ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 189, No. 2, August, pp. 336-343, 1978
The Interaction of Calcium and Procaine with Hepatocyte and Hepatoma Tissue Culture Cell Plasma Membranes Studied by Fluorescence Spectroscopy’ SHIRLEY Department
of Biochemistry,
CHENG, University
H. M. McQUEEN, of Southern
California
AND School
DANIEL of Medicine,
LEVY” Los Angeles,
California
90033 Received June 20, 1977; revised November
23, 1977
The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) has been used to investigate the properties of plasma membranes derived from normal hepatocytes and from hepatoma tissue culture (HTC) cells as well as used to study the effects of Cal+ and procaine on these membrane syst,ems. The interaction of ANS with hepatocyt,e plasma membranes (50 nmol/mg protein; K,, = 120 PM) resulted in a marked enhancement of fluorescence and a 20-nm blue shift. Both Cal+ and procaine further increased the fluorescence intensity. Binding studies showed no alteration in the number of ANS binding sites but a significant decrease in Ku (40-50 PM). Procaine was also shown to completely displace Ca” from the membrane. The interaction of ANS with HTC cell plasma membranes again resulted in an enhancement in fluorescence intensity but with different binding properties (102 nmol/mg protein; KU = 74 PM) from the hepatocyte system. The addition of Ca” resulted in the formation of high and low affinity ANS binding sites as shown by Scatchard plot analysis with K,, values of 15 PM and 50 PM. The effect of procaine on ANS fluorescence in the normal and transformed cell membranes was indistinguishable; however, in the latter system procaine only displaced 60% of the bound Ca”‘. These studies suggest several structural and binding alterations between plasma membranes derived from hepatocytes and HTC cells.
The surface of mammalian cells has been shown to play a critical role in the regulation of cellular activity through processes such as binding of hormones, drugs, and ions. The characterization of a variety of membrane systems has been the subject of extensive investigations (l-5). In malignant cells, altered cellular behavior can be understood in part in terms of modifications in plasma membrane structure and function (6-8). The interaction of Ca”+ and anesthetics such as procaine with plasma membranes has been shown to have profound effects on these complex systems, such as the regulation of membrane fluidity and the transmembrane control of surface receptors ’ This investigation was supported by a research grant (CA 14089) and a Cancer Research Training Fellowship for S.C. (CA 05297), from the National Institutes of Health, and a grant from the American Diabetes Association, Southern California Affiliate. ’ To whom correspondence should be addressed. 336 0003-9861/78/1892-0336$02.00/O Copyright 0 1078by Academic Press, Inc. All rights of reproduction in any form reserved.
(9-17). The effect of anesthetics on Ca’+membrane interaction has also been documented (9, 15). The technique of fluorescence spectroscopy has been used to probe many parameters of membrane structure using a variety of extrinsic probe molecules (18). The anionic probe, l-anilinonaphthalene-B-sulfonate (ANS),” has been used to study the effect of cations such as Ca’+ on the properties of several membrane systems (19-24). The effects of anesthetics on membrane structure have also been examined using fluorescence techniques (23, 25-27). In this study we report the use of ANS as a fluorescent probe to investigate the properties of plasma membranes derived from normal hepatocytes and from HTC cells and the effects of Ca”+ and procaine on these complex systems. A preliminary report of this ’ Abbreviations used: ANS, l-anilinonapbthalene8-sulfonate; HTC, hepatoma tissue culture.
EFFECT
OF PROCAINE
AND
work has been presented in abstract form cm. MATERIALS
AND
METHODS
Procaine-HCl and ANS were obtained from Sigma Chemical Co. ANS was recrystallized several times from a saturated MgClr solution as previously described (29). All other reagents were of analytical grade. 4”Ca” was purchased from International Chemical and Nuclear Corporation as CaCIL in 0.5 N HCl (20.8 mCi/mg calcium). Millipore filters, type HAWP, with pore size of 0.45 pm, were obtained from Millipore Corporation. of plasma membranes. Hepatocyte Isolation plasma membranes were isolated from the livers of male Sprague-Dawley rats fed ad libitum, using a sucrose density gradient procedure, according to the method of Neville (30), as modified by Ray (31), and by the aqueous two-phase polymer system developed by Lesko et al. (32). Protein concentrations were established by the method of Lowry et al. (33), as modified by Hartree (34). Membrane purity from the two procedures afforded similar results as adjudged from the activities of marker enzymes for plasma membranes (5’.nucleotidase (32)), endoplasmic reticulum (glucose-Gphosphatase (35)) and mitochondria (succinate dehydrogenase (36)). HTC cells were grown as suspension cultures in Swim’s 77 medium supplemented with 5% fetal calf serum and 5% calf serum as previously described (37). Under these conditions the cells had a doubling time of 24 h. Isolation of HTC cell plasma membranes was effected by suspending the cells (8 x 10’) in 20 ml of 1 mM NaHCO., buffer, pH 7.5, containing 75 mM NaCl and 2 mM CaCL. The cells were allowed to stand on ice for 10 min and then homogenized in a Dounce homogenizer (Kontes Glass Co.) with the A-pestle (30-40 strokes) until 85% of the cells were broken, as determined by phase-contrast microscopy. The homogenate was diluted to 40 ml and subjected to a lowspeed spin (163~; 10 min). The post-nuclear supernatant was centrifuged at 