β-endorphin: thermodynamics of the binding reaction with rat brain membranes

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 217, No. 1, August, pp. 89-86, 1982

@-Endorphin: Thermodynamics of the Binding Reaction with Rat Brain Membranes’

PIERRE NICOLAS, R. GLENN HAMMONDS, JR., SOPHIE GOMEZ, AND CHOH HA0 L12

Hormone Research Laboratory, University of Caltimia, San Francisco, California 94149

Received January 11, 1982, and in revised form March 10, 1982

The binding of human /3-endorphin to rat brain membranes was studied at various temperatures in the absence and presence of sodium. Between 0 and 3O”C, 100 mM sodium depresses the binding of &,-endorphin by reducing its affinity for binding sites 7- to lo-fold but has no effect on the total number of binding sites present in the membranes preparation. Kinetic studies show that the decrease in affinity in sodium is largely accounted for by a lo-fold decrease in the association rate constant within the whole range of temperatures examined. High versus low temperatures markedly enhance the binding of &-endorphin’by increasing its affinity for receptors, both in the presence and absence of sodium, without change in the number of receptors. This enhancement in affinity is due to an increase in the association rate constant between 0 and 3O”C, coupled with a slight increase in the respective dissociation rate constants. Temperature variations do not interact with the influence of sodium on the fi-endorphin receptor. The integrity of the tritiated peptide was assessed at each temperature used. No more than 6% of degraded material was found at the highest temperature of incubation examined. Large positive values of the standard enthalpy and entropy changes were observed both in the absence and presence of sodium indicating an entropically driven binding reaction in both cases. The dependence of binding affinity upon sodium and temperature strongly suggests that hydrophobic interactions play a major role in stabilizing the hormone-receptor complex while electrostatic and/or hydrogen-bonded interactions might play a more specific role in recognition processes.

/?-Endorphin (1) exhibits opiate-like properties both in wivo and in vitro (l-8). It possesses the highest affinity for brain opiate receptors (9-11) and is the most active opioid when injected directly into the brain (4). /3-Endorphin is also the only naturally occurring peptide which exhibits potent analgesic activity by intravenous

i This work was supported by the National Insti- tute of Mental Health (MH-30245), the National In- stitute of Health (GM-2997) and the Hormone Re- search Foundation. P.N. is a European Molecular Biology Organization Fellow.

‘Author to whom all correspondence should be sent.

injection (5). While enkephalin and alkaloid opiate binding properties have been extensively studied, the properties of @- EP3 binding are less well known. The unique pharmacological profile of a P-EP warrants a closer examination of its interaction with brain opiate receptors.

Temperature and sodium differentially effect the binding of alkaloid opiates of varying agonist character, while enke-

3 Abbreviations used: &EP, human /3-endorphin; OHAc, acetic acid; EtOH, ethanol; HPLC, high-per- formance liquid chromatography; PrOH. propanol-1; MetOH, methanol; NaCl, sodium chloride; ‘H-&,-EP, tritiated &,-EP.

ooo3-9861/82/090080-07%o2.oo/0 Copyright Q 1982 by Academic Press, Inc. All righta of reproduction in any form reserved.

80

@ENDORPHIN BINDING THERMODYNAMICS 81

phalins response to sodium and temperature variations seems unrelated (12-18). Temperature variations interact with the influence of sodium on the alkaloid opiate receptor in a complex fashion (15). Infor- mation on the effect of temperature and sodium on p-EP binding would be helpful in characterizing the /3-EP receptor(s), particularly in regard to the possibility of an overlap between the peptide and the alkaloid binding site(s). It has been estab- lished that sodium ions can discriminate between alkaloid agonists and antagonists (12-18) and sodium has been reported to decrease the binding of P-EP to brain membrane homogenates (9, 11). However, it was not demonstrated whether this inhibition reflects a change in affinity or in the number of binding sites or both (11). An apparent inhibition could reflect an alteration of the kinetics which could pro- duce changes in the time required to reach equilibrium (9). Finally, none of these data assessed the possibility that temperature variations could interact with the influence of sodium as reported in the case of alkaloid opiates (15). Such data are needed to elucidate the mechanism of action of this ion toward P-EP binding and to compare it with the one postulated for alkaloid opiates. This would reveal the inability of sodium to discriminate between P-EP homologs exhibiting differences in their intrinsic activity as recently reported (19, 20).

