of chemistry vol. 256, no. 4, 1643-1650.1981 m of ... journal of biolooical chemistry vol. 256, no....

THE JOURNAL OF BIOLOOICAL CHEMISTRY Vol. 256, No. 4, Issue of February 25, pp. 1643-1650.1981 Printed m U.S.A.

The Effect of Bilayer Thickness and n-Alkanes on the Activity of the (Ca2+ + Mg2')-dependent ATPase of Sarcoplasmic Reticulum*

(Received for publication, April 14, 1980, and in revised form, September 23, 1980)

Axel Johannsson, Christopher A. Keightley, Gerry A. Smith, Christopher D. Richards$, T. Robin Hesketh, and James C. Metcalfe From the Department of Biochemistry, Tennis Court Road, Cambridge CB2 IQW, United Kingdom and the $Division of Neurophysiology and Neuropharmacology, National Institute for Medical Research, Mill Hill, London NW7, United Kingdom

The activities of the (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum supported by a series of phosphatidylcholines (PC) with unsaturated (cis 9) fatty acyl chains (di(n:l)PC) varying in length from n = 12 to n = 23 were determined by the lipid titration technique (Warren, G. B., Toon, P. A., Birdsall, N. J. M., Lee, A. G., and Metcalfe, J. C. (1974) Biochemistry 13, 5501- 5507). The ATPase activity at 37°C increased with lipid chain length in the PC bilayer from 0.06 pmol min" mg" of protein (n = 12) to a maximum of 16 (n = 20) and decreased to 12 for complexes of the ATPase with di(23:l)PC. All ATPase activity changes with PC chain length were reversible by lipid exchange titrations. Bilayers containing mixtures of two di(n:l)PCs supported ATPase activities which corresponded approximately to the average chain length of the lipid mixture. The data indicate that the major factor determining ATPase activity in these complexes is the thickness of the lipid bilayer. The di(n:l)PC-ATPase complexes were treated with decane, previously shown to increase bilayer thickness in black lipid membranes of egg PC (Fettiplace, R., Andrews, D. M., and Haydon, D. A. (1971) J Membr. Biol. 5, 277-296). The addition of optimal proportions of decane to di( 12 : l)PC ATPase complexes caused a 500-fold increase in activity to 29 pmol min" mg" of protein at 37°C. For other complexes (n = 14 to l8), the ATPase activity first increased and subsequently decreased as the proportion of decane was increased. For complexes with the longer chain PCs (n = 20 or 23) decane only caused a progressive decrease in activity with increasing concentration. Maximal activity for mixtures of each complex with n- decane was obtained at the same total proportion by weight of hydrocarbon (lipid alkyl chains and n-decane) per PC molecule, equivalent to di(20: 1)PC without decane. These effects confirm quantitatively that the major factor determining the maximal activity in each bilayer is its thickness. However, the maximal activity which could be generated by adding decane to each di(n: 1)PC-ATPase complex increased as n decreased. This effect is attributed to the enhanced fluidity of bilayers where high proportions of decane are required to produce the optimal bilayer thickness equivalent to di(2O:l)PC. All decane effects were reversible on addition of bovine serum albumin, and models for the lipid-

* This work was supported by grants from the SRC to JCM and by a British Council Scholarship to AJ. This paper was written during tenure by JCM of a John E. Fogarty International Center Scholar- ship-in-Residence of the National Institutes of Health, from June to October, 1979. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

protein interactions and the effects of decane in the various complexes are proposed. The data and the models are qualitatively consistent with the mechanism proposed by Haydon et aZ. (Haydon, D. A., Hendry, B. M., Levinson, S. R., and Requena, J. (1977) Nature 268, 356-358) for the local anesthetic action of the alkanes, although the ATPase activity is much less sensitive to bilayer thickness than the function of gramicidin A or the Na+ channel in axons.

Although it has been known for some time that a major class of membrane proteins, which probably includes all transport proteins, span the lipid bilayer (4), there has been no systematic examination of the effect of lipid chain length on the function of any transmembrane protein. The length of the lipid chains and, therefore, the thickness of the bilayer will presumably affect the interactions of both the chains and the headgroups of the lipid molecules directly apposed to a transmembrane protein. It might, therefore, be expected that the range of bilayer thickness for the optimal function of this class of proteins will be limited.

We have examined the effect on (Ca2+ + Mg")-dependent ATPase of systematically varying the chain length of cis-9 unsaturated phosphatidylcholines (di(n:l)PC)' in complexes with the ATPase. In addition, all di(n:l)PC.ATPase complexes were treated with increasing proportions of decane, which has been shown previously to increase the thickness of black lipid membranes of phosphatidylcholines and mono- glycerides with chain lengths from 14 to 24 carbons (3, 18). The results are interpreted in terms of simple models of the lipid-protein interaction, assuming that the decane is mainly located at the center of the bilayer (5) and that it complements the phospholipid chains by extending the hydrophobic hydrocarbon region and increasing the separation between the polar headgroups on the opposite sides of the bilayer. The models are compared with the proposal of Haydon et al. (3) that a thickening of the lipid part of an axon membrane through adsorption of alkane reduces the stability of the ionic channels formed during electrical excitation by analogy with the well characterized effect of bilayer thickness on gramicidin A, where it has been shown that the conductance of a black lipid f i m containing gramicidin A decreases rapidly as the thickness and hydrocarbon content of the f i increases ( 6 ) .

