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305 - Structural dynamics of prothrombin interacting with mixed monolayers of phosphatidyl serine and phosphatidyl choline
Bioelectrocke~rzistry J_ Electronnnl. @
Elsevier
nnd Chem.
Sequoia
Bioener,oetics 104
SA_,
(1979)
6 (1979)
537-5-p
537-541
Lausanne
-
Printed
in Italy
305 - Struetural~ Dynamics of Prothrombin Interacting with Mixed Monolagers of Phosphatidyl
and Phosphatidyl Choline
Serine
by M_ F. LECOMPTE * and I. R. MILLER Department Israel Manuscript
of Membrane received
August
Research, 6th
\\‘eizmann
Institute
of Science,
Rehovot,
1979
The conversion path of prothrombin to thrombin goes through its membrane-bound complexes with the proteolytic enzyme Factor X, and with the conversion-acceerating protein Factor V_lA3 In these complexes the interaction of the proteins with negatively-charged phospholipids, e.g. phosphatidyl serine (PS) in the presence of Ca”+, plays a crucial role and indeed the membranes can be replaced by phosphatidyl The maximal biological actw-ity observed serine-containing liposomes. is not on the surface of stron_gly-bondin, e surfaces of pure PS but on phospholipid mixtures containing around 25 O/$, PS_” It has been suggested that the biological activity on a phospholipid surface of this or other composition is related to the conformation4 but it may be also related to the freedom of motion of the adsorbed protein molecules_ This possibility was considered by investigating the electrodic behaviour of fully compressed spread lipid monolayers containing IOO o/o PS and ag 7’0 PS + 75 o/o phosphatidyl choline (PC) interacting with prothrombin and with its Fragment I. The freedom of motion and the conformational constraints on the ,availabi!ity of hidden groups were inferred from the from the_ osi-reduction of the cystinesv’ pseudocapacitance .resulting groups of the adsorbed prothrombin and of Fragment I as measured by n-c. polarography5~6~s with a mercury electrode contacting the manolayer from the gaseous phase. The concentration of the prothrombin (PT) and of its Fragment I adsorbed to monolayers of phosphatidyl serine was obtained fror;n the surface radiation of the respective protein 3H labelled in its sialic acid residuem6 The ma_ximal surface concentration of the prothrdmbin adsorbed in the presence of no Ca”+ or low Ca2+ concentration (
Paris-Nerd,
Laboratoire
Villetaneuse,
de Recherches
France
sur les 1\Iacromolkules,
Uni-
Lecompte and Miller
535
masimal number of adsorbed molecules covers about 50 o/o of the area. The capacitance at -0-5 Tr relative to the ~V-calomel electrode of the surface layer varies under these conditions from about 1.5 pF/crn” for the pure lipid monolayer to about 6.5 pF/cm”- at masimal adsorption_ Since the capacitance of a condensed monolayer of prothrombin at -0.5 V is about 12 @F/cm”, the degree of penetration or displacement of the lipid by the protein as obtained From the assumption of the parallel arrangement of the lipid and the protein monolayer is 3 0 =
(6.5 -
I.~)/(Iz -
1-5) z
0-45
It is seen from this that almost all the prothrombin adsorbed up to this concentration penetrates or partly displaces the lipid monolayer_ Further adsorption of prothrombin up to S x ro-l”~mole/cmB- (- zoo AZ/molecule) at increased concentrations of Ca”+ does not alter the capacitance any more, indicating that no additional perturbation of the hydrophobic side of the surface structure adjacent to the electrode surface is incurred. The masimal capacitance values are approached below concentrations of 2 &cm3The area of the pseudocapacitance peak to about 0.7 V or its integral JCdU over the whole potential span (insert in Fig. 2) gives. the number of charges transferred durin,= the cystine-cysteine oAxi-reduction. If the electrode process is a kinetic one, the number of charges transferred increases when lowering the frequency of the applied a-c. potential is lowered, until