Characterization of the properties of a binding equilibrium can be extended by examining the effects of temperature on both kinetic and equilibrium constants. This will provide both the enthalpy and entropy of the binding reaction and, therefore, a knowledge of the driving force(s) involved in this process. A comparison of thermodynamic data among alkaloid and peptide opioids and between P-EP analogs and homologs should allow a better understanding of their respective binding sites. This would also provide a framework for interpretation of the effects of chemical modifications within the @-EP sequence which alter binding and/or analgesic po- tency, as affinity will reflect changes in these components. Thus, thermodynamic

analysis of the binding properties of fi-EP analogs and homologs may contribute to our understanding of the molecular mechanism involved in these changes.

This communication describes a system- atic analysis of ph-EP binding to rat brain membrane homogenates at various temperatures and sodium concentrations. Binding capacities, binding affinities, and kinetic constants obtained under each set of experimental conditions used are pre- sented together with an examination of the stability of 3H-/3h-EP.

MATERIALS AND METHODS

Human @EP was synthesized by the solid-phase method as described (21). Myelin basic protein was isolated from bovine brain as reported (22). 3H-j3i,- EP (50 Ci/mmol) prepared as described (23) and stored at 4°C in 5% HOAc, 5% EtOH, was repurified by HPLC on a Whatman 5-pm Cl8 column using iso- cratic conditions (18% PrOH-1, 0.5 M pyridine ace- tate, pH 4.4). The peak tubes were combined, lyoph- ilized to l/4 the initial volume to remove pyridine, diluted in 2% EtOH, 5% HOAc, and stored in small aliquots in liquid nitrogen. The tritiated peptide was diluted into the binding buffer just prior to addition to the assay mixture (see below).

Rat brain membranes were prepared from decer- ebellate whole brain of male 180-g Sprague-Dawley rats by four cycles of polytron homogenization and centrifugation (30 min at 10,OOOg) at 4°C. The buffer used was 50 mM Tris-Cl, pH 7.4. The yield of washed membranes equivalent to 10 brains was dispersed in 100 ml of 50 mM Tris-Cl, pH 7.4, 20% glycerol, and stored at -80°C. The final protein concentration of this extract was 5.7 mg/ml as determined by the method of Lowry et al. (24) using bovine serum albumin as a standard.

Binding was performed in plastic tubes (12 X 75 mm, polyethylene) at a final volume of 2 ml in 50 mM

Tris-Cl buffer, pH 7.5 (corrected to the desired temperature of incubation), plus 0.1% bovine serum albumin, 0.01% bacitracin, and with or without 100 mM

NaCI. Appropriate constant amounts (final concentrations ranging from 0.1 to 1 nM depending on temperature and sodium concentration) of 3H-/3h-EP was added (in 50 ~1) together with 50 pl of cold /3,,-EP at varying final concentrations (0.1 to 100 nM). After preincubation for 5 min at the desired temperature, binding was initiated by addition of 0.1 ml of the membranes suspension (0.57 mg of protein) and the tubes were incubated at a constant temperature (kO.5”C) for the desired time period with four equally spaced stirrings. The binding reaction was termi- nated by rapid vacuum filtration through myelin ba-

82 NICOLAS ET AL.

Time (mud Time (min)

FIG. 1. Rate of association of eH-&,-EP to rat brain membranes at various temperatures either in the absence (A) or in the presence (B) of 100 mM NaCl. Tritiated &EP (0.45 nM) was incubated with membranes (0.57 mg of protein) at temperatures indicated in the presence or absence of a 200-fold excess of unlabeled peptide. Specific binding was measured as a function of time. Each value was the mean of duplicate determinations.

sic protein-coated Whatman glass fiber filters (G/F B). The filters were washed two times with 15 ml of cold (2Y) 50 mM Tris-Cl, pH 7.4,0.1% bovine serum albumin and transferred to vials containing 5 ml of PCS scintillation fluid (Amersham). After overnight incubation at room temperature, radioactivity was measured by liquid scintillation counting. Specific binding was considered to be the difference in radioactivity trapped on the filters in the absence and presence of 200-fold molar excess of unlabeled peptide. All determinations were performed in duplicate and the standard error of the replicates is approxi- mately 3%. All binding experiments were performed with the same preparation of brain membranes and of tritiated and cold fir,-EP.