EXPERIMENTAL PROCEDURES

Materials-Cis-A9-fatty acids were obtained from Sigma or synthesized from nonanedioic monoethyl ester (Aldrich) and the auDro-

* The abbreviations used are: PC, phosphatidylcholine; SR, sarcoplasmic reticulum; BSA, bovine serum albumin; Temao. 2.2.6.6-tetra-

..

methylpiperidine-1-oxyl.

1643

1644 Bilayer Thickness, n-Alkanes, and (Caz+ + MgZ+)-ATPase Activity

priate alkyl iodide (Sigma). The di(n:l)PCs were synthesized by the method of Robles and Van den Berg (7). All of the PCs gave a single spot on thin layer chromatography (Silica Gel G; ch1oroform:metha- no1water:acetic acid, 2515:4:2 or chloroform:methanol:water:7 N NHsOH, 6530:3:1). Gas-liquid chromatographic analysis of the fatty acid methyl esters derived from the PCs indicated that the chain composition was at least 98% homogeneous in all preparations. Po- tassium cholate (Sigma) was recrystallized from ethanol, and the n- alkanes (highest quality grade) were obtained from Koch-Light; bovine serum albumin (Fraction V) was obtained from Sigma and extracted twice with chloroform:methanol, 2:l. 2,2,6,6-Tetramethyl- piperidine-1-oxy1 was synthesized as described previously (8).

Sarcoplasmic reticulum was prepared from rabbit skeletal muscle as described previously (13). The (CaZf + Mg2')-ATPase accounts for at least 70% of the total SR protein on polyacrylamide gels and was purified to 95% together with about 30 molecules of SR phospholipid per molecule of ATPase which are necessary for full ATPase activity, by treating SR with cholate and centrifuging into a sucrose gradient. This ATPase preparation, which is described in detail elsewhere (I), has an activity of 10 to 12 pmol min" mg" of protein (IU) at 37"C, determined by the double-enzyme coupled assay (9). Protein was estimated by the microbiuret method of Goa (10). All SR and ATPase preparations were frozen in liquid NP and stored at -2OOC in sucrose buffer (250 mM sucrose, 1 M KCI, 50 mM K2HP04/KH2P0, pH 8.0).

Methods-Lipid titrations of the purified SR-ATPase with individual di(n:l)PCs to give di(n:l)PC.ATPase complexes were performed by the procedures described previously (1). Briefly, a large excess of the lipid to be tested, dispersed in cholate, and sonicated to homogeneity, was mixed with ATPase (usually to give a final ratio of 0.5 mg of cholate per mg of total phospholipid). The complexes were incubated, usually on ice, until the ATPase activity came to equilib- rium when assayed by diluting small aliquots of the incubation mixture at least 200-fold in the assay medium (2.0 ml). The data which demonstrate the equilibration of the small proportion of en- dogenous SR lipid with the large excess of added PC in the lipid bilayer containing the ATPase molecules, and the dissociation of almost all of the cholate from these complexes after dilution, have been described previously (1,9). In the present series of experiments, complexes of di(l4:I)PC. and di(l81)PCwATPases were isolated by centrifugation, and analysis of the lipid chain composition by gas- liquid chromatography of the transesterified fatty acids showed that there was 98% equilibration of the phospholipid pools in the complexes. Similar titration procedures were used for titrations with mixtures of PCs or of PCs with decane after cosonication in cholate to homogeneity. In all experiments the concentration of decane in the assay medium was 10- to 100-fold higher than the solubility of decane in the assay medium (12), so that at least 90 to 99% of the decane was either partitioned in the complexes or present as a separate phase. Reversal of the effects of decane on ATPase activity was achieved by the addition of 200 mg ml" of BSA to give a final BSA concentration in the assay medium of 10 mg ml". The experimental details necessary to reproduce each experiment are given in the figure legends.

The binding of Tempo to di(23:l)PC at a molar ratio of 1:100 was assayed from the ESR spectra of the spin label (13) recorded on a Varian E3 spectrometer, and the temperature was monitored contin- uously during the experiment by a thermistor inserted immediately above the cavity. The samples were inserted in 1.0-mm (inner diameter) glass capillaries and the power level adjusted to ensure that local heating did not affect the observed transition temperature of the lipid.

Light scattering (90') at 550 nm by aqueous phospholipid suspen- sions and fluorescence measurements of carboxyfluorescein were made in a Perkin-Elmer MFP 44-B fluorescence spectrophotometer.

RESULTS

Effect of di(n:I)PC Chain Length on (Ca" + M$')-dependent ATPase Actiuzty-Lipid titrations with di(n:l)PCs with chain lengths from n = 12 to n = 23 give specific activities a t 37°C which increase from 0.06 IU for n = 12 to a maximum of 16.0 IU a t n = 20 and decrease to 12.0 IU a t n = 23 (Fig. 1). The lower activities supported by PCs with nonoptimal chain lengths can be fully reactivated by back titration with di(20: 1)PC. An example of the reversibility of the activity changes with PCs is shown in Fig. 2, where a complex inhibited by titration with di(l2:l)PC is reactivated by back titration with di(l8:l)PC. Experiments where ATPase complexes are made

by titration with binary mixtures of PCs with different chain lengths in which the proportions of the two PCs are varied show changes in activity similar to those in Fig. 1 when plotted against the average chain length in each mixture. In Fig. 3 mixtures of di(l21)PC and di (231)PC are compared in their

