The small changes in the UV spectrum and the retention of activity lead to a model in which enolase, in the presence of high concentrations of Mn2+, dissociates into native monomers; upo
Trang 1Use of hydrostatic pressure to produce ‘native’ monomers of yeast enolase
M Judith Kornblatt1, Reinhard Lange2,* and Claude Balny2,*
1
Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada;2INSERM Unite 128, IFR 122, Montpellier, France
The effects of hydrostatic pressure on yeast enolase have
been studied in the presence of 1 mMMn2+ When
com-pared with apo-enolase, and Mg-enolase, the Mn-enzyme
differs from the others in three ways Exposure to
hydro-static pressure does not inactivate the enzyme If the
experiments are performed in the presence of 1 mMMg2+,
or with apo-enzyme, the enzyme is inactivated [Kornblatt,
M.J., Lange R., Balny C (1998) Eur J Biochem 251, 775–
780] The UV spectra of the high pressure forms of the
Mg2+- and apo-forms of enolase are identical and distinct
from the spectrum of the form obtained in the presence of
1 mMMn2+; this suggests that Mn2+remains bound to the
high pressure form of enolase With Mn-enolase, the various
spectral changes do not occur in the same pressure range,
indicating that multiple processes are occurring Pressure
experiments were performed as a function of [Mn2+] and
[protein] One of the changes in the UV spectra shows a dependence on protein concentration, indicating that eno-lase is dissociating into monomers The small changes in the
UV spectrum and the retention of activity lead to a model
in which enolase, in the presence of high concentrations of
Mn2+, dissociates into native monomers; upon release of pressure, the enzyme is fully active Although further spectral changes occur at higher pressures, there is no inactivation as long as Mn2+remains bound We propose that the relatively small and polar nature of the subunit interface of yeast enolase, including the presence of several salt bridges, is responsible for the ability of hydrostatic pressure to disso-ciate this enzyme into monomers with a native-like structure Keywords: dissociation; enolase; hydrostatic pressure; native monomers
Many enzymes normally exist as oligomeric proteins In
some cases, the enzyme is a regulatory enzyme; allosteric
kinetics require multiple subunits In other cases, the active
site is at the interface of the subunits, with two subunits each
contributing residues In many cases, however, it is not
obvious what role is played by the oligomeric structure
Attempts to study the relationship between oligomeric state
and the structure and function of the protein usually involve
dissociating the protein into its subunits and then
compar-ing the properties of the monomeric and oligomeric forms
Often, the resulting monomers are catalytically inactive
Because tertiary and quaternary structure are maintained by
similar forces, agents, such as temperature and chemical
denaturants, that disrupt quaternary structure may also
disrupt tertiary structure Thus, when faced with inactive
monomers of an active oligomeric protein, it is difficult to
know if the conformation of the monomer has been altered
or if the quaternary structure is, in some way, necessary for
activity
Hydrostatic pressure is a useful tool for studying protein structure and function If an equilibrium system, AÐ B, is subjected to pressure, the equilibrium will be displaced in the direction of the system that occupies the smaller volume In the case of a solution of a protein, hydrostatic pressure may change the conformation, promote binding or dissociation
of a ligand, denature the protein or dissociate an oligomeric protein [1–3] Factors that contribute to differences in volume between an oligomer and its subunits include removal of packing defects, hydration of buried surfaces, and disruption of salt bridges As a general rule (although there are exceptions), the pressure required to dissociate an oligomeric protein is less than that required to denature monomeric proteins It therefore seems reasonable to expect that pressure could dissociate oligomeric proteins while having relatively little effect on the secondary and tertiary structure of the resulting monomers In spite of this expectation, most monomers produced by hydrostatic pressure have been inactive [1]
Yeast enolase (EC 4.2.1.11), which catalyzes the inter-conversion of 2-phosphoglyceric acid and phosphoenol-pyruvate, is a homodimer High resolution X-ray structures are available for the yeast enzyme [4–6], as well as enolase from lobster [7], Escherichia coli [8] and Trypanosoma brucei [9] Each subunit has two domains; the larger domain is an a/b barrel, while the smaller is a mixture of b-sheet and a-helices The dimer interface includes two helices in the large domain and two b-strands in the small domain The active site is at the bottom of the barrel and is totally
Correspondence to M J Kornblatt, Department of Chemistry and
Biochemistry, Concordia University, 7141 Sherbrooke W, Montreal,
Que H4B 1R6 Canada Fax: +1 514 848 2868,
E-mail: judithk@vax2.concordia.ca
Enzyme: enolase, EC 4.2.1.11
*Current address: Universite´ Montpellier 2, EA3763, Place Euge`ne
Bataillon, 34095 Montpellier cedex 5, France.