10,OOOgfor 30 min. The resultant pellet was applied in the homogenizing buffer, adjusted to 10% sucrose (6 ml), to the top of two discontinuous sucrose gradients consisting of 8 ml of 39%, 8 ml 37%, 7 ml 33% and 7 ml 29%. The tubes were centrifuged at 112,500g for 2 h in a Beckman SW 27 rotor. The material at the 33-37% interface was collected, diluted to 50 ml with the homogenizing medium, and centrifuged for 30 min at 27,578g. Membrane purity was estimated by enzyme marker assays for 5’-nucleotidase (32), NADH-diaphorase (38), glucase-6-phosphatase (35), and succinate dehydrogenase (36). HTC cell plasma membranes were also thin sectioned and analyzed by electron microscopy. Fluorescence measurements. Fluorescence measurements were made on a Perkin-Elmer Model MPF4 spectrophotometer at a 90” angle to the exciting beam with 6-run slit widths for both excitation and
Ca”
ON PLASMA
MEMBRANES
337
emission channels and a 390~nm cutoff filter. For all studies the excitation wavelength for ANS was 365 nm. All measurements were carried out at 25 + O.l”C, using a thermostated cuvette holder. Light scattering effects were insignificant under the conditions used in these studies. Plasma membranes (0.1 mg/ml) were suspended in 0.25 M sucrose, 5 mM Tris-HCI, pH 7.4, and titrated with aliquots of 5 IIIM ANS. Fluorescence spectra were also obtained of membranes suspended in 0.1 M Tris-HCl (pH 7.4). Binding of ANS to the membrane system was analyzed by a double reciprocal plot (39) and Scatchard plot (40,41) to afford average apparent dissociation constants (K,,) and number of ANS binding sites. Lines were fitted by linear regression analysis. The binding characteristics of ANS were also determined in the presence of Ca” and procaine. The effect of procaine on Ca” binding was evaluated by the addition of the anesthetic to the membrane pretreated with Ca”’ and ANS. Fluorescence spectra were also obtained by the addition of Ca”+ and procaine to ANS-treated membranes. Calcium binding assay. 4’Ca’+ binding to the membrane preparations was measured by a Millipore filtration technique as previously described (42). The effect of procaine on ?a”’ binding was determined by the subsequent addition of the anesthetic to the Ca’)+treated membranes. After a 10 min incubation at 37°C the residual Ca” was determined by Millipore filtration. RESULTS
Hepatocyte
and HTC Plasma Membranes
Plasma membranes were isolated from liver and HTC cells. Analysis of the enzymatic activities of this cell fraction is shown in Table I. Both preparations were significantly enriched in 5’-nucleotidase activity, and were substantially free of contamination from mitochondria and endoplasmic reticulum. HTC cell plasma membranes appeared to show a slight enrichment in endoplasmic reticulum when assayed by glucase-6-phosphatase. This may have been caused by a nonspecific reaction of plasma membrane associated alkaline phosphatase. When the preparation was evaluated using NADH-diaphorase, another enzyme marker for endoplasmic reticulum, the preparation showed greatly diminished amounts, suggesting very slight contamination by this subcellular fraction. Electron microscopy of plasma membranes derived from HTC cells (Fig. 1) confirmed the enzymatic data, indicating the absence of significant amounts of other subcellular organelles.
CHENG,
McQUBBN, TABLE
ENZYMATIC
ANALYSIS
OF HEPATOCYTE
AND 1
AND HTC CELL PLASMA
mogenate
HTC cell homogenate
HTC cell plasma membrane
1.6 1.1 2.5
36 0.10 1.2
0.55 0.62 0.15 250
6.72 0.12 0.25 35
from HTC
cells. Length
5’-Nucleotidase (pmoles/30 min/mg protein) Succinate dehydrogenase (,amoles/h/mg protein) Glucose-Gphosphatase ((.rmoles/30 min/mg protein) NADH-diaphorase (pmoles/min/mg protein)
micrograph
of plasma membranes
Fluorescence and Binding Studies of Hepatocyte Plasma Membranes The addition of a hepatocyte plasma membrane suspension to an aqueous ANS solution which exhibits minimal fluorescence (quantum yield: 0.004) resulted in a large enhancement of fluorescence and a significant blue shift of the emission maximum. Figure 2 shows the emission spectrum of 20 PM ANS in 0.25 M sucrose, 5 mM Tris-HCl, pH 7.4. The addition of the plasma membrane resulted in a 2.5fold increase in fluorescence and a 20-nm blue shift. The additional of 5 mu Ca2+ resulted in a further 3-fold increase in fluorescence intensity and an additional blue shift from 500 nm to 470 nm (Fig. 2). Similar results
MEMBRANES
Hepatocyte plasma membrane
Liver
FIG. 1. Electron denotes 0.5 pm.
LEVY
ho-
derived
of bar
were obtained in 100 mM Tris-HCl (pH 7.4). The apparent dissociation constant (KU) of ANS binding to the hepatocyte plasma membrane was calculated from a Scatchard plot (Fig. 3) to be 120 f 5 PM. The KD value was shown to be dependent on the Ca2+ concentration as shown in Fig. 4 where a maximal effect on the KD value was reached at a Cal+ concentration of approximately 1.0 mM. The Scatchard plot (Fig. 3) showed the number of ANS binding sites on the hepatocyte plasma membrane to be 50 + 5 nmol/mg membrane protein. Furthermore, the addition of Ca2+ had no significant effect on the number of ANS binding sites (Fig. 3, Table II). The addition of procaine to ANS-treated
EFFECT
OF PROCAINE
AND
Ca”+ ON PLASMA
339
MEMBRANES
1
WAVELENGTH,
“m
FIG. 2. Fluorescence emission spectra of ANS bound to hepatocyte plasma membranes in the presence and absence of Ca”+ at 25 + O.l”C. Plasma membranes (100 pg/ml) were suspended in 0.25 M sucrose, 5 mM Tris-HCl (pH 7.4), and treated with 20 PM ANS with 5 mM Ca’+ (- - -); without Ca”+ (----). ANS in sucrose-Tris buffer (- - .-).
I IO
I
I 20
I
I
40
5.0
COCI,, mM FIG. 4. The effect of Ca” concentration on the Kn value for the binding of ANS to hepatocyte plasma membranes.