37°C

15

ATPase

ACTIVITY

( I U )

10

/. i

L 0 ' ~ l ~ ~ n n . 6 # # n l 12 14 16 18 20 22

CHAIN LENGTH f n 1 FIG. 1. Activity of di(n:l)PC-ATPase at 37°C. SR-ATPase

(0.23 mg of protein) was added to cosonicated mixtures of 0.80 mg of di(n:l)PC and 0.40 mg of cholate, each sample in a final volume of 40 1.1 of sucrose buffer containing 5 mM MgATP. The samples were incubated at 0°C for 15 min, then diluted 10-fold into sucrose buffer, and incubated for 2 h after which 8-pl aliquots were assayed at 37°C in 2.0 ml of assay medium. SR-lipid comprised leas than 6% of the total phospholipid in all the incubations. In a separate series of experiments, back titrating with di(l8lfPC or di(201)PC as described in Fig. 2, all inhibitions were shown to be completely reversible to within 10% of the activity of the di(l8l)PC.ATPase or di(201)PC- ATPase complexes. Addition of EGTA to 5 mM in the assay medium abolished more than 95% of the ATPase activity.

37°C

ATPase ACTIVITY diCl8:llPC-ATPase .--.--.- 0

-a- c)

* /

Q t 20 40 TIME (rnin)

FIG. 2. Lipid titration of SR-ATPase with di(l2:l)PC and back titration with di(l8:l)PC. SR-ATPase (0.23 mg of protein) was added to a cosonicated mixture of 0.40 mg of di(l2:l)PC and 0.20 mg of cholate at 0°C in a final volume of 40 1.1 of sucrose buffer containing 5 mM MgATP. Aliquots (2 pl each) were removed at intervals and assayed in 2.0 ml of assay medium at 37°C (W). After 5 min (t) 6 pl of the incubation of SR-ATPase with di(lZ1)PC were added to 0.40 mg of di(l81)PC and 0.20 mg of cholate in a final volume of 46 pl of sucrose buffer at 0°C. The activity of 8 p1 aliquots was assayed as above ( O " 4 ) . A control experiment is shown where SR-ATPase (0.03 mg of protein) was added to 0.40 mg of di(l81)PC and 0.20 mg of cholate in a final volume of 40 pl of sucrose buffer at 0°C and assayed as above (M). The activity of untreated SR- ATPase was 12 IU (*).

Bilayer Thickness, n-Alkanes, and (Ca2+ + Mg2+)-ATPase Activity 1645

20 - ATPase

ACTIVITY

( I U )

15 -

10 -

5 -

12 14 16 18 20 22

2c

ATPase ACTIVITY (IU)

1C

AVERAGED CHAIN LENGTH I n )

b

di(l2:l)PC -ATPase

r -ATPase .ATPase

without with PCmixing PCrnixing

FIG. 3. Lipid titrations of SR-ATPase with two di(n:l)PCs and the effect of lipid mixing. a, activity of (di(l2:l)PC + di(23: 1)PC) .ATPase complexes at 37°C. SR-ATPase (0.23 mg of protein) was added to cosonicated mixtures of di(l2:l)PC + di(23:l)PC in varying proportions (0.80 mg of total lipid) and cholate (0.40 mg), each sample in a final volume of 40 1.11 of sucrose buffer containing 5 m~ MgATP. The samples were incubated at 0°C for 15 min and then diluted 10-fold into sucrose buffer and incubated for 2 h after which 8-pl aliquots were assayed at 37°C in 2.0 ml of assay medium. ATPase

effect on ATPase activity with the individual PC-ATPase complexes. The activities of the mixtures are a little higher than for the corresponding PC-ATPases, which might indicate a slight preference of the ATPase for the longer chain PC in the mixture or reflect the enhanced fluidity of a bilayer composed of mixed chain lengths (14). However, the absolute values of the ATPase activities of the individual complexes can vary by up to 25% using different ATPase preparations, and the main conclusion to be drawn is that the activity of the ATPase complexes with binary PC mixtures is determined to a good approximation by the average chain length of the mixed PC bilayer. Similar results were obtained with other PC mixtures (see Fig. 3). This conclusion clearly depends on the assumption that the two lipids (e.g. di(l2:l)PC and di(23: 1)PC) are mixed more or less ideally in the membrane particles. Two approaches were used to demonstrate this experi- mentally. Separate complexes of di( 12:l)PC-ATPase and di(23:l)PC-ATPase were made as described in Fig. 1. Mixtures of the separate complexes (di(l2:1)PC:di(23:1)PC, 1:3, w/w) were made in the assay medium without further addition of