Degradation of *H-&,-EP was estimated by chromatography on Water Cl8 Sep-Pak columns, follow- ing a procedure similar to that of Gay and Lahti (25). A 0.5-ml aliquot from the binding experiment was made 5% in acetic acid by addition of glacial acetic acid after completion of the binding equilibrium, then boiled for 10 min, chilled on ice, and centrifuged for 5 min in an Eppendorf 5412 microfuge (15,600~). The supernatant was applied to a Sep-Pak previously washed with 2 ml methanol, 5 ml HrO, and 5 ml 5% acetic acid. Degraded material was eluted with 3 ml of 5% acetic acid, 10% PrOH-2, and intact with 5 ml of 5% acetic acid, 30% PrOH-2 directly into scintillation vials. Then 10 ml of PCS was added and radioactivity measured by liquid scintillation. Degra-

lo 20 30403060 Time (min ) Time (min )

FIG. 2. Kinetics of association of 8H-j3,,-EP to rat brain membranes at various temperatures in the absence (A) and presence (B) of 100 rnrd NaCl. The data from Fig. 1 were plotted and analyzed according to the equation given by Maelicke et al. (26). RH,, equilibrium concentration of bound hormone-receptor complex; Ht, total &,-EP added; Rt, total concentration of receptor (determined from Scatchard analysis); RH, concentration of bound hormone-receptor complex at time t.

P-ENDORPHIN BINDING THERMODYNAMICS 83

TABLE I RESULTS AND DISCUSSION

KD’s AND KINETICS CONSTANTS OF @,,-EP BINDING TO RAT BRAIN MEMBRANES AT VARIOUS TEMPER-

ATURES IN THE ABSENCE AND PRESENCE OF 100 mM NaCl

3H-Ph-EP binding to rat brain membranes as a function of time obtained at various temperatures, either in the absence or presence of 100 IIIM NaCl, are shown in Fig. 1. The time required to reach equilibrium is dependent on the temperature of incubation and, at each temperature studied, on the presence or absence of sodium. Considerable attention should be devoted to these changes in time equilibrium in designing binding experiments which compare effects of ions or effecters. Association rate constants (k,) of 3H-/3h- EP to the binding sites were determined in each case by analyzing data obtained from the time-course binding studies as described (26). The data obtained (Fig. 2) are consistent with a single k1 at any temperature studied both with and without 100 mM NaCl (Table I). From these data, it is apparent that 100 InM NaCl results in a lo-fold decrease of the association rate constant within the whole range of temperatures examined. Increasing the temperature of incubation produces a con- tinuous increase of the association rate constant (Table I), both in the presence and absence of sodium.

Temper- ature (“Cl

k, (M-l- min-‘)

k-1 (min-‘)

0

5 10

15

20

24

30

35 40

3.40 2.44 1.46

(15.00)b 1.15

(11.75) 0.69

(6.31) 0.61

(4.07) 0.48

(2.89) 0.59 0.75

6.27 X 10’ 2.13 X lo-’

1.55 x lo7 2.26 X lo-’

(1.43 x 106)b (2.14 X lo-‘)*

4.42 X 10’ 2.71 X lo-’ (3.31 x 106) (1.26 X 10-2) 8.14 x lo7 3.90 x 1o-2

(8.12 x 106) (2.34 X 10-2)

(1.81 X 108) 13.57 x 1o-2

a Dissociation rate constants were calculated from known values of KD.

*Values in parentheses were obtained in the presence of 100 mM NaCI.

dation (the fraction of total activity eluted from the column contributed by the 10% PrOH-2 fraction) ranged from 2 to 6% at 0-35°C.

