f PE n islated spectrin (Fig. 1C). PE suspensin was fund t quench up t 50% f tryptphan flurescence f spectrin at ph and up t 75% flurescence at ph 5.5 (Sikrski et al. 1987a). In the wide range f lys- PE cncentratins (up t 78 //ml/1, lipid t prtein rati f ~ 1600) practically x u. u. LIPIDiPROTEIN [ml/mll O O LYSO-PC [fiml/l] O U LIPOPROTEIN Iml/ml] LYSO-PS [nml/l] Figure 1. The effect f lysphsphatidylchline (A), lysphsphatidylserine (B) and lysphsphatidylethanlamine (C) n the intrinsic flurescence quenching f islated red bld cell spectrin. Spectrin was islated by gel filtratin n Sepharse CL 4B clumn equilibrated with 20 mml/1 Na 2 HP0 4, 50 mml/1 NaCl, 0.1 mml/1 EDTA, 1 mml/1 NaN 3, 0.1 mml/1 2- mercaptethanl, ph 7.4. Spectrin cncentratin in the sample was in (A) 27 nml/1 and in (B) and (C) 50 nml/1. The excitatin and emissin wavelength was 290 and 337 nm respectively. O u. LIPOPROTEIN Iml/mll K LYSO-PE [fjml/l]

4 60 Michalak et al. n changes f the tryptphan flurescence f spectrin were bserved (Fig. 1C). At lipid: prtein rati f 1400 mre than 35% f initial spectrin flurescence was quenched by PE suspensins (Sikrski et al. 1987a). # x LIPOPROTEIN [ml/ml] Figure 2. The effect f snicated dileyl-phsphatidylethanlamine n the intrinsic flurescence f spectrin. Experiment was carried ut in the clumn buffer (Fig. 1 legend) -- r with 0.15 ml/1 NaCl in this buffer -A-. Fr cmparisn, the effect f natural PE (filled symbls and straight lines) in the same buffers is shwn PE [uml/l] The effect f lys-pe n PE suspensin-spectrin interactin was rather small exhibiting a tendency t increase at lw (up t abut 4% at 50 /iml/1) and t decrease (up t abut 3% at 160 /tml/1) at higher cncentratins f lys-pe f the PE effect n the flurescence f spectrin (results nt shwn). In the case f lys-ps, n effect culd be detected f the flurescence f PE-spectrin cmplex (data nt shwn). The abve data might suggest a rle f the presence f bth fatty acid chains and cnsequently micelle/bilayer/hii phase structure (Cullis and de Kruijff, 1978) fr the interactin f PE with spectrin. T test this hypthesis the effect f snicated dileyl-pe suspensin n the intrinsic flurescence f spectrin was analyzed. The results are shwn in Fig. 2. At lw inic strength, at lw DOPE cncentratins quenching f the intrinsic flurescence f spectrin by DOPE was abut 10% and was similar t the effect induced by natural PE (Fig. 2). At higher DOPE cncentratins, n quenching and even an increase f the flurescence culd be bserved. At 0.2 ml/1 NaCl, at lw (up 30 /iml/1) DOPE cncentratins ~ 10% increase and then ( //ml/1 DOPE) 10% quenching and at higher DOPE cncentratins up t 50% increase f flurescence was bserved. This increase was nt due t the light scattering effect since the flurescence f each sample was crrected fr light scattering (see Materials and Methds). Fr cmparisn the data n the effect f natural PE suspensin at the same cncentratins and the same inic cnditins are shwn (Fig. 2). In spite small differences between the effects at lw and mderate inic strength, presented abve data may indicate a distinct effect f DOPE, HII phase frming lipid [bilayer - HII phase transitin temperature