C

activity is plotted against the average chain length for each mixture (W), and this profile is compared to that obtained from individual di(n:l)PC.ATPase complexes as shown in Fig. 1 (---). Activities are also shown for (di(l2:l)PC + di(lB:l)PC).ATPase complexes prepared and assayed exactly as described above ( O " 0 ) . b, activity of ATPase complexes with different proportions of di(l2:l)PC and di(231)PC and the effect of lipid mixing. Complexes of SR-ATPase and di(l2:1)PC, di(23:1)PC, or (di(12:1)PC:di(23:1)PC, 1:3, w/w) were prepared and diluted into assay medium as described in a. The resulting activities are shown by (i), (ii), and (iu). Alternatively di(12: 1)PC.ATPase or di(231)PC .ATPase (prepared in assay media) was mixed in a 1:3 ratio, and the resulting ATPase activity is shown by (iii). c, 90' light scattering at 550 nm of di(l21)PC, di(23:1)PC, or (di(lZ:I)PC:di(23:1)PC, 1:3, w/w) as a function of temperature. Five microliters of a suspension of PC and cholate (50 mg/ml of PC + 25 mg/ml of cholate) were added to 4 ml of 100 mM triethanolamine hydrochloride adjusted to pH 7.2 with KOH at 25°C. 90' light scattering (arbitrary units) was measured in a stirred temperature controlled cuvette as a function of temperature. Di( 12:l)PC (O), di(23: l)PC (A), di(l2:1)PC:di(23:1)PC, 1:3, w/w, mixed before (0) or 2 h after dilution into the TREACl buffer (W). (The final cholate concentration, 0.03 mg/ml, was shown to have no effect on the transition temperature of di(23:l)PC.)

cholate (see legend to Fig. 3b). The activity of this mixture was 9.3 IU compared with a calculated mean value for this mixture of separate complexes of 9.0 IU, which is entirely consistent with the presence of two distinct populations of membrane particles. However, if the binary lipid complex is prepared by mixing both lipids (in the same proportion of 3:1, w/w) with the ATPase in the presence of cholate before dilution into the assay medium as described in the legend to Fig. 3a, then the activity was 19.0 IU (see Fig. 3b). We emphasize that the only difference in protocol between these two experiments is whether the lipids are mixed before, or after, treatment with cholate. The large difference in activity (19.0 IU cf. 9.3 IU) is, therefore, attributable to the well characterized effect of cholate in equilibrating PC pools with the ATPase (1). To provide direct physical evidence that the di(l2:l)PC and di(23:l)PC lipids form a mixed lipid bilayer after equilibration with cholate, we made use of the lipid phase transition in di(23:l)PC-ATPase between 18 and 24°C (defined in Fig. 5). In Fig. 3c the 90' light scattering at 550 nm of di(23:l)PC shows an inflexion at the transition temper-

1646 Bilayer Thickness, n-Alkanes, and (Ca" + Mg2+)-ATPase Activity

ature, whereas there is no evidence for a phase transition in di(l2:l)PC from the light scattering between 8°C and 35°C. When di(l2:l)PC was added to di(23:l)PC (1:3) in the presence of cholate to allow equilibration of the lipids before removal of cholate from the mixture by dilution (see legend to Fig. 3c) no phase transition in the lipid mixture was observed from 8 to 35°C (Fig. 3c). This demonstrates that there are virtually no separate di(23:l)PC particles (<5%) after this treatment. However, if the cholate is removed before mixing the lipids, the phase transition in the di(23:l)PC component is observed as in the absence of di(l2:l)PC (Fig. 3c). Both of these experimental approaches demonstrate 'extensive mixing of these two lipids as a result of their exposure to cholate and that the two PCs do not segregate significantly after the cholate is removed.

The thickness of lipid bilayers increases slightly as the temperature is lowered, although the effect is small (15). If the ATPase activity was very sensitive to bilayer thickness, we might expect the optimal chain length for ATPase activity to become shorter with decreasing temperature. When the activity profies for the ATPase as a function of the PC chain length were compared to di(2O:l)PC.ATPase at 37"C, 25"C, and lO"C, there was no detectable shift in the optimal chain length within the resolution of the experiment. Since the change in bilayer thickness over this temperature range is approximately 7% (15) or less than three carbon-carbon bond lengths for a di(201)PC bilayer this result is consistent with the view that the bilayer thickness is a primary determinant of activity. The data in Fig. 4 indicate that the peak in activity with di(2O:l)PC.ATPase becomes more pronounced as the temperature is reduced, with respect to both di(l8:l)PC. and di(23:l)PC .ATPase complexes, indicating that the activation energy is minimized at the optimal chain length for activity.

The activity of the di(23:l)PC.ATPas.e complex decreased sharply between 25°C and 1O"C, suggesting that there might be a phase transition in this temperature range, which was c o n f i e d by assaying binding of the spin label Tempo to the lipid. The transition occurs between 18°C and 24°C (Fig. 5, inset). Arrhenius plots for the activity of di(23:l)PC-ATPase indicate an inflexion in the curve at approximately 22°C (Fig. 5). However, similar plots for di(l4:l)PC -ATPase (not shown) indicate a less pronounced inflexion at about 28"C, similar to

2ot &." , , , , \ , 10 c

0 12 14 16 18 x) 22

CHAIN LENGTH (n) FIG. 4. Activity of di(n:l)PC-ATPase relative to di(20:l)PC-

ATPase (100%) at 37"C, 25°C. and 10°C. SR-ATPase (0.23 mg of protein) was added to cosonicated mixtures of 0.60 mg of di(n:l)PC and 0.30 mg of cholate, each sample in a final volume of 40 pl of sucrose buffer containing 5 m~ MgATP. The samples were incubated at 0°C for 30 min, then diluted 5-fold into sucrose buffer and incubated for 2 to 4 h after which aliquots of between 4 and 25 pl were assayed at the temperature indicated in 2.0 ml of assay medium.

that reported previously for di( 18:l)PC .ATPase (17), neither of which can be attributed to the primary phase transitions in these lipids, which occur below 0°C (16). This amplifies our previous observations that inflexions occur in the Arrhenius plots for the activity of transmembrane proteins for reasons other than primary phase transitions within the lipid bilayer