(Received 7 June 2004, revised 2 August 2004, accepted 6 August 2004)
Trang 2interface contains elements of secondary structure which
should be relatively unperturbed by mild treatments, we
have been puzzled by our inability to produce active
monomers Brewer, in his extensive studies on the
dissoci-ation of yeast enolase [14], dissociated the enzyme by diluting
it into millimolar solutions of EDTA These monomers,
formed in the absence of a divalent cation, were inactive Our
studies using hydrostatic pressure indicated that the Mg2+
was lost during pressure-induced dissociation [12]; these
monomers were also inactive We proposed that, in order to
maintain the structure of the active site, divalent cation and,
perhaps, substrate had to be bound to the enzyme
This article presents a study of the pressure-induced
dissociation of yeast enolase in the presence of Mn2+ We
demonstrate the ability of hydrostatic pressure to produce
native monomers – monomers which have undergone no
apparent conformational changes and maintain enzymatic
activity upon return to low pressure
Materials and methods
Buffer for all experiments contained 25 mM Mes, 25 mM
Tris, pH 7.1 The pH of this buffer is relatively insensitive to
pressure Mg2+ or Mn2+ was present at the stated
concentration; EDTA was present at 1/10th the
concentra-tion of the Mg2+or Mn2+ EDTA binds divalent cations
such as Co2+, Ni2+, Cu2+and Zn2+more strongly than it
binds Mn2+and Mg2+ EDTA was added to the buffer in
the hope of minimizing the free concentration of other
divalent cations Yeast enolase (Sigma) was dialyzed against
buffer prior to use For the experiments that compared
apo-enolase with apo-enolase containing Mg2+or varying
concen-trations of Mn2+, the enzyme was dialyzed against buffer
and then passed through a small chelex column (Bio-Rad,
Hercules, CA, USA) in order to remove divalent cations
The stated concentrations of EDTA, Mg2+ or Mn2+
were then added The concentration of yeast enolase
was determined from its absorbance at 280 nm, using,
e¼ 0.9 mLÆmg)1Æcm)1 [19] and a molecular mass of
94 000 Da Protein concentrations are expressed as the
concentration of dimeric enzyme Enzyme activity was
measured by following the change in absorbance at 240 nm,
due to the production of phosphoenol pyruvate; the
concentration of 2-phosphoglyceric acid (Sigma) in the
assay was 1 mM Pressure dissociation and pressure
inacti-vation experiments were performed at 15C
UV spectra were recorded under pressure, using a Varian
Cary 3 spectrophotometer (Varian, Australia) interfaced to
a high pressure bomb After correction for increased
absorption due to compression, the 4th derivatives of the
spectra were calculated, as described previously [20] Data
collection was optimized in order to maintain spectral
quality When the protein concentration was 9 l
changes were occurring and the length of time necessary for equilibrium to be reached One sample was used for each complete pressure curve At low pressures (the range where
no spectral changes are occurring) the pressure was held for 5–10 min, the spectrum was recorded and the pressure then increased to the next value In the pressure range where spectral changes were occurring, pressure was held for
45 min prior to recording the spectrum At high pressures (spectral changes are complete), the time at pressure was decreased to 10 min At the end of the experiment, the pressure was lowered to 5–10 MPa and, after 10 min, a final spectrum was recorded At 2.2 lM enolase, 22 min was required to record each spectrum; the time at pressure was reduced to 7 min at low and high pressures and 37 min in the region where spectral changes were occurring In all cases, in the range where changes were occurring, the total time at pressure (hold time plus recording time) was at least 53 min The spectra were analyzed using four parameters (Fig 1): (a) the absorbance value at 296 nm, after correction for pressure; (b) D1¼ (maximum value of the 4th derivative in the region of 291 nm) – (minimum value of the 4th derivative in the region of 295–296 nm); (c) D2¼ (maxi-mum value of the 4th derivative at 287–288 nm)) (mini-mum value at 283–284 nm); (iv) D3¼ (value of the 4th derivative at 276.6 nm) – (value at 279.6 nm)