at very low frequencies the total equilibrium value of the charge transferred is obtained_’ Working at a fixed frequency the area of the pseudocapacitance peak is a function of the surface concentration of the cystine-containing protein molecules, as well as of their conformation and their freedom of movement in the surface. In Fig. I (upper two curves, right ordinate), the number of cystine residues inferred from the charge transferred, as obtained from the integral 1 CdU over the peak is plotted against the lo,m of the concentration of Ca”f when prothrombin is adsorbed on a prosphatidyl serine monolayer_ The curve obtained at Pt concentration > 5 pg/cm3, at which the protein approaches surface saturation, as measured by surface radioactivity of tritium-labelled PT at all the Ca”+-concentrations, is above that obtained at 3.2 pg/cm3, Bearing in mind that the background capacitance, which measures degree of penetration, reaches its limiting value below 2 pg/cm3, we have to conclude that the pseudocapacitance is related to the total prothrombin molecules on the surface and not just to those that have penetrated the lipid layer. Such behaviour can be esplained only if there is a free exchange between the penetrating molecules and those adsorbed on the lipid-water interface_ It is, therefore, instructive to plot the number of cystines reduced per adsorbed PT molecule as a function of Ca2+ concentration. This is done in Fig. I and about 0.4 cystines undergo ox&reduction per PT molecule up to 10-l mfU Ca2+ as measured by capacitive a-c. current at So Hertz. At higher concentrations of Ca”+ this number starts decreasing_ This indicates that the high concentrations of. Ca”+ cause an immobilisation of the prothrombin molecules bound to the phosphatidyl serine monolayer.
Structural Dynamics of Protbrombin
-3
-2
-1
0
53b
1
log Cat* Fig. I. Interaction of cystine residues of prothrombin adsorbed on PS containiug phosphohpid monolayer with the mercury electrode_ Upper curves: number of moles of cystine~per cm* undergoing redos reaction through a Ps-containing monolayer on the electrode at So-Herz a~ inferred from the pseudocapacitance peak area. Lower curve: number of reacting cystino residue per adsorbed prothrombin molecule_ 0 - at surface saturation by prothrombin and e at prothrombin con_centrationof 3.2 pg/cnts - for a pure PS monolayer, /-- at surface saturation on a monolayer containing 75 yO-PC and 25 oh PS. . 6
%
.._
The behaviour of prothrcmbin Fragment I is entirely di$erentl.- At low Ca”+ concentrations ( (Io-2 m&Q it produces enormous pseudocapacitance peaks when adsorbed on PS monolayers. The amGunti:,adK sorbed under these conditions is very small, e.g. at 10-s Ca2* the- amotintadsorbed at bulk concentration of 3 x 10-m moles/ems at which the overal) capacitance as well as the pseudocapacitance peaks have reached-thtiir ma,ximal values, the surface concentration of Fragment I is only about The size of the pseudocapacitance peak under-the 5 X ro-13 moIes/cm-2. same conditions indicates that between about 2.0 and 2-5 x 107’~ moles/ cm2 of S-S groups undergo the redox reaction or almost al& the: five cystines of Fragment I are available for the electrode process. As +videtit from Fig. 2, this number decreases sharply with increasing concentratioti of Ca2f until in the mUimolar, region only a negligible fractions of cystine
Lecompte
540
and
MilIer
N
-0 t2=
zd
i
L-l-
c
06 34
-
02 -2
log Co Fig.
0
-1
2+
2.
Interaction of cystine residues of Fragment I. adsorbed on a PS monolayer. with the mercury electrode_ Number of moles of cystine per cm’ participatin, = in the electrode reaction at So Hera 0. Insert : Capacitance Number of reacting cystine groups per adsorbed Fragment I &against potcntia1 relative to N-calomel electrode_ Lower curl-e pure PS monolayer. Upper curl-e as affected by interaction with prothrombin or Fragment I_ The pseudocapacitance peak is shaded.