The Effect of Decane on di(n:l)PC. ATPase Complexes- The data in Figs. 1 and 3 suggested that there was an optimal bilayer thickness required to support maximal ATPase activity. Since alkanes have been reported to cause substantial thickening of the bilayer in black lipid films (2), we examined their effects on the di(n:l)PC.ATPase complexes. Decane causes a 500-fold activation of di(lZ:l)PC.ATPase, with a progressive increase in activity up to about 0.36 mg of decane per mg of PC, after which activity remains constant up to 0.4 mg of decane per mg" PC (Fig. 6). The amount of decane required for optimal activity is almost exactly equivalent to increasing the chain length to n = 20, assuming that decane has the same density in the bilayer as the alkyl chains of the lipid. The effect of the decane was reversed (>go%) by the addition of 10 mg rn" of BSA to the assay medium (not shown). Complete binding of the decane by BSA corresponds to 0.15 molecules per molecule of BSA. Similar experiments with the other di(n:l)PC-ATPase complexes up to n = 18 show that decane fiit activates and subsequently inhibits the complexes as the proportion of decane is increased (Fig. 6). For complexes where n = 20 or 23, decane only causes inhibition of activity as the proportion in the bilayer is increased (Fig. 6). It is clear from Fig. 6 that the proportion of decane required to achieve maximal activity in each complex decreases as the value of n increases. The equivalent chain length of the combined lipid chains and the optimal proportion of decane is close to n = 20 for all complexes (Table I).

(17).

r

10 30 50

1

3 2 3 3 3 4 35

I / T (K-' . IO~)

FIG. 5. Arrhenius plots of the ATPase activities of di(23: 1)PC. and di(l8:l)PC.ATPase. SR-ATPase (0.23 mg of protein) was added to cosonicated mixtures of 0.80 mg of di(n:l)PC and 0.40 mg of cholate, each sample in a final volume of 40 pl of sucrose buffer containing 5 m~ MgATP. The samples were incubated at 0°C for 40 min and then diluted 5-fold into sucrose buffer and incubated for 1 to 4 h after which aliquots of between 4 and 25 p1 were assayed over the temperature range 12-42OC in 2.0 ml of assay medium. Inset, fraction ( f ) of Tempo (approximately 1 mole '% of lipid) bound to di(23:l)PC (40 mg/ml in 100 m~ triethanolamine buffer, pH 7.2) as a function of temperature. The parameter fwas determined as in Ref. 17.

Bilayer Thickness, n-Alkanes, and (Ca2+ + Mg2')-ATPase Activity 1647

ATP- 1 ACTIVITY

- b

V 0. 0 a2 0.4 0 a2

mg decarwr per m~ diIn:l)PC 0.4

FIG. 6. The effect of decane on the activity of di(n:lPC-ATP- ases at 37°C. SR-ATPase (0.23 mg of protein) was added to freshly sonicated dispersions of 0.80 mg of di(n:l)PC and 0.40 mg of cholate with varying proportions of decane from 0 to 0.32 mg, each sample in a final volume of 40 pl of sucrose buffer containing 5 m~ MgATP. The samples were incubated for 10 min at 23°C and then diluted 10- fold into sucrose buffer and incubated for a further 5 min after which 8-1.11 aliquots were assayed at 37°C in 2.0 ml of assay medium.

TABLE I The calculated equivalent PC chain lengths (n) of the optimal proportions by weight of decane and di(n:I)PC for maximum

ATPase activities a t 37°C

di(n:l)PC.ATPase cane (w/w Eypp chain length Equivalent Optimal de-

PC) (nl - - , ~ " ,

12:l 0.35 833 19.8 131 0.30 838 19.9 14:l 0.25 841 20.0 151 0.24 869 20.7 161 0.19 868 20.6

(160) (0.19) (870) (20.7) 18:l 0.08 848 20.2 20:l 0.0 84 1 20.0

12:1/1&1 (1:l w/w)" 0.20 841 20.0

a An ATPase complex containing equal proportions by weight of di(121)PC and di(l8:1)PC, with an average chain length in the mixture of n = 14.6.

Experiments in which decane was added to complexes of the ATPase with binary mixtures of PCs also show maximal activity at the same equivalent chain length (Table I) and inhibition of activity when the bilayer is overthickened. It should be noted that although ATPase activities in the presence of up to 0.3 mg of decane per mg of PC were constant for at least 1 h, the activity of complexes at higher decane proportions was less stable, which could be attributed to a slow separation of part of the decane from the bilayer to form a separate phase. This separation had no irreversible effects on the ATPase activity which reverted to the level observed in the absence of decane after the addition of BSA.

The results of the experiments with decane and di(l2:l)PC lipids are interpreted in terms of the effect of decane on the thickness of the di(n:l)PC bilayers. Extensive evidence from work on black lipid membranes has shown that the presence of substantial proportions of alkanes, including a-decane, do not destroy the basic bilayer structure for lipids with chain lengths between 14 and 22 carbons (2, 12, 18). In particular the formation of micellar structures in the bilayer by decane would cause collapse of the electrical resistance. However, direct experimental data for the effect of decane on the di(12: 1)PC or di(l3:l)PC bilayers is not available, and experiments were .performed to determine the effect of n-decane at up to 0.5 mg per mg of PC on the retention of the impermeant dye carboxyfluorescein by liposomes.