TABLE
Y
1
3.0
II
CHARACTERISTICSOF ANS BINDINGTOHEPATOCYTE PLASMAMEMBRANES.EFFECTSOFC~"+ AND PROCAINE~ Binding sites (nmol/mg protein)
Additions to hepatocyte plasma membranes
50 f 5 54 + 4 60 f 5
120 f 5 40 f 2 54 f 5
None 5 mM Ca? 20 mM Procaine
a Plasma membranes (0.1 mg/ml) were suspended in 0.25 M sucrose, 5 mM Tris-HCl (pH 7.4) and titrated with ANS. The values reported are the mean f SE. FIG. 3. Scatchard plot analysis of ANS binding to hepatocyte plasma membranes in the presence (A-A) and absence (W---O) of 5 mM Ca”+. The dissociation constants and number of binding sites are shown in Table II.
hepatocyte plasma membranes resulted in an increase in fluorescence intensity with only a slight blue shift. Scatchard plot analysis gave a Ko value of 54 -+ 5 PM with only a slight change in the number of ANS binding sites (Table II). When procaine was added to membranes pretreated with 5 mM Ca”+, a net decrease in fluorescence was observed as shown in Fig. 5. The effect of the anesthetic on Ca”’ binding is demonstrated in Fig. 6. The fluorescence data are obtained by subtracting the fluorescence contribution from procaine in the absence of Ca2’ from that obtained in the presence of Ca2+ (Fig. 5). At 10 mM procaine, the
I
1
0
IO
I
20 PROCAINE,
L
30
I
40 mM
FIG. 5. Effect of procaine on membrane-bound ANS fluorescence in the presence (A-A) and absence (U) of 5 mM Ca’+.
340
CHENG,
McQlJEEN,
fluorescence of membrane-bound ANS in the presence of 5 mM Ca’+ is 50% of its initial value. The direct measurerpent of membrane-bound 45Ca2+by Millipore filtration as a function of procaine concentration is also shown in Fig. 6 where the addition of 10 mM procaine resulted in the displacement of 50% of the Ca’+. Binding studies afforded a value of 14 + 2 nmol Ca”+/mg membrane protein. This data suggest a close correlation between the fluorescence and radioactive assays for Ca2+ binding.
AND
7. A Scatchard plot analysis (Fig. 8) indicated a Ku value of 74 +- 2 pM with the number of ANS binding sites to be 102 + 3 nmol/mg membrane protein (Table III). As
W
,”
The addition of HTC cell plasma membranes to an aqueous ANS solution resulted in a 2-fold increase in fluorescence and emission maximum shift from 525 to 495 nm. The addition of 2.5 mM Ca2+ resulted in a 20-nm blue shift and a further 2-fold increase in fluorescence, while 5 mM Ca2+ afforded a 2.3-fold increase as shown in Fig.
z” z m
-60
:
-40
2 c-l
30
W
Fluorescence Studies of HTC Cell Plasma Membranes
-80
LEVY
=: W
20
E ZJ -I L
IO
WAVELENGTH,
nm
7. Fluorescence emission spectra of ANS bound to HTC cell plasma membranes in the presence and absence of Ca” at 25 + O.l”C. Plasma membranes (100 pg/ml) were suspended in 0.25 M sucrose, 5 mM Tris-HCl (pH 7.4) and treated with 20 l&M ANS with 2.5 mM Ca’+ (. .); with 5 mM Ca” Ca” C---L without Ca’+ (-); ANS in sucrose-Tris buffer (-.-.-). FIG.
;
z 20
0
IO
20 PROCAINE,
30
40 mM
FIG. 6. The effect of procaine on Ca’+ binding to hepatocyte plasma membranes (U) and HTC plasma membranes (M) as measured by ANS fluorescence. The effect of procaine on Ca’+ binding to hepatocyte plasma membranes (A-A) and HTC plasma membranes (a-n) as measured by Millipore filtration using 4’Cas+.
FIG. 8. Scatchard plot analysis of ANS binding to HTC cell plasma membranes in the presence (M) and absence (M) of 5 mM Ca’+. The dissociation constants and binding site numbers are shown in Table III.
TABLE CHARACTERISTICS
OF ANS
BINDING
CELL
III PLASMA
MEMBRANES.
EFFECTS
K, (PM) 74 f 2 50 + 3
OF Ca’+O
High affinity
Low affinity
Addition to HTC cell plasma membranes
None 5 mM ca’+
TO HTC
Binding sites (nmol/mg protein)
Binding sites (nmol/mg protein) 102 f 3 127 + 5
15 -t 1
’ Plasma membranes (0.1 mg/ml) were suspended in 0.25 M sucrose, 5 mM Tris-HCl with ANS. The values reported are the mean k S.E.
90 * 3 (pH 7.4) and titrated
EFFECT
OF PROCAINE
AND
opposed to hepatocyte plasma membranes, the addition of 5 mM Ca2+ induced the appearance of a high affinity binding site with a KU of 15 f 1 PM and number of ANS binding sites to be 90 + 3 nmol/mg membrane protein (Fig. 8). The number of ANS binding sites in the low affinity class was shown to be 127 f 5 nmol/mg membrane protein with a Ku value of 50 Ifr 3 PM (Table III). The addition of procaine to ANStreated HTC cell plasma membranes resulted in an enhancement of fluorescence identical to that observed for hepatocyte membranes. The effect of procaine on Ca2+ binding by HTC cell plasma membranes was carried out as previously described for hepatocyte plasma membranes. As assayed by ANS fluorescence, 40 mM procaine resulted in the displacement of only 60% of the membrane associated Cast as opposed to complete displacement in the hepatocyte system (Fig. 6). Direct measurement of 45Ca2+ binding by Millipore filtration as a function of procaine concentration afforded values which were in excellent agreement with the fluorescence studies. In the absence of the anesthetic, binding studies afforded a value of 20 f 2 nmol Ca”‘/mg HTC cell membrane protein. IXKXJSSION
In this report the anionic fluorescent probe, ANS, has been used to investigate membrane properties of normal and neoplastic plasma membranes. ANS is thought to bind to polar-apolar interfaces with the chromophoric group extending into the hydrocarbon core and the sulfonate group located in the plane of the membrane polarhead-groups (43, 44). Evidence also suggests that ANS may bind to protein components of the membrane (18, 45). The interaction of the probe with normal hepatocyte plasma membranes resulted in a large enhancement in fluorescence and a blue shift in the emission maximum indicative of the binding of the probe to nonpolar sites on the membrane. The effect of Ca” on this system could be understood in terms of a suppression of electrostatic repulsion between ANS and the negative surface phosphate and/or carbohydrate groups by the binding of the cation or by
Ca”’
ON PLASMA
MEMBRANES
341