residue is available for the electrode process per adsorbed Fragment I. Thus Ca”’ induces conformational stabilization of Fragment I and probably also of the intact prothrombin. This is in keeping with the view that Ca”+ induces a slow structural change in the Fra,gnent I or in the prothrombin before the fast binding reaction to the phospholipid surface.* However, while the fraction of S-S groups per adsorbed Fragment I molecules undergoing electrode process at So Hertz between 0.1: and 2 m.31 Ca” is negligible, their fraction per adsorbed prothrombin is still appreciable (vaqkg between 0-4 and 0-1 S-S groups per molecule)_ This indicates that under these conditions it is not one of the five cystines of Fragment I that interacts with the electrode through the lipid layer but that it is either the prothrombin or the Fragment II. On the other
Structural
Dynamics
of Prothrombin
-=&&I
hand the very high electrodic activity of the Fragment I cystines acrossthe lipid show an open conformation of Fragment I after its cleavage from the prothrombin, which is then reverted by addition of Ca”+. Also the degree of freedom of the bound molecules to move, to rotate, or to exchange depends both on the charge of the phospholipid surface and on the Ca2+ concentration_ This right balance between the adsorption and freedom to accomodate on the membrane surface with respect to Factor Tr and X, may be of major importance in the prothrombin to thrombin transformation. The enhancement of prothrombin to thrombin transformation by phospholipids was found to be maximal when the phospholipid contained about 25 o/O PS and Ca2+ concentration was in the millimolar region. The effect of the concentration of Ca*+ in the presence of phospholipids of different composition has not been investigated as yet_ However, the present results indicate that a correlated effect of C&2+ concentration and of phospholipid composition is to be expected.
References J_\V_ SUTTLE and CX. JACKSOS. PI~ysioZ.Re-J. 57 (1977) I B-F_-1. ZWAAL, Biochinr. Biophys. Acta 515 (197s) 163 E-W. DAVIE and I<. FUJIKAWA, Amzrc. Rev. Biochenr. 44 (1975) igg G.L. XELSESIUEP, J_ BioZ. Chem. 251 (1976) 5.64s 1-R. MILLER, Experientia SIL$@. 18 (rg7r) 477 M-F. LECOMPTE and 1-R. MILLER, Adv. Chew Ser. (in press) 0. PAVLOVIC and 1-R. MILLER, Experiedia SxppZ. 18 (1971) 513 1-R. MILLER, J_ RISHPOX and _-I.TESNENBAUM, Bioelectrochewt. Bioexirg. (1976)
PS
3
Elsevier
nnd Chem.
Sequoia
Bioener,oetics 104
SA_,
(1979)
6 (1979)
537-5-p
537-541
Lausanne
-
Printed
in Italy
305 - Struetural~ Dynamics of Prothrombin Interacting with Mixed Monolagers of Phosphatidyl
and Phosphatidyl Choline
Serine
by M_ F. LECOMPTE * and I. R. MILLER Department Israel Manuscript
of Membrane received
August
Research, 6th
\\‘eizmann
Institute
of Science,
Rehovot,
1979
The conversion path of prothrombin to thrombin goes through its membrane-bound complexes with the proteolytic enzyme Factor X, and with the conversion-acceerating protein Factor V_lA3 In these complexes the interaction of the proteins with negatively-charged phospholipids, e.g. phosphatidyl serine (PS) in the presence of Ca”+, plays a crucial role and indeed the membranes can be replaced by phosphatidyl The maximal biological actw-ity observed serine-containing liposomes. is not on the surface of stron_gly-bondin, e surfaces of pure PS but on phospholipid mixtures containing around 25 O/$, PS_” It has been suggested that the biological activity on a phospholipid surface of this or other composition is related to the conformation4 but it may be also related to the freedom of motion of the adsorbed protein molecules_ This possibility was considered by investigating the electrodic behaviour of fully compressed spread lipid monolayers containing IOO o/o PS and ag 7’0 PS + 75 o/o phosphatidyl choline (PC) interacting with prothrombin and with its Fragment I. The freedom of motion and the conformational constraints on the ,availabi!ity of hidden