It can be seen from the data in Table I1 that di(l2:1)PC, di(l3:1)PC, and di(l8:l)PC show a substantial included volume in the presence or absence of decane whereas di(8:O)PC, which forms micelles rather than bilayers, shows no significant included volume in the presence or absence of decane. These results clearly indicate that di(l2:lfPC and di(l3:l)PC retain a bilayer structure in the presence of high proportions of decane. The isolated liposomes containing carboxyfluorescein formed by these lipids in the presence or absence of n-decane were observed under the light microscope and ranged in size up to about 2 pm in diameter. Seventy-two per cent of the di( 12:l)PC-decane liposomes were retained on a 0.22-pm Mil- lipore filter, compared with <lo% of a similar di(8:O)PC-decane mixture.

The data in Fig. 6 also suggest that a second factor influ- ences the maximum activity generated by the optimal proportion of decane in the di(n:l)PC-ATPase complexes. The maximum activity with decane decreases from 29 IU for di(12 1)PC complexes to 18 IU for di(l8:l)PC complexes (Fig. 7). Thus, although the maximum activity generated by the addition of decane corresponds to the same equivalent chain length of n = 20, the activity increases with the proportion of decane required. This suggests that the highest activities are generated by bilayers which are exceptionally fluid through a

TABLE I1 Retention of carboxyfluorescein by PCs * decane

A solution containing 2 mM carboxyfluorescein in 100 mM triethanolamine hydrochloride adjusted to pH 7.2 with KOH was added to PC f 0.5 mg of decane per mg of PC, and vortex mixed to a homogeneous suspension (20 mg/ml of PC). Samples (100 pl) of each suspension were passed through a Sephadex G-50 column. Liposomes or micelles were eluted in the void volume of the column and were well separated from the free carboxyfluorescein. The amount of carboxyfluorescein in each fraction was quantitated by measurement of the fluorescence intensity.

PC Capture volume (% of total)

di(8:O) di(8:O) + decane di(12:l) di(12:l) + decane di(13:l) di(13:l) + decane di(18:l) di(18:l) + decane

t0.005 <0.005

0.53 0.89 0.79 2.9 1.7 0.68

30- .37-C ATPase

(IU)

'* ACTIVITY \

"*

\

12 14 16 18 M 22 CHAIN LENGTH (n)

FIG. 7. Maximum ATPase activities with or without decane as a function of PC chain length. The maximum activities obtained from di(n:l)PC-ATPases with optimal proportions of decane at 37OC (t".). (C"O), refer to activities obtained without added decane.

1648 Bilayer Thickness, n-Alkanes, and (Ca2+ + MgZ+)-ATPase Activity

combination of short lipid chains and high proportions of decane, as discussed later.

I t is clear from Figs. 6 and 7 that any fluidity increase which may result from the presence of decane at optimal PC chain length a t 37°C (n = 20) is ineffective in increasing activity compared with the inhibition caused by the increase in bilayer thickness. To observe significant fluidity effects on activity, it is necessary either to use decane with suboptimal chain lengths as described or to reduce the temperature to create a more rigid bilayer which is susceptible to increases in fluidity. This can be demonstrated most strikingly by taking di(23: 1)PC-ATPase complexes to 20"C, just below the transition temperature, and treating the complex with increasing proportions of decane. I t can be seen from Fig. 8a that an increase of about 8% in the total hydrocarbon content of the bilayer by

ATPase

dd23:l)PC.ATPase

+ BSA " " " " " _ _

0- 0 a2 a4

mg decane per rng PC

1 37°C

'\ m,

t/

r/ '.

I 1

0 a2 a4

FIG. 8. The effect of decane on PC- ATPase complexes below the PC phase transition temperature. a, di(231)PC.ATPas.e at 20°C. SR-ATPase (0.23 mg of protein) was added to freshly sonicated dispersions of 0.80 mg of di(231)PC and 0.40 mg of cholate with varying proportions of decane from 0 to 0.32 mg, each sample in a final volume of 40 pl of sucrose buffer containing 5 m~ MgATP. The samples were incubated for 10 min at 23OC after which 4-p1 aliquots were assayed for 2 to 10 min in 2.0 ml of assay medium at 20°C (M). BSA (100 p1 of 200 mg ml" in triethanolamine buffer, pH

activity recorded (0-"0). b. di(l6O)PC.ATPase at 37°C. SR-ATP- 7.2) was subsequently added to the assay medium and the resulting

ase (0.23 mg of protein) was added to freshly sonicated dispersions of 0.80 mg of di(l6O)PC and 0.40 mg of cholate with varying proportions of decane from 0 to 0.32 mg, each sample in a final volume of 40 4 of sucrose buffer containing 5 mM MgATP. The samples were incubated for 10 min at 38°C then diluted 10-fold into sucrose buffer and incubated for a further 1 min after which aliquots were assayed at 37°C in 2.0 ml of assay medium.

mg decane per mg PC

decane produces a 2-fold activation, followed by a sharp inhibition of activity a t higher proportions of decane. I t should be emphasized that this activating effect of decane on the di(23:l)PC.ATPase complex is not seen a t 37"C, and that even below the transition temperature, the inhibition of activity due to overthickening of the bilayer rapidly predominates the presumed fluidity effect. A similar effect can be demonstrated at 37°C with di-saturated PC. ATPase complexes with transition temperatures above 37"C, so that the bilayer is relatively rigid even a t 37°C. Treatment of di(l6:O)PC .ATP- ase with decane at 37°C causes an increase in activity to a maximal value of 20 IU (Fig. 86) which is only slightly less than the maximal value of 23.0 IU for di(l6:l)PC-ATPase with optimal proportions of decane (Fig. 7). Thus the difference between the activities of the two complexes, which is almost 2-fold in the absence of decane, is greatly reduced when there is a fluid region of decane within the bilayers.