an ion induced conformational change. This enhancement of fluorescence could be due to a decrease in the KD value of ANS, a decrease in binding site polarity with a resultant increase in quantum yield or an increase in the number of ANS binding sites. In previous studies the effects of ions have been attributed to an increase in ANS binding capacity facilitated by suppression of eleckostatic repulsion (18, 19). This report suggests, however, that Ca’+ does not alter the number of ANS binding sites but does have a significant effect on the KU for ANS (Table II). Several reports have shown that local anesthetics cause ANS fluorescence enhancement in the presence of several membrane systems (22, 46-48). This effect may again be understood in terms of a) shielding of anionic membrane constituents which would facilitate the penetration of the anionic probe (ANS) into the hydrophobic region of the membrane, or b) alterations in membrane binding sites resulting from architectural modifications induced by the anesthetic. Local anesthetics have been shown to interact electrostatically with phospholipids (9, 15). Hydrophobic interactions have also been shown to play a significant role (49). Binding studies in the hepatocyte system again suggest that fluorescence enhancement results from a decrease in Ku and not from an alteration in the number of ANS binding sites (Table 1). The effect of anesthetics on Ca2+ binding has been previously reported (9, 15). This report shows that 40 mM procaine can completely displace Cae+ from the hepatocyte membrane system as measured by ANS fluorescence as well as by 4”Ca”+ binding (Fig. 6). The interaction of ANS with HTC cell plasma membranes suggested the existence of significant alterations in membrane structure compared to normal hepatocytes. The fluorescence intensity and emission maximum were quite similar to the hepatocyte system; however, binding studies showed that there were approximately twice the number of ANS binding sites and a 38% decrease in the Ku value (Table III), suggesting significant differences in the environment of the ANS binding sites in the
342
CHENG,
McQUEKN,
two systems, possibly as a result of altered lipid and/or protein composition. The interaction of Ca2+ with the HTC cell membrane system resulted not only in a decrease in the Ku value, as observed for hepatocyte plasma membranes but in an increase in the number of ANS binding sites as well as the appearance of a new class of high affinity binding sites (Fig. 8), suggesting that Ca2’ affects the HTC cell plasma membrane architecture in a significantly different way than observed for normal hepatocytes. The appearance of additional ANS binding sites as well as a new class of binding sites may be due to cation induced conformational changes resulting in the deshielding of previously inaccessible sites. The effect of procaine on ANS fluorescence in the two systems was indistinguishable; however, the ability to displace Ca2+ from the HTC cell membrane system was reduced by 40%, suggesting that Ca2+ was either bound more tightly or at sites inaccessible to the anesthetic. The effect of Ca”+ and anesthetics on plasma membrane structure and membrane associated cytoskeletal proteins has been demonstrated (9-17). The results presented in this report indicate that (a) ANS is a sensitive probe of membrane architecture and can be used to elucidate the effects of Ca’+ and anesthetics such as procaine on complex membrane systems, and (b) that significant differences in membrane dynamics exist between hepatocytes and HTC cell plasma membranes.
LEVY
Publishing Co., Amsterdam. 9. SEEMAN, P. (1972) Pharmacol. Rev. 24, 583-655. 10. POSTE, G., PAPAHADJOPOULOS, D., JACOBSON, K., AND VAIL, W. J. (1975) Biochim. Biophys. Acta
394, 520-539. 11. ADAMS, D., MARKES, CARRAWAY, K. L.
M. E., LEIVO, W. J., AND (1976) Biochim. Biophys.
Acta 426, 38-45. 12. SCHNEPEL, G. H., HECNER, D., AND SCHUMMER, U. (1974) Biochim. Biophys. Acta 367, 67-74. 13. ROTH, S., AND SEEMAN, P. (1971) Nature New Biol. 231,284-285. 14. POSTE, G., AND REEVE, P. (1972) Exp. Cell Res. 72,556-560. 15. PAPAHADJOPOULOS, D. (1972) Biochim. Biophys. Acta 265, 169-186. 16. POSTE, G., PAPAHADJOPOULOS, D., AND NICOLSON, G. (1975) Proc. Nat. Acad. Sci. USA 72, 4430-4434. 17. NICOLSON, G. L., SMITH, J. R., AND POSTE, G.
(1976) J. Cell Biol. 68.395-402. 18. AZZI, A. (1975) Quart. Reu. Biophys. 8, 237-316. 19. KRISHNAN, K. S., AND BALARAM, P. (1976) Arch. Biochem. Biophys. 174.420-430. 20. VANDERKOOI, J., AND MARTONOSI, A. (1969) Arch. Biochem. Biophys. 133, 153-163. 21. RUBALCAVA, B., MUNOZ, D. M., AND GITLER;~. 8, 2742-2749. (1969) Biochemistry 22. GOMPERTS, B., LANTELME, F., AND STOCK, R.
(1970) J. Membrane 23. MADEIRA,
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V. M. C., AND ANTUNES-MADEIRA,
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24. DIONISI, O., GALEOTTI, STAN, P., AND AZZI,
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28. LEVY, D., AND CHENG, S. (1976) Federation Proc. 35, 1451. 29. DODD, G. H., AND RADDA, G. K. (1969) Biochem. J 114,407-417. 30. NEVILLE, D. M. (1960) J. Biophys. Biochem. Cy-
tol. 8,413-422. 31. RAY, T. K. (1970) Biochim. Biophys. Acta 196, 1-9. 32. LESKO, L., DONLON, M., MARINETTI, G. V., AND HARE, G. D. (1973) Biochim. Biophys. Acta 311, 173-179. 33. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL. R. J. (1951) J. Biol. Chem. 193, 265-275. 34. HARTREE, E. F. (1972) Anal. Biochem. 48, 422-427. 35. ARSON, N., AND TOUSTER, 0. (1974) in Methods in Enzymology (Fleischer, S., and Packer, L., eds.), Vol. 31, Part A, pp. 90-102, Academic
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Press, New York. 36. KING, T. E. (7967) in. Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., eds.), Vol. 10, pp. 322-331, Academic Press, New York. 37. MACKENZIE, C. W., III, AND STELLWAGEN, R. H. (1974) J. Biol. Chem. 249,5755-5762. 38. WALLACH, D. F. H., AND KAMAT, V. B. (1966) in Methods in Enzymology (Neufeld, E. F., and Ginsburg, V., eds.), Vol. 8, pp. 165-166, Academic Press, New York. 39. CHEUNG, H. C., AND MORALES, M. F. (1969) Biochemistry 8,2177-2182. 40. AZZI, A., GHERARDINI, P., AND SANTATO, M. (1971) J. Biol. Chem. 246, 2035-2042. 41. AZZI, A. (1974) in Methods in Enzymology (Fleischer, S., and Packer, L., eds.), Vol. 32, pp. 234-246, Academic Press, New York.