groups were inferred from the from the_ osi-reduction of the cystinesv’ pseudocapacitance .resulting groups of the adsorbed prothrombin and of Fragment I as measured by n-c. polarography5~6~s with a mercury electrode contacting the manolayer from the gaseous phase. The concentration of the prothrombin (PT) and of its Fragment I adsorbed to monolayers of phosphatidyl serine was obtained fror;n the surface radiation of the respective protein 3H labelled in its sialic acid residuem6 The ma_ximal surface concentration of the prothrdmbin adsorbed in the presence of no Ca”+ or low Ca2+ concentration (
Paris-Nerd,
Laboratoire
Villetaneuse,
de Recherches
France
sur les 1\Iacromolkules,
Uni-
Lecompte and Miller
535
masimal number of adsorbed molecules covers about 50 o/o of the area. The capacitance at -0-5 Tr relative to the ~V-calomel electrode of the surface layer varies under these conditions from about 1.5 pF/crn” for the pure lipid monolayer to about 6.5 pF/cm”- at masimal adsorption_ Since the capacitance of a condensed monolayer of prothrombin at -0.5 V is about 12 @F/cm”, the degree of penetration or displacement of the lipid by the protein as obtained From the assumption of the parallel arrangement of the lipid and the protein monolayer is 3 0 =
(6.5 -
I.~)/(Iz -
1-5) z
0-45
It is seen from this that almost all the prothrombin adsorbed up to this concentration penetrates or partly displaces the lipid monolayer_ Further adsorption of prothrombin up to S x ro-l”~mole/cmB- (- zoo AZ/molecule) at increased concentrations of Ca”+ does not alter the capacitance any more, indicating that no additional perturbation of the hydrophobic side of the surface structure adjacent to the electrode surface is incurred. The masimal capacitance values are approached below concentrations of 2 &cm3The area of the pseudocapacitance peak to about 0.7 V or its integral JCdU over the whole potential span (insert in Fig. 2) gives. the number of charges transferred durin,= the cystine-cysteine oAxi-reduction. If the electrode process is a kinetic one, the number of charges transferred increases when lowering the frequency of the applied a-c. potential is lowered, until at very low frequencies the total equilibrium value of the charge transferred is obtained_’ Working at a fixed frequency the area of the pseudocapacitance peak is a function of the surface concentration of the cystine-containing protein molecules, as well as of their conformation and their freedom of movement in the surface. In Fig. I (upper two curves, right ordinate), the number of cystine residues inferred from the charge transferred, as obtained from the integral 1 CdU over the peak is plotted against the lo,m of the concentration of Ca”f when prothrombin is adsorbed on a prosphatidyl serine monolayer_ The curve obtained at Pt concentration > 5 pg/cm3, at which the protein approaches surface saturation, as measured by surface radioactivity of tritium-labelled PT at all the Ca”+-concentrations, is above that obtained at 3.2 pg/cm3, Bearing in mind that the background capacitance, which measures degree of penetration, reaches its limiting value below 2 pg/cm3, we have to conclude that the pseudocapacitance is related to the total prothrombin molecules on the surface and not just to those that have penetrated the lipid layer. Such behaviour can be esplained only if there is a free exchange between the penetrating molecules and those adsorbed on the lipid-water interface_ It is, therefore, instructive to plot the number of cystines reduced per adsorbed PT molecule as a function of Ca2+ concentration. This is done in Fig. I and about 0.4 cystines undergo ox&reduction per PT molecule up to 10-l mfU Ca2+ as measured by capacitive a-c. current at So Hertz. At higher concentrations of Ca”+ this number starts decreasing_ This indicates that the high concentrations of. Ca”+ cause an immobilisation of the prothrombin molecules bound to the phosphatidyl serine monolayer.