In order to compare different experiments and different spectral changes, the values of these parameters were normalized as follows The low pressure and high pressure values for each parameter were determined from the spectra Values for intermediate pressures were expressed
as a fraction of the total change in that parameter that occurred between low and high pressures The values of D2 were used to calculate Kdfor dissociation of the dimer into monomers, assuming that the low pressure value of D2 is that of dimeric enzyme and the high pressure value of D2
is that of the monomer: Kd¼ 4[enolase](fraction mono-meric)2/(fraction dimeric)
As¶(ln Kd)/¶P ¼ –DV/RT, a plot of ln Kdvs pressure gives DV, the volume change for the process, and Kd at 0.1 MPa
Pressure inactivation of enolase was measured by subjecting dilute solutions of enolase to pressure for varying times, returning to 0.1 MPa, and immediately assaying the sample for enzymatic activity Samples used for equilibrium inactivation experiments contained 0.04 mgÆmL)1 BSA The albumin was added in order to minimize losses of activity due to absorption of enolase to the sides of the cuvette during the 45 min incubation under pressure Results
Exposure of yeast enolase to hydrostatic pressure in the range of 0.1–240 MPa dissociates the enzyme reversibly into
Trang 3monomers [11–13]; there is no spectral evidence for
denaturation occurring in these samples There are a
number of small changes that occur in the UV spectrum
of the protein Figure 1 shows the zero order UV spectra of
the protein at low and high pressures and the 4th derivatives
of the same spectra Changes in the UV spectra of Fig 1A,
which are small, are magnified by calculating the 4th
derivative; in addition, it is easier to quantify the changes in
the UV spectra if one uses the 4th derivatives In our
analysis, we use the following spectral characteristics: (a)
changes in the zero order spectra at 296 nm; (b) changes in
three regions of the 4th derivative of the spectra, as indicated
on Fig 1B The changes in these three regions – D1, D2,
D3 – were calculated as described in Material and methods
These spectral changes are fully and rapidly (within 10 min)
reversible upon return to 5–10 MPa The changes in the UV
spectra that occur during exposure to hydrostatic pressure
indicate that changes are occurring in the environment of at
least some of the aromatic residues This is consistent with
earlier work [11] showing changes in the intrinsic
fluores-cence emission spectrum of enolase during pressure-induced
dissociation
In an earlier study [12], we concluded that Mg2+
disso-ciates from enolase during pressure-induced dissociation of
enolase into monomers; the resulting monomers were inactive We reasoned that the monomers produced by hydrostatic pressure might maintain their native structure and activity if the divalent cation remained bound We therefore compared the spectral changes occurring under pressure with Mg2+, Mn2+or no divalent cation present The spectra in Fig 1 were recorded in the presence of
Mn2+ Figure 2 shows the spectra of the low pressure (Fig 2A) and high pressure (Fig 2B) forms of apo-, Mg2+ -and Mn2+-enolase The low pressure form is fully dimeric; based on previous experiments, we assume that the high pressure forms are monomeric The presence or absence of divalent cation has a small effect on the spectrum of dimeric enzyme Although the Mg2+ and Mn2+ forms have identical spectra at low pressure, they do not have the same high pressure spectra; this is most apparent for the parameter, D1 With the Mn2+enzyme, D1 decreases very slightly at high pressure; with the Mg2+form, there is a noticeable increase in D1 at high pressure Figure 2B shows that the spectra of the apo- and Mg2+forms of enolase at high pressure are identical and differ from that of the Mn2+ enzyme As the spectrum of Mn2+-enolase differs from the other two spectra, we conclude that Mn2+remains bound
to the high pressure form of the enzyme
Fig 2 Fourth derivative spectra of the apo-, Mg2+and Mn2+forms of yeast enolase at high and low pressures Enolase was passed through a small chelex column and diluted, with chelexed buffer, to 10 m M Additions were then made such that the samples contained 1 m M
EDTA (apo-form, solid line), 1 m M Mg2+and 0.1 m M EDTA (dotted line) or 1 m M Mn2+and 0.1 m M EDTA (dashed line) (A) Spectra were recorded at 10 MPa (B) Spectra were recorded at 220 MPa.