The Effect of Other n-Alkanes-The modulation of the activities of di(l2:l)PC .ATPase and di(l8:l)PC. ATPase by the incorporation of optimal amounts of a homologous series of alkanes is summarized in Fig. 9a (37°C) and Fig. 96 (20°C). The effect on the activity of the ATPase (activation of the di(l2:l)PC complex and inhibition of the di(l8:l)PC complex) diminished as the chain length of the alkane is increased from 10 to 16. This diminution of the effect of the alkanes on ATPase activity closely parallels their decreasing effect on black lipid membrane thickness (Fig. 9b), due to their decreasing solubility in the bilayer with increasing chain length (18). Alkanes shorter than decane have a significant solubility in water (12) and may also evaporate at 37°C in an open cuvette over the assay period, and data for these are not included. The change in enzyme activity with increasing alkane chain length is sharper at 20°C than at 37"C, presumably because partition of the alkanes shows a greater dependence on the

30 - a

ATPase ACTIVITY (IU)

15

6

3

0 L " - - O L - - " J 10 12 14 16 1 0 1 2 1 4 1 6

Chain length Or alkane

FIG. 9. The effect of n-alkanes on the activity of di(l2:l)PC and di(l8:l)PC-ATPase. a, SR-ATPase (0.23 mg of protein) was added to freshly sonicated dispersions of 0.80 mg of di(l8:l)PC (H) or di(l2:l)PC (W) with 0.40 mg of cholate and 0.32 mg of alkane, each sample in a final volume of 40 pl of sucrose buffer containing 5 n" MgATP. The samples were incubated for 10 min at 23°C and then diluted IO-fold into sucrose buffer and incubated for a further 5 min after which 8-4 aliquots were assayed at 37OC in 2.0 ml of assay medium. b, protocol as a except that the 10-fold dilution into sucrose buffer was omitted, and 4-pl aliquots of the incubations were assayed in 2.0 ml of assay medium at 20°C. The effect on bilayer thickness of varying the chain length of the alkane solvent used in the formation of monolein bilayers from Ref. 18 is also indicated (- - -).

Bilayer Thickness, n-Alkanes, and (Ca2+ + Mg2+)-ATPase Activity 1649

fluidity of the host lipid membrane for the longer chain alkanes as the temperature decreases.

DISCUSSION

Bilayer Thickness-It seems clear from the effects of lipid chain length and the modulations in activity caused by decane and other alkanes that the major factor determining the activity of the ATPase is the thickness of the bilayer. Assum- ing a uniform density of the hydrocarbon region (lipid chains + decane), the optimal bilayer thickness over the range 10"- 37°C appears to be equivalent to that of di(ZO:l)PC, irrespec- tive of the proportions of decane and PC, or mixture of PCs required. The invariance of the optimal chain length over the same temperature range is consistent with the very s m d change in bilayer thickness which occurs (15).

A simple model to account for the data is shown in Fig. 10. The plain regions of the ATPase molecule represent the hydrophobic surface of the protein which interacts only with the hydrocarbon chains in lipid bilayers of optimal thickness (Fig. loa). It seems reasonable to suppose that the dimensions of the hydrophobic region of the protein are precisely defined, as implied in the model, from the limited information available for the amino acid sequences of transmembrane proteins. Glycophorin contains a sequence of about 32 nonpolar residues, which would span a hydrophobic bilayer region of 48 8, thick if structured as an a-helix (19). Bacteriorhodopsin also appears to have defined sequences of hydrophobic residues (20), with very little of the protein extending beyond the lipid headgroups, and with seven transmembrane helices each about 35 to 40 8, in length (21). For the ATPase of sarcoplasmic reticulum, it is likely that a substantial part of the protein carrying the MgATP2- binding site extends beyond the bilayer surface (22), which presumably has a normal proportion of polar amino acid residues (23) to interact with the medium and the polar headgroups of the lipid molecules next to the protein (shaded in Fig. 10). It seems likely that such polar regions on the protein must exist on both sides of the bilayer, and it is also probable that any specificity in the

a

C

interactions of the lipid headgroups with the protein are determined by the interaction of one or more of the lipids with the appropriate sites defined by the polar residues within the shaded area. The differences in ATPase activity supported by lipids with the same chain structure but different polar headgroups are consistent with the view that these polar interactions play a significant role in determining the ATPase conformation and hence the enzymatic activity (24). The polar areas of the protein surface may, therefore, define the dimensions of a hydrophobic collar around the protein. In inactive complexes (e.g. di(l2:l)PC. ATPase in Fig. lob), displacement of the lipid headgroups from the polar areas of the protein surface toward the hydrophobic surface is presumed to be responsible for the loss of an active protein conformation. The addition of decane to the two complexes in Fig. 10, a and b, has opposite effects on activity, as illustrated in Fig. 10, c and d. The lipid hydrocarbon chains in the overthickened di(20: l)PC .ATPase bilayer are now forced to interact with the polar shaded area of the protein surface, and at the same time the polar lipid headgroups are displaced from any sites in which they may normally act to support ATPase activity, and the complex is inactivated. The di(l2:l)PC. ATPase complex, in contrast, is brought to the optimal interaction of the headgroups with the protein by addition of the appropriate proportion of decane which is preferentially localized at the center of the bilayer (5).