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ON PLASMA
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42. SHLATZ, L., AND MARINETTI, G. V. (1972) Biochim. Biophys. Acta 290,70-83. 43. LESSLAUER, W., CAIN, J. E., AND BLASIE, J. K. (1972) Proc. Nut. Acad. Sci. USA 69,1499-1503. 44. WAGGONER, A. S., AND STRYER, L. (1970) Proc. Nat. Acad. Sci. USA 67,579-589. 45. AUGUSTIN, J., AND HASSELBACH, W. (1973) Eur. J. Biochem. 35, 114-121. 46. FEINSTEIN, M. B., SPERO, L., AND FELSENFELD, H. (1970) FEBS Lett. 6, 245-248. 47. CHANCE, B., AZZI, A., MELA, L., RADDA, G., AND VAINCO, H. (1969) FEBS Lett. 3, 10-13. 48. KOBLIN, D. D., KAUFMAN, S. A., AND WANG, H. H. (1973) Biochem. Biophys. Res. Commun. 53, 1077-1083. 49. FERNANDEZ, M. S., AND CERBON, J. (1973) Biochim. Biophys. Acta 298,8-14.
The Interaction of Calcium and Procaine with Hepatocyte and Hepatoma Tissue Culture Cell Plasma Membranes Studied by Fluorescence Spectroscopy’ SHIRLEY Department
of Biochemistry,
CHENG, University
H. M. McQUEEN, of Southern
California
AND School
DANIEL of Medicine,
LEVY” Los Angeles,
California
90033 Received June 20, 1977; revised November
23, 1977
The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) has been used to investigate the properties of plasma membranes derived from normal hepatocytes and from hepatoma tissue culture (HTC) cells as well as used to study the effects of Cal+ and procaine on these membrane syst,ems. The interaction of ANS with hepatocyt,e plasma membranes (50 nmol/mg protein; K,, = 120 PM) resulted in a marked enhancement of fluorescence and a 20-nm blue shift. Both Cal+ and procaine further increased the fluorescence intensity. Binding studies showed no alteration in the number of ANS binding sites but a significant decrease in Ku (40-50 PM). Procaine was also shown to completely displace Ca” from the membrane. The interaction of ANS with HTC cell plasma membranes again resulted in an enhancement in fluorescence intensity but with different binding properties (102 nmol/mg protein; KU = 74 PM) from the hepatocyte system. The addition of Ca” resulted in the formation of high and low affinity ANS binding sites as shown by Scatchard plot analysis with K,, values of 15 PM and 50 PM. The effect of procaine on ANS fluorescence in the normal and transformed cell membranes was indistinguishable; however, in the latter system procaine only displaced 60% of the bound Ca”‘. These studies suggest several structural and binding alterations between plasma membranes derived from hepatocytes and HTC cells.
The surface of mammalian cells has been shown to play a critical role in the regulation of cellular activity through processes such as binding of hormones, drugs, and ions. The characterization of a variety of membrane systems has been the subject of extensive investigations (l-5). In malignant cells, altered cellular behavior can be understood in part in terms of modifications in plasma membrane structure and function (6-8). The interaction of Ca”+ and anesthetics such as procaine with plasma membranes has been shown to have profound effects on these complex systems, such as the regulation of membrane fluidity and the transmembrane control of surface receptors ’ This investigation was supported by a research grant (CA 14089) and a Cancer Research Training Fellowship for S.C. (CA 05297), from the National Institutes of Health, and a grant from the American Diabetes Association, Southern California Affiliate. ’ To whom correspondence should be addressed. 336 0003-9861/78/1892-0336$02.00/O Copyright 0 1078by Academic Press, Inc. All rights of reproduction in any form reserved.
(9-17). The effect of anesthetics on Ca’+membrane interaction has also been documented (9, 15). The technique of fluorescence spectroscopy has been used to probe many parameters of membrane structure using a variety of extrinsic probe molecules (18). The anionic probe, l-anilinonaphthalene-B-sulfonate (ANS),” has been used to study the effect of cations such as Ca’+ on the properties of several membrane systems (19-24). The effects of anesthetics on membrane structure have also been examined using fluorescence techniques (23, 25-27). In this study we report the use of ANS as a fluorescent probe to investigate the properties of plasma membranes derived from normal hepatocytes and from HTC cells and the effects of Ca”+ and procaine on these complex systems. A preliminary report of this ’ Abbreviations used: ANS, l-anilinonapbthalene8-sulfonate; HTC, hepatoma tissue culture.