Fig 1 UV spectra of yeast enolase at high and low pressures The
spectrum of yeast enolase, 10.6 m M , in Mes/Tris buffer containing
1 m M Mn2+and 0.1 m M EDTA was recorded at 10 (continuous line)
and 220 (dashed line) MPa (A) The zero order spectra, which have
been corrected for the volume change due to pressure (B) The 4th
derivative of the spectra shown in (A); the three arrows indicate the
parameters that were used to analyze the changes that occur upon
exposure to pressure.
Trang 4the sample, no loss of activity was observed at 240 MPa,
even when the time at pressure was increased to 15 min
Either the high pressure form of Mn-enolase is active or
its activity is completely recovered within 1.5 min at
atmospheric pressure
Pressure experiments performed in the presence of 1 mM
Mn2+differed from those in the presence of 1 mMMg2+in
another significant manner When Mg2+was the cation, all
the spectral changes (D1, D2, D3, absorbance at 296 nm),
occurred in the same pressure range This is also observed
with apo-enzyme When Mn2+was the cation, the various
spectral changes did not all occur at once (Fig 3), indicating
the existence of multiple processes In order to determine if
any of the spectral changes monitor dissociation, the
pressure experiments were performed at three protein
concentrations – 2.2 lM, 9.4 lMand 53 lM; all three were
performed at 1 mMMn2+ Changes in D2 showed a clear
dependence on protein concentration (Fig 4A) This
indi-cates that exposure to hydrostatic pressure does dissociate
the enzyme into monomers and that D2 monitors the
dissociation In addition, if D2 is used to calculate Kdfor
dissociation as a function of pressure, data from all three
protein concentrations fall on the same line (Fig 4B) From
this data, we calculate that Kdat 10 MPa is 4.5· 10)9and
53 l sample (not shown) This suggests that these spectral changes only occur in the monomeric form of the protein
We have now established that yeast enolase, in the presence of 1 mMMn2+, is dissociated by pressure and that the spectral parameter D2 is a measure of dissociation The differences seen in the high pressure spectra of Fig 2B indicate that the Mn2+remains bound to the monomers Upon release of pressure, the enzyme is fully active
As the Mn2+ concentration is decreased, the high pressure spectra approach that of apo-enolase (Fig 5) and inactivation occurs Does this inactivation parallel dissoci-ation? An equilibrium pressure-inactivation experiment was performed with 6 nMenolase and 25mM Mn2+ Using the data shown in Fig 4B, we can calculate that, at 6 nM, the
Fig 3 Effects of pressure on the spectral parameters A solution of
enolase, 2.2 l M , containing 1 m M Mn2+, was subjected to increasing
pressure; spectra were recorded, analyzed, and normalized as described
in Materials and methods The parameters are D3 (s), D2 (d) and
absorbance at 296 nm (.).
Fig 4 Changes inD2 depend on protein concentration (A) A solution
of enolase, containing 1 m M Mn 2+ , was subjected to increasing pres-sure; spectra were recorded, analyzed, and normalized as described in Materials and methods The concentrations of enolase are 2.2 lM (s), 9.4 lM (d), 53 lM (.) (B) The data shown in (A) were used to calculate K d , as described in Materials and methods Enolase con-centrations were 2.2 l (s), 9.4 l (.) and 53 l (d).