16

Bound (PM) Bound (pM )

Scatchard analysis of replicate saturation binding experiments conducted at

FIG. 3. Scatchard analysis of &,-EP binding to rat brain membranes at various temperatures in the absence (A) and presence (B) of 100 mM NaCl. The amount of membranes was 0.57 mg of protein in all experiments. Values are the mean of duplicate incubations and slope of the plots were determined by linear regression analysis.

84 NICOLAS ET AL.

various temperatures, either in the presence (Fig. 3B) or in the absence (Fig. 3A) of 100 mM NaCl, suggest no difference in the maximum binding capacity. In all cases, the total binding capacity ranged between 0.39 and 0.42 pmol of j3h-EP bound/ mg of protein. It is also clear that a single affinity class model is sufficient to char- acterize the interaction of Ph-EP with rat brain membranes under all the various experimental conditions examined.

&-Endorphin binding is markedly sodium and temperature dependent. Be- tween 10 and 3O”C, sodium at a concentration of 100 mM depresses the binding of &,-EP by reducing its affinity for binding sites 7- to lo-fold without changing the number of binding sites (Fig. 3A, Table I). The ‘7- to lo-fold decline in the binding affinity in the presence of sodium is largely accounted for by a lo-fold decrease in the association rate constant at each temperature, with little decrease of the corre- sponding calculated dissociation rate constants (Table I). This is in contrast with kinetics data previously obtained for alkaloid opiate agonists (14). The decrease of the binding affinity due to sodium cor- responds to a small change in the free energy of binding, 1 to 1.5 kcal- M-l out of 13 kcal- M-’ at 20°C. Such small change might reasonably be attributed to disrup- tion or weakening of one or a few nonco- valent interactions as a result of an alteration of the tissue state in the presence of sodium, and/or a direct nonspecific effect of ionic strength on nonbonded interactions.

High-versus-low temperatures markedly enhanced the binding of Pi,-EP by increasing its affinity for receptors both with and without sodium (Fig. 3). The increase in binding is not due to a change in the number of binding sites, and the effects are very similar either in the absence or presence of 100 mM NaCl on a quantitative basis (Table I). There is a ‘7-fold enhancement in affinity from 0 to 30°C in the absence of sodium and a 5-fold increase in binding between 10 and 30°C in the presence of 100 mM sodium. This enhancement in affinity comes from a 13-fold increase in the association rate constant coupled

3.2 3.3 3.4 3.5 3.6 3.7 J

l/T PK x IO3 )

FIG. 4. Arrhenius plots of rate constants-temperature data for Bh-EP binding to rat brain membranes in the absence (0) and presence (0) of 100 mM NaCl. Inset: Variation of the free energy changes (AGO) of &EP-receptor binding with temperature in absence (0) and presence (0) of 100 mM NaCl.

with a very slight increase of the dissociation rate constant (1.9-fold) between 0 and 30°C in the absence of sodium (Table I). Arrhenius plots for the association rate (kr) in the presence or absence of sodium are linear with almost identical slopes (Fig. 4) reflecting similar energies of activation (E,) and enthalpy of activation (AW). Calculation of the thermodynamic parameters of activation in the presence and absence of sodium (Table II) shows that the difference in the free energy of activation (AG+), reflected by the difference in rate of association, is due almost exclusively to a difference in the entropy of activation (AS+) between these two experimental conditions. Therefore, the faster rate of association of P,,-EP with rat brain membranes in the absence of sodium is due to a larger positive entropy of activation. This might reflect an increase in the steric hindrance (worse accessibility) for association of the molecule in the presence of sodium. The similarities in the enthalpy of activation suggest that changes in the energy of intermolecular attraction or repulsion are similar in the presence or absence of sodium, over the range of temperatures studied. These results indicate that, in striking contrast with alkaloid

/9-ENDORPHIN BINDING THERMODYNAMICS 85

TABLE II

THERMODYNAMICAL PARAMETERS OF RATES FOR &-EP BINDING TO RAT BRAIN MEMBRANES IN THE ABSENCE AND PRESENCE OF 100 mM NaCl

NaCl Cm@

AG+ (kcal * M-‘)