EFFECT
OF PROCAINE
AND
work has been presented in abstract form cm. MATERIALS
AND
METHODS
Procaine-HCl and ANS were obtained from Sigma Chemical Co. ANS was recrystallized several times from a saturated MgClr solution as previously described (29). All other reagents were of analytical grade. 4”Ca” was purchased from International Chemical and Nuclear Corporation as CaCIL in 0.5 N HCl (20.8 mCi/mg calcium). Millipore filters, type HAWP, with pore size of 0.45 pm, were obtained from Millipore Corporation. of plasma membranes. Hepatocyte Isolation plasma membranes were isolated from the livers of male Sprague-Dawley rats fed ad libitum, using a sucrose density gradient procedure, according to the method of Neville (30), as modified by Ray (31), and by the aqueous two-phase polymer system developed by Lesko et al. (32). Protein concentrations were established by the method of Lowry et al. (33), as modified by Hartree (34). Membrane purity from the two procedures afforded similar results as adjudged from the activities of marker enzymes for plasma membranes (5’.nucleotidase (32)), endoplasmic reticulum (glucose-Gphosphatase (35)) and mitochondria (succinate dehydrogenase (36)). HTC cells were grown as suspension cultures in Swim’s 77 medium supplemented with 5% fetal calf serum and 5% calf serum as previously described (37). Under these conditions the cells had a doubling time of 24 h. Isolation of HTC cell plasma membranes was effected by suspending the cells (8 x 10’) in 20 ml of 1 mM NaHCO., buffer, pH 7.5, containing 75 mM NaCl and 2 mM CaCL. The cells were allowed to stand on ice for 10 min and then homogenized in a Dounce homogenizer (Kontes Glass Co.) with the A-pestle (30-40 strokes) until 85% of the cells were broken, as determined by phase-contrast microscopy. The homogenate was diluted to 40 ml and subjected to a lowspeed spin (163~; 10 min). The post-nuclear supernatant was centrifuged at 10,OOOgfor 30 min. The resultant pellet was applied in the homogenizing buffer, adjusted to 10% sucrose (6 ml), to the top of two discontinuous sucrose gradients consisting of 8 ml of 39%, 8 ml 37%, 7 ml 33% and 7 ml 29%. The tubes were centrifuged at 112,500g for 2 h in a Beckman SW 27 rotor. The material at the 33-37% interface was collected, diluted to 50 ml with the homogenizing medium, and centrifuged for 30 min at 27,578g. Membrane purity was estimated by enzyme marker assays for 5’-nucleotidase (32), NADH-diaphorase (38), glucase-6-phosphatase (35), and succinate dehydrogenase (36). HTC cell plasma membranes were also thin sectioned and analyzed by electron microscopy. Fluorescence measurements. Fluorescence measurements were made on a Perkin-Elmer Model MPF4 spectrophotometer at a 90” angle to the exciting beam with 6-run slit widths for both excitation and
Ca”
ON PLASMA
MEMBRANES
337
emission channels and a 390~nm cutoff filter. For all studies the excitation wavelength for ANS was 365 nm. All measurements were carried out at 25 + O.l”C, using a thermostated cuvette holder. Light scattering effects were insignificant under the conditions used in these studies. Plasma membranes (0.1 mg/ml) were suspended in 0.25 M sucrose, 5 mM Tris-HCI, pH 7.4, and titrated with aliquots of 5 IIIM ANS. Fluorescence spectra were also obtained of membranes suspended in 0.1 M Tris-HCl (pH 7.4). Binding of ANS to the membrane system was analyzed by a double reciprocal plot (39) and Scatchard plot (40,41) to afford average apparent dissociation constants (K,,) and number of ANS binding sites. Lines were fitted by linear regression analysis. The binding characteristics of ANS were also determined in the presence of Ca” and procaine. The effect of procaine on Ca” binding was evaluated by the addition of the anesthetic to the membrane pretreated with Ca”’ and ANS. Fluorescence spectra were also obtained by the addition of Ca”+ and procaine to ANS-treated membranes. Calcium binding assay. 4’Ca’+ binding to the membrane preparations was measured by a Millipore filtration technique as previously described (42). The effect of procaine on ?a”’ binding was determined by the subsequent addition of the anesthetic to the Ca’)+treated membranes. After a 10 min incubation at 37°C the residual Ca” was determined by Millipore filtration. RESULTS
Hepatocyte
and HTC Plasma Membranes
Plasma membranes were isolated from liver and HTC cells. Analysis of the enzymatic activities of this cell fraction is shown in Table I. Both preparations were significantly enriched in 5’-nucleotidase activity, and were substantially free of contamination from mitochondria and endoplasmic reticulum. HTC cell plasma membranes appeared to show a slight enrichment in endoplasmic reticulum when assayed by glucase-6-phosphatase. This may have been caused by a nonspecific reaction of plasma membrane associated alkaline phosphatase. When the preparation was evaluated using NADH-diaphorase, another enzyme marker for endoplasmic reticulum, the preparation showed greatly diminished amounts, suggesting very slight contamination by this subcellular fraction. Electron microscopy of plasma membranes derived from HTC cells (Fig. 1) confirmed the enzymatic data, indicating the absence of significant amounts of other subcellular organelles.
CHENG,
McQUBBN, TABLE
ENZYMATIC
ANALYSIS
OF HEPATOCYTE
AND 1
AND HTC CELL PLASMA
mogenate
HTC cell homogenate
HTC cell plasma membrane
1.6 1.1 2.5
36 0.10 1.2
0.55 0.62 0.15 250
6.72 0.12 0.25 35
from HTC
cells. Length
5’-Nucleotidase (pmoles/30 min/mg protein) Succinate dehydrogenase (,amoles/h/mg protein) Glucose-Gphosphatase ((.rmoles/30 min/mg protein) NADH-diaphorase (pmoles/min/mg protein)
micrograph
of plasma membranes
Fluorescence and Binding Studies of Hepatocyte Plasma Membranes The addition of a hepatocyte plasma membrane suspension to an aqueous ANS solution which exhibits minimal fluorescence (quantum yield: 0.004) resulted in a large enhancement of fluorescence and a significant blue shift of the emission maximum. Figure 2 shows the emission spectrum of 20 PM ANS in 0.25 M sucrose, 5 mM Tris-HCl, pH 7.4. The addition of the plasma membrane resulted in a 2.5fold increase in fluorescence and a 20-nm blue shift. The additional of 5 mu Ca2+ resulted in a further 3-fold increase in fluorescence intensity and an additional blue shift from 500 nm to 470 nm (Fig. 2). Similar results
MEMBRANES
Hepatocyte plasma membrane
Liver
FIG. 1. Electron denotes 0.5 pm.
LEVY
ho-
derived
of bar
were obtained in 100 mM Tris-HCl (pH 7.4). The apparent dissociation constant (KU) of ANS binding to the hepatocyte plasma membrane was calculated from a Scatchard plot (Fig. 3) to be 120 f 5 PM. The KD value was shown to be dependent on the Ca2+ concentration as shown in Fig. 4 where a maximal effect on the KD value was reached at a Cal+ concentration of approximately 1.0 mM. The Scatchard plot (Fig. 3) showed the number of ANS binding sites on the hepatocyte plasma membrane to be 50 + 5 nmol/mg membrane protein. Furthermore, the addition of Ca2+ had no significant effect on the number of ANS binding sites (Fig. 3, Table II). The addition of procaine to ANS-treated
EFFECT
OF PROCAINE
AND
Ca”+ ON PLASMA
339
MEMBRANES
1
WAVELENGTH,
“m
FIG. 2. Fluorescence emission spectra of ANS bound to hepatocyte plasma membranes in the presence and absence of Ca”+ at 25 + O.l”C. Plasma membranes (100 pg/ml) were suspended in 0.25 M sucrose, 5 mM Tris-HCl (pH 7.4), and treated with 20 PM ANS with 5 mM Ca’+ (- - -); without Ca”+ (----). ANS in sucrose-Tris buffer (- - .-).