5 Spectrin Interactin with Nnbilayer Phsphlipids 61 ~ 12 C (Ellens et al. 1986)] n islated spectrin intrinsic flurescence frm that f natural PE suspensins. In cnclusin the results bth f lys-pe and DOPE interactin with spectrin might indicate an imprtance f bilayer structure f PE aggregates fr this interactin. The plymrphic state f lipid has nt been taken int the cnsideratin in the descriptin f physilgical significance f membrane skeletn - membrane bilayer interactin (Mmbers et al. 1980; Maksymiv et al. 1987; Sikrski et al. 1987a; Bitbl et al. 1989). It shuld be nted again that abve cnclusin cncerns nly PE species, since n essential changes in the effect f lys- PC and lys-ps n intrisic flurescence f spectrin cmpare t thse f PC and PS suspensins were bserved. It was als fund previusly that erythrcyte spectrin bund many amphipatic, micelle-frming cmpunds as fatty acids, brminated fatty acids and catinic and aninic detergents (Isenberg et al. 1981; Sikrski et al. 1987b; Kahana et al. 1992). Further studies n the physilgical significance f PE - spectrin interactin and in particular, the effect f plymrphic state f this phsphlipid suspensin are t be cnducted. Acknwledgements. This research was supprted by KBN Grant N t A.F.S. and frm Medical Schl Research Fund. The authrs thank Mr A. Pla fr excellent technical assistance. References Bartlett G. R. (1959): Phsphrus assay in clumn chrmatgraphy. J. Bil. Chem. 234, Bennett V. (1990): Spectrin-based membrane skeletn: a multiptential adaptr between plasma membrane and cytplasm. Physil. Rev. 70, Bitbl M., Dempsey C, Watts A., Devaux P. F (1989): Weak interactin f spectrin with phsphatidylchline-phsphatidylserine multilayers; a 2 H and 31 P NMR study. FEBS Lett, 44, Bradfrd M. M. (1976): A rapid and sensitive methd fr the quantitatin f micrgram quantities f prtein utilizing the principle f prtein dye binding. Anal. Bichem. 72, Cullis P. R., de Kruijff B. (1978): Lipid plymrphism and the functinal rles f lipid in the bilgical membrane. Bichim. Biphys. Acta 507, Derick L. H., Liu S. C, Chishti A. H., Palek J. (1992): Prtein immunlcalizatin in the spread erythrcyte membrane skeletn. Eur. J. Cell Bil. 57, Ellens H., Bentz J., Szka F. C. (1986): ph-induced destabilizatin f phsphatidylethanlamine-cntaining lipsmes: rle f bilayer cntact. Bichemistry USA 25, Gdman S. R., Krebs K. E., Whitfield C. F., Riederer B. M., Zagn I. S (1988): Spectrin and related mlecules. CRC Crit. Rev. Bichem. 23, Haest C. W. M., Plasa G., Kamp D., Deuticke B. (1978): Spectrin as a stabilizer f the phsphlipid asymmetry in the human erythrcyte membrane. Bichim. Biphys. Acta 509, 21 32

62 Michalak et al. Isenberg H., Kenna J. G., Green N. M., Gratzer W. B. (1981): Binding f hydrphbic ligands t spectrin. FEBS Lett. 129, 109 112 Julian R. L., Kimelberg H. K., Papahadjpuls D.

6 62 Michalak et al. Isenberg H., Kenna J. G., Green N. M., Gratzer W. B. (1981): Binding f hydrphbic ligands t spectrin. FEBS Lett. 129, Julian R. L., Kimelberg H. K., Papahadjpuls D. (1971): Synergistic effects f a membrane prtein (spectrin) and Ca n the permeability f phsphlipid vesicles. Bichim. Biphys. Acta 241, Kahana E., Pinder J. C., Smith K. S., Gratzer W. B. (1992): Flurescence quenching f spectrin and ther red cell membrane cytskeletal prteins. Bichem. J. 282, Maksymiv R., Sui S. F., Gaub H., Sackmann E. (1987): Electrstatic cupling f spectrin dimers t phsphatidylserine cntaining lipid lamellae. Bichemistry USA 26, Michalak K., Bbrwska M., Sikrski A. F. (1993): Interactin f bvine erythrcyte spectrin with aminphsphlipid lipsmes. Gen. Physil. Biphys. 12, Mmbers C, De Gier J., Demel R. A., and Van Deenen L. L. M. (1980): Spectrinphsphlipid interactin. A mnlayer study. Bichim. Biphys. Acta 603, Sahr K. E., Laurila P., Ktula L., Scarpa A., Cupal E., Let T., Linnenbach A. J., Winkelman J. C, Speicher D. W., Marchesi V. T, Curtis P. J., Frget B. G. (1990): The cmplete cdna and plypeptide sequences f human erythrid a-spectrin. J. Bil. Chem. 265, Sikrski A. F., Kuczek M. (1985): Labelling f erythrcyte spectrin in situ with phenylisthicyanate. Bichim. Biphys. Acta 820, Sikrski A. F., Michalak K., Bbrwska M. (1987a): Interactin f spectrin with phsphlipids. Quenching f spectrin intrinsic flrescence by phsphlipid suspensins. Bichim. Biphys. Acta 904, Sikrski A. F., Michalak K., Bbrwska M., Kzubek A. (1987b): Interactin f spectrin with sme amphipatic cmpunds. Studia Biphys. 121, Winkelman J. C, Chang J. G., Tse W. T., Scarpa A. L., Marchesi V. T., Frget B. G. (1990): Full length sequence f the cdna fr human erythrid /3-spectrin. J. Bil. Chem. 265, Final versin accepted December 29, 1993

Interaction of Bovine Erythrocyte Spectrin with Aminophospholipid Liposomes

Gen. Physiol. Biophys. (1993), 12, 163 170 163 Interaction of Bovine Erythrocyte Spectrin with Aminophospholipid Liposomes K. MICHALAK 1, M. BBRWSKA 1 and A. F. SIKRSKI 2 1 Department of Biophysics, Academy