Structural Dynamics of Protbrombin
-3
-2
-1
0
53b
1
log Cat* Fig. I. Interaction of cystine residues of prothrombin adsorbed on PS containiug phosphohpid monolayer with the mercury electrode_ Upper curves: number of moles of cystine~per cm* undergoing redos reaction through a Ps-containing monolayer on the electrode at So-Herz a~ inferred from the pseudocapacitance peak area. Lower curve: number of reacting cystino residue per adsorbed prothrombin molecule_ 0 - at surface saturation by prothrombin and e at prothrombin con_centrationof 3.2 pg/cnts - for a pure PS monolayer, /-- at surface saturation on a monolayer containing 75 yO-PC and 25 oh PS. . 6
%
.._
The behaviour of prothrcmbin Fragment I is entirely di$erentl.- At low Ca”+ concentrations ( (Io-2 m&Q it produces enormous pseudocapacitance peaks when adsorbed on PS monolayers. The amGunti:,adK sorbed under these conditions is very small, e.g. at 10-s Ca2* the- amotintadsorbed at bulk concentration of 3 x 10-m moles/ems at which the overal) capacitance as well as the pseudocapacitance peaks have reached-thtiir ma,ximal values, the surface concentration of Fragment I is only about The size of the pseudocapacitance peak under-the 5 X ro-13 moIes/cm-2. same conditions indicates that between about 2.0 and 2-5 x 107’~ moles/ cm2 of S-S groups undergo the redox reaction or almost al& the: five cystines of Fragment I are available for the electrode process. As +videtit from Fig. 2, this number decreases sharply with increasing concentratioti of Ca2f until in the mUimolar, region only a negligible fractions of cystine
Lecompte
540
and
MilIer
N
-0 t2=
zd
i
L-l-
c
06 34
-
02 -2
log Co Fig.
0
-1
2+
2.
Interaction of cystine residues of Fragment I. adsorbed on a PS monolayer. with the mercury electrode_ Number of moles of cystine per cm’ participatin, = in the electrode reaction at So Hera 0. Insert : Capacitance Number of reacting cystine groups per adsorbed Fragment I &against potcntia1 relative to N-calomel electrode_ Lower curl-e pure PS monolayer. Upper curl-e as affected by interaction with prothrombin or Fragment I_ The pseudocapacitance peak is shaded.
residue is available for the electrode process per adsorbed Fragment I. Thus Ca”’ induces conformational stabilization of Fragment I and probably also of the intact prothrombin. This is in keeping with the view that Ca”+ induces a slow structural change in the Fra,gnent I or in the prothrombin before the fast binding reaction to the phospholipid surface.* However, while the fraction of S-S groups per adsorbed Fragment I molecules undergoing electrode process at So Hertz between 0.1: and 2 m.31 Ca” is negligible, their fraction per adsorbed prothrombin is still appreciable (vaqkg between 0-4 and 0-1 S-S groups per molecule)_ This indicates that under these conditions it is not one of the five cystines of Fragment I that interacts with the electrode through the lipid layer but that it is either the prothrombin or the Fragment II. On the other
Structural
Dynamics
of Prothrombin
-=&&I
hand the very high electrodic activity of the Fragment I cystines acrossthe lipid show an open conformation of Fragment I after its cleavage from the prothrombin, which is then reverted by addition of Ca”+. Also the degree of freedom of the bound molecules to move, to rotate, or to exchange depends both on the charge of the phospholipid surface and on the Ca2+ concentration_ This right balance between the adsorption and freedom to accomodate on the membrane surface with respect to Factor Tr and X, may be of major importance in the prothrombin to thrombin transformation. The enhancement of prothrombin to thrombin transformation by phospholipids was found to be maximal when the phospholipid contained about 25 o/O PS and Ca2+ concentration was in the millimolar region. The effect of the concentration of Ca*+ in the presence of phospholipids of different composition has not been investigated as yet_ However, the present results indicate that a correlated effect of C&2+ concentration and of phospholipid composition is to be expected.
References J_\V_ SUTTLE and CX. JACKSOS. PI~ysioZ.Re-J. 57 (1977) I B-F_-1. ZWAAL, Biochinr. Biophys. Acta 515 (197s) 163 E-W. DAVIE and I<. FUJIKAWA, Amzrc. Rev. Biochenr. 44 (1975) igg G.L. XELSESIUEP, J_ BioZ. Chem. 251 (1976) 5.64s 1-R. MILLER, Experientia SIL$@. 18 (rg7r) 477 M-F. LECOMPTE and 1-R. MILLER, Adv. Chew Ser. (in press) 0. PAVLOVIC and 1-R. MILLER, Experiedia SxppZ. 18 (1971) 513 1-R. MILLER, J_ RISHPOX and _-I.TESNENBAUM, Bioelectrochewt. Bioexirg. (1976)
PS
3