I IO
I
I 20
I
I
40
5.0
COCI,, mM FIG. 4. The effect of Ca” concentration on the Kn value for the binding of ANS to hepatocyte plasma membranes.
TABLE
Y
1
3.0
II
CHARACTERISTICSOF ANS BINDINGTOHEPATOCYTE PLASMAMEMBRANES.EFFECTSOFC~"+ AND PROCAINE~ Binding sites (nmol/mg protein)
Additions to hepatocyte plasma membranes
50 f 5 54 + 4 60 f 5
120 f 5 40 f 2 54 f 5
None 5 mM Ca? 20 mM Procaine
a Plasma membranes (0.1 mg/ml) were suspended in 0.25 M sucrose, 5 mM Tris-HCl (pH 7.4) and titrated with ANS. The values reported are the mean f SE. FIG. 3. Scatchard plot analysis of ANS binding to hepatocyte plasma membranes in the presence (A-A) and absence (W---O) of 5 mM Ca”+. The dissociation constants and number of binding sites are shown in Table II.
hepatocyte plasma membranes resulted in an increase in fluorescence intensity with only a slight blue shift. Scatchard plot analysis gave a Ko value of 54 -+ 5 PM with only a slight change in the number of ANS binding sites (Table II). When procaine was added to membranes pretreated with 5 mM Ca”+, a net decrease in fluorescence was observed as shown in Fig. 5. The effect of the anesthetic on Ca”’ binding is demonstrated in Fig. 6. The fluorescence data are obtained by subtracting the fluorescence contribution from procaine in the absence of Ca2’ from that obtained in the presence of Ca2+ (Fig. 5). At 10 mM procaine, the
I
1
0
IO
I
20 PROCAINE,
L
30
I
40 mM
FIG. 5. Effect of procaine on membrane-bound ANS fluorescence in the presence (A-A) and absence (U) of 5 mM Ca’+.
340
CHENG,
McQlJEEN,
fluorescence of membrane-bound ANS in the presence of 5 mM Ca’+ is 50% of its initial value. The direct measurerpent of membrane-bound 45Ca2+by Millipore filtration as a function of procaine concentration is also shown in Fig. 6 where the addition of 10 mM procaine resulted in the displacement of 50% of the Ca’+. Binding studies afforded a value of 14 + 2 nmol Ca”+/mg membrane protein. This data suggest a close correlation between the fluorescence and radioactive assays for Ca2+ binding.
AND
7. A Scatchard plot analysis (Fig. 8) indicated a Ku value of 74 +- 2 pM with the number of ANS binding sites to be 102 + 3 nmol/mg membrane protein (Table III). As
W
,”
The addition of HTC cell plasma membranes to an aqueous ANS solution resulted in a 2-fold increase in fluorescence and emission maximum shift from 525 to 495 nm. The addition of 2.5 mM Ca2+ resulted in a 20-nm blue shift and a further 2-fold increase in fluorescence, while 5 mM Ca2+ afforded a 2.3-fold increase as shown in Fig.
z” z m
-60
:
-40
2 c-l
30
W
Fluorescence Studies of HTC Cell Plasma Membranes
-80
LEVY
=: W
20
E ZJ -I L
IO
WAVELENGTH,
nm
7. Fluorescence emission spectra of ANS bound to HTC cell plasma membranes in the presence and absence of Ca” at 25 + O.l”C. Plasma membranes (100 pg/ml) were suspended in 0.25 M sucrose, 5 mM Tris-HCl (pH 7.4) and treated with 20 l&M ANS with 2.5 mM Ca’+ (. .); with 5 mM Ca” Ca” C---L without Ca’+ (-); ANS in sucrose-Tris buffer (-.-.-). FIG.
;
z 20
0
IO
20 PROCAINE,
30
40 mM
FIG. 6. The effect of procaine on Ca’+ binding to hepatocyte plasma membranes (U) and HTC plasma membranes (M) as measured by ANS fluorescence. The effect of procaine on Ca’+ binding to hepatocyte plasma membranes (A-A) and HTC plasma membranes (a-n) as measured by Millipore filtration using 4’Cas+.
FIG. 8. Scatchard plot analysis of ANS binding to HTC cell plasma membranes in the presence (M) and absence (M) of 5 mM Ca’+. The dissociation constants and binding site numbers are shown in Table III.
TABLE CHARACTERISTICS
OF ANS
BINDING
CELL
III PLASMA
MEMBRANES.
EFFECTS
K, (PM) 74 f 2 50 + 3
OF Ca’+O
High affinity
Low affinity
Addition to HTC cell plasma membranes
None 5 mM ca’+
TO HTC
Binding sites (nmol/mg protein)
Binding sites (nmol/mg protein) 102 f 3 127 + 5
15 -t 1
’ Plasma membranes (0.1 mg/ml) were suspended in 0.25 M sucrose, 5 mM Tris-HCl with ANS. The values reported are the mean k S.E.