This model is qualitatively consistent with the mechanism proposed by Haydon et al. (3, 11) for the local anesthetic action of the alkanes on the Na' channel, in that it demonstrates that the function of a transport protein is affected by the thickness of the bilayer in which it is operating. If we assume that the lipid bilayer in membranes is normally at or close to the optimal thickness for the function of the transmembrane proteins it supports, the present data are consistent with a general inhibition of function of this class of proteins by agents like the alkanes which increase bilayer thickness. However, the change in ATPase activity with lipid chain length is not very sharp. It is necessary to change the bilayer

b

FIG. 10. Models of di(20:l)PC. ATPase (a and c) and di(l2:l)PC- ATPase (b and d ) with and without decane. The shaded areas of the ATP- ase represent regions of the protein with polar residues on the surface defining the limits of the hydrophobic (unshaded) surface with which the lipid chains and

~ decane interact. The linear dimensions of the lipid headgroups and chains are

+decane ] 1 -decane drawn approximately to scale. Although the decane is shown to be situated entirely between the two monolayers, most of the decane probably thickens the bilayer by intercalating between the acyl chains. We emphasize that the diagram is not intended to imply that the decane forms a discrete layer between the two monolayers, but is merely a schematic representation.

d

1650 Bilayer Thickness, n-Alkanes, and (Ca” + MgZi)-ATPase Activity

thickness by an amount equivalent to f 5 carbon-carbon bonds to cause a 50% decrease in maximal ATPase activity at 37°C. This is equivalent to a change in bilayer thickness of about 7 A, compared with the estimate of Haydon et al. (3) that a 1.6 A increase in bilayer thickness is sufficient to cause a 50% decrease in the amplitude of the squid axon potential at about 20°C and an approximately similar reduction in the conductance of gramicidin channels. It may be that the geometry of the passive sodium channel is inherently more sensitive to changes in the dimensions of the bilayer than the (Ca2+ + Mg2+)-ATPase which depends on its enzymatic activity to drive ion transport. It is an essential feature of the model in Fig. 10 that, in order to modulate activity, the alkane interacts directly with the hydrophobic surface of the protein at the center of the bilayer to displace the lipids interacting with the protein in a direction perpendicular to the plane of the bilayer. This mechanism may differ in molecular terms from the “dimpling” effect suggested by Haydon et al. (3) to cause conformational distortion of the Na+ channel through the action of surface tension in the thickened bilayer, which does not necessarily require interaction of the alkane with the channel.

The decreasing effect of the long chain alkanes on the ATPase activity of the complexes coincides quite closely with the data from black lipid films for the effect of the alkanes on bilayer thickness in cholesterol-free bilayers (12, la), which decreases as the partition of the alkane into the bilayer decreases and is negligible for hydrocarbons longer than hex- adecane.

Bilayer Fluidity-In a fluid PC bilayer of optimal chain length at the physiological temperature of 37”C, the effects of fluidity changes on activity due to the addition of decane are small compared with the inhibitory effect of overthickening the bilayer. To observe the effect on activity of increased fluidity in the bilayer due to the presence of decane at 37”C, it is either necessary to have a rigid lipid bilayer by using a PC below its transition temperature, as shown for di( 16:O)PC - ATPase, or to create a “superfluid” bilayer by a combination of very short lipid chains and decane. Neither of these bilayers correspond to the normal lipid environment of the ATPase in native sarcoplasmic reticulum, where the average chain length is approximately 17 to 18 carbons (calculated from Ref. 25) and about 67% of the chains are unsaturated, and the bilayer is, therefore, well above any phase transition at physiological temperatures.

Fluidity effects due to decane were observed at lower temperatures, where the di(n:l)PC bilayers were more ordered, and particularly for di(23:l)PC below the transition temperature (Fig. 8). The evidence which suggests that the activating effects of decane in the more ordered lipid bilayers can be attributed to an increase in fluidity which it introduces comes from I3C-NMR TI relaxation measurements (26-28). The TI relaxation time for the terminal methyls of decane at 31°C is 5.8 s, compared with a value for the terminal methyls of di(12: 0)PC of 3.85 sand 3.88 s for di(l8:l)PC at 52”C, indicating the faster molecular motion of the decane chain. The effect of decane partitioned mainly at the center of the bilayer is to create a region of extended fluidity, approximately compara- ble to that which normally exists only at the ends of the PC

chains at the center of the bilayer (see Fig. 10d). The structure is equivalent in effect to breaking the chains of longchain PC molecules at n = 12, thereby increasing the molecular motion of both the phospholipid molecules and the severed hydrocarbon chains.

We conclude that increases in fluidity due to extraneous agents are unlikely to be of major importance in determining activity in the native lipid bilayer at physiological temperatures, unless it is possible to achieve an increase in fluidity with agents which do not also thicken the bilayer.

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of chemistry vol. 256, no. 4, 1643-1650.1981 m of ... journal of biolooical chemistry vol. 256, no....

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