AH+ (kcal * M-l)

AS+ (Cal/m. “K)

E, (kcal * M-l)

0 9.30 13.2 13.6 13.95 100 10.80 12.7 6.4 13.20

Note. Energy of activation (E.) was calculated from the slope of the Arrhenius plots where the slope equals -E./R. Enthalpy of activation (AW) was calculated from the equation AH+ = E, - RT. Free energy of activation was calculated from the equation AG+ = -RT In kl + RT In (kT/h), where k is the Boltzman’s constant and h is the Plank’s constant. All calculations are given at 20°C.

opiates (15), temperature variations do not interact with the influence of sodium on /3-EP receptor. This is strongly reinforced by the parallelism which is observed in the variation of the free energy changes of P-EP-receptor binding with temperature, in the absence and presence of sodium (Fig. 4). Therefore, the resulting effects of both temperature and sodium are simply additive over that range of temperatures studied.

Standard enthalpy (MO) and entropy (AS%) changes accompanying binding re- actions in the presence and absence of 100 mM NaCl were determined from analysis of van? Hoff plots of In KA (where KA is the equilibrium association constant in M-‘) versus l/T, using KA determined from saturation experiments conducted at dif- ferent temperatures. Linearity of these plots (Fig. 5) indicates that the enthalpy

changes were independent of temperature and there is no evidence of change in heat capacities between 0 and 30°C. However, above 3O”C, binding affinity decreases with temperature in the absence of sodium. It is not known whether this decrease reflects degradation of the tritiated peptide, membrane phase transition, or changes in driving forces of the binding equilibrium. Large positive values are obtained both for AH’ (+ll.O and 15.5 kcal. M-‘, respectively, in the absence and presence of sodium) and for AS”, (+79.7 and 90.5 Cal. M-l * OK-‘, respectively, in the absence and presence of 100 mM sodium). At ZO”C, free energy of binding is equal to -12.4 and -11.0 kcal. M-‘, respectively, in the absence and presence of 100 mM NaCl. There- fore, in both cases, binding reaction is strongly entropy driven. A very large entropy change counterbalances a large pos-

?i fi

l/T P’K x IO?

19 iB’ / . .

IS- \ .

\

I I I I I 32 33 3.4 35 3.6

l/T (OK x 103)

FIG. 5. van’t Hoff plots of the dependence of the equilibrium association constant K*(M-‘) on temperature for &,-EP binding to rat brain membranes in the absence (A) and presence (B) of 100 mM NaCl. Enthalpy changes were determined by linear regression analysis of the data.

86 NICOLAS ET AL.

itive unfavorable enthalpy change. As ionic interactions could only account for a very slightly positive, but usually neg- ative value of U” and positive value of AS” (27), such large positive values of both entropy and enthalpy changes with and without sodium could arise only from hydrophobic interactions (by hydrophobic interactions we are referring to the partial withdrawal of the nonpolar groups from water and not to any further interaction between these nonpolar groups them- selves). The fact that AS are positive is further evidence that the driving forces for the formation of the transition state are hydrophobic since, for example, a neg- ative value would be expected if there were more hydrogen bonds in the transition state than the reactants.

Physicochemical interpretations of the entropy and the enthalpy changes are more or less speculative for such a complex binding system. However, some molecular events can be postulated which can account for the thermodynamic changes observed. In the case of ,&EP, it is assumed that part of the molecule interacts with membrane lipids once bound to the receptor (28). This kind of hydrophobic interaction may well provide both large enthalpy and entropy changes such as those observed here, while other forces with smaller net energy contributions such as ionic interactions, hydrogen bonding, and van der Wals interactions may also be involved in a more specific process (recognition).

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10. LAW, P. Y., HOUGHTON, R. A., LOH, H. H., AND LI, C. H. (1980) in Endogenous and Exogenous Opiate Agonists and Antagonists (Way, E. L., ed.), pp. 225-228, Pergamon Press, New York.

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β-endorphin: thermodynamics of the binding reaction with rat brain membranes

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