90 * 3 (pH 7.4) and titrated
EFFECT
OF PROCAINE
AND
opposed to hepatocyte plasma membranes, the addition of 5 mM Ca2+ induced the appearance of a high affinity binding site with a KU of 15 f 1 PM and number of ANS binding sites to be 90 + 3 nmol/mg membrane protein (Fig. 8). The number of ANS binding sites in the low affinity class was shown to be 127 f 5 nmol/mg membrane protein with a Ku value of 50 Ifr 3 PM (Table III). The addition of procaine to ANStreated HTC cell plasma membranes resulted in an enhancement of fluorescence identical to that observed for hepatocyte membranes. The effect of procaine on Ca2+ binding by HTC cell plasma membranes was carried out as previously described for hepatocyte plasma membranes. As assayed by ANS fluorescence, 40 mM procaine resulted in the displacement of only 60% of the membrane associated Cast as opposed to complete displacement in the hepatocyte system (Fig. 6). Direct measurement of 45Ca2+ binding by Millipore filtration as a function of procaine concentration afforded values which were in excellent agreement with the fluorescence studies. In the absence of the anesthetic, binding studies afforded a value of 20 f 2 nmol Ca”‘/mg HTC cell membrane protein. IXKXJSSION
In this report the anionic fluorescent probe, ANS, has been used to investigate membrane properties of normal and neoplastic plasma membranes. ANS is thought to bind to polar-apolar interfaces with the chromophoric group extending into the hydrocarbon core and the sulfonate group located in the plane of the membrane polarhead-groups (43, 44). Evidence also suggests that ANS may bind to protein components of the membrane (18, 45). The interaction of the probe with normal hepatocyte plasma membranes resulted in a large enhancement in fluorescence and a blue shift in the emission maximum indicative of the binding of the probe to nonpolar sites on the membrane. The effect of Ca” on this system could be understood in terms of a suppression of electrostatic repulsion between ANS and the negative surface phosphate and/or carbohydrate groups by the binding of the cation or by
Ca”’
ON PLASMA
MEMBRANES
341
an ion induced conformational change. This enhancement of fluorescence could be due to a decrease in the KD value of ANS, a decrease in binding site polarity with a resultant increase in quantum yield or an increase in the number of ANS binding sites. In previous studies the effects of ions have been attributed to an increase in ANS binding capacity facilitated by suppression of eleckostatic repulsion (18, 19). This report suggests, however, that Ca’+ does not alter the number of ANS binding sites but does have a significant effect on the KU for ANS (Table II). Several reports have shown that local anesthetics cause ANS fluorescence enhancement in the presence of several membrane systems (22, 46-48). This effect may again be understood in terms of a) shielding of anionic membrane constituents which would facilitate the penetration of the anionic probe (ANS) into the hydrophobic region of the membrane, or b) alterations in membrane binding sites resulting from architectural modifications induced by the anesthetic. Local anesthetics have been shown to interact electrostatically with phospholipids (9, 15). Hydrophobic interactions have also been shown to play a significant role (49). Binding studies in the hepatocyte system again suggest that fluorescence enhancement results from a decrease in Ku and not from an alteration in the number of ANS binding sites (Table 1). The effect of anesthetics on Ca2+ binding has been previously reported (9, 15). This report shows that 40 mM procaine can completely displace Cae+ from the hepatocyte membrane system as measured by ANS fluorescence as well as by 4”Ca”+ binding (Fig. 6). The interaction of ANS with HTC cell plasma membranes suggested the existence of significant alterations in membrane structure compared to normal hepatocytes. The fluorescence intensity and emission maximum were quite similar to the hepatocyte system; however, binding studies showed that there were approximately twice the number of ANS binding sites and a 38% decrease in the Ku value (Table III), suggesting significant differences in the environment of the ANS binding sites in the
342
CHENG,
McQUEKN,
two systems, possibly as a result of altered lipid and/or protein composition. The interaction of Ca2+ with the HTC cell membrane system resulted not only in a decrease in the Ku value, as observed for hepatocyte plasma membranes but in an increase in the number of ANS binding sites as well as the appearance of a new class of high affinity binding sites (Fig. 8), suggesting that Ca2’ affects the HTC cell plasma membrane architecture in a significantly different way than observed for normal hepatocytes. The appearance of additional ANS binding sites as well as a new class of binding sites may be due to cation induced conformational changes resulting in the deshielding of previously inaccessible sites. The effect of procaine on ANS fluorescence in the two systems was indistinguishable; however, the ability to displace Ca2+ from the HTC cell membrane system was reduced by 40%, suggesting that Ca2+ was either bound more tightly or at sites inaccessible to the anesthetic. The effect of Ca”+ and anesthetics on plasma membrane structure and membrane associated cytoskeletal proteins has been demonstrated (9-17). The results presented in this report indicate that (a) ANS is a sensitive probe of membrane architecture and can be used to elucidate the effects of Ca’+ and anesthetics such as procaine on complex membrane systems, and (b) that significant differences in membrane dynamics exist between hepatocytes and HTC cell plasma membranes.
LEVY
Publishing Co., Amsterdam. 9. SEEMAN, P. (1972) Pharmacol. Rev. 24, 583-655. 10. POSTE, G., PAPAHADJOPOULOS, D., JACOBSON, K., AND VAIL, W. J. (1975) Biochim. Biophys. Acta
394, 520-539. 11. ADAMS, D., MARKES, CARRAWAY, K. L.
M. E., LEIVO, W. J., AND (1976) Biochim. Biophys.
Acta 426, 38-45. 12. SCHNEPEL, G. H., HECNER, D., AND SCHUMMER, U. (1974) Biochim. Biophys. Acta 367, 67-74. 13. ROTH, S., AND SEEMAN, P. (1971) Nature New Biol. 231,284-285. 14. POSTE, G., AND REEVE, P. (1972) Exp. Cell Res. 72,556-560. 15. PAPAHADJOPOULOS, D. (1972) Biochim. Biophys. Acta 265, 169-186. 16. POSTE, G., PAPAHADJOPOULOS, D., AND NICOLSON, G. (1975) Proc. Nat. Acad. Sci. USA 72, 4430-4434. 17. NICOLSON, G. L., SMITH, J. R., AND POSTE, G.
(1976) J. Cell Biol. 68.395-402. 18. AZZI, A. (1975) Quart. Reu. Biophys. 8, 237-316. 19. KRISHNAN, K. S., AND BALARAM, P. (1976) Arch. Biochem. Biophys. 174.420-430. 20. VANDERKOOI, J., AND MARTONOSI, A. (1969) Arch. Biochem. Biophys. 133, 153-163. 21. RUBALCAVA, B., MUNOZ, D. M., AND GITLER;~. 8, 2742-2749. (1969) Biochemistry 22. GOMPERTS, B., LANTELME, F., AND STOCK, R.
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OF PROCAINE
AND
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