Trang 5concentration used for the pressure-inactivation
experi-ments, the enzyme would be 95% dissociated by 90 MPa
However, inactivation does not begin until 120 MPa (8%
inactivation), with 50% inactivation occurring at about
170 MPa Dissociation is not accompanied by loss of
activity The loss of activity that occurs at the higher
pressures is reversible, with 90% of the initial activity
recovered within 12 min at 10 MPa
Pressure dissociates enolase
Depending on the identity and concentration of the divalent
cation, two different forms of the monomer are produced
One, which we call native, has spectral properties almost
identical to that of the dimeric enzyme and still has the
cation bound Upon return to 0.1 MPa, it is fully active
The second has lost the divalent cation, has greater spectral
differences and is inactive upon return to 0.1 MPa We do
not know if 1.5 min after return to 10 MPa, the enzyme is
still monomeric At 9 nMenzyme, 95% reassociation within
1.5 min would mean that the rate constant for reassociation
was 1· 107s)1ÆM )1 Although this is fast, it is within the
range of observed rate constants for protein–protein
reactions [21] What we do know is that the presence of
bound Mn2+stabilizes the monomer such that either it is
fully active or requires nothing more than reassociation to
be active We will use the term native monomer to refer to
the form of the enzyme produced by dissociation under
pressure that is fully active on return to 0.1 MPa
Our results can be summarized by the following model (Fig 6) With Mg2+as the divalent cation, dissociation, loss
of Mg2+, inactivation, and conformational changes in the monomer all occur in one step (step 1) When Mn2+is the cation, dissociation occurs (step 2) to produce monomers which still have Mn2+bound and are fully active upon return to 0.1 MPa As the pressure is raised still higher, conformational changes occur in the monomer Depending
on the concentration of Mn2+and the Kdof the monomer for Mn2+, Mn2+and activity may be retained (step 3) or
Mn2+may be lost (step 4), yielding inactive monomers As both the empty Mn2+site and the free Mn2+would be hydrated, it is not surprising that dissociation of the Mn2+
is promoted by hydrostatic pressure
Based on this model, we predicted that, at high Mg2+ concentrations, the Mg2+ form of the enzyme would behave as the Mn2+– i.e the monomers formed initially would retain Mg2+and activity This prediction has been confirmed Pressure-inactivation experiments were per-formed as a function of the Mg2+concentration Exposure
of 3 nM enolase to 220 MPa for 4 min results in almost complete inactivation of the enzyme when the sample contains 0.45 mMMg2+ If, however, the sample contains
5 mM Mg2+, there was only a 13% loss of activity Increasing the time at 220 MPa or increasing the pressure to
260 or 300 MPa did not result in any further loss of activity Discussion
A large number of oligomeric enzymes, including phospho-fructokinase [22], hexokinase [23], lactate dehydrogenase [24], and creatine kinase [25], have been examined by using pressure In these examples, and others, pressure both dissociated and inactivated the protein In addition, recov-ery of activity was a slow process and was often incomplete
We are aware of only two other studies reporting the production by hydrostatic pressure of native monomers Two of the partial activities of carbamoyl-phosphate synthetase were largely unaffected when the dimeric enzyme was dissociated [26]; in this experiment, assays were begun within 15 s of returning to 0.1 MPa Based on electropho-resis under pressure and activity staining of the gels, hydroxylamine oxidoreductase is dissociated but not inac-tivated by pressure [27]
We believe that the ability to produce native monomers
of enolase depends on at least two factors: the properties of
Fig 5 Changes in spectral parameters as a function of [Mn 2+ ]
Apo-enolase, prepared by passing a sample of enolase through a small
chelex column, was used to prepare samples containing varying
con-centrations of Mn 2+ Protein concentration was 2.2 l M Spectra were
recorded for each sample after 10 min at 10 MPa and after 45 min at
2200 MPa The fourth derivatives were calculated as described in
Materials and methods The changes in spectral parameters D1 (j)
and D2 (h), are expressed as the ratio of the high pressure to low
pressure values.
Fig 6 Model for the effects of hydrostatic pressure on yeast enolase Species in bold are enzymatically active; monomer and monomer* indicate different conformations of the monomer.
Trang 6percentage surface area buried at the interface vary from
protein to protein; as a general rule, the greater the
percentage buried, the greater the degree of hydrophobicity
Both Tsai et al [28] and Janin et al [29] suggest that in
oligomers with large interfaces, the isolated monomers
would be unstable, due to the exposure of the large
hydrophobic surface to solvent The subunit interface of
yeast enolase is small by the criteria of Tsai et al with only
13% of the surface buried [4] In addition, there are a large
number of polar groups at the interface, many of which
participate in subunit-subunit hydrogen bonds or
electro-static interactions Enolase monomers appear to be
relatively stable under pressure Even the inactive
apo-monomers, formed in the presence of low Mg2+or Mn2+
and held at 240 MPa for 45 min or more, rapidly and
completely recover both activity and spectral properties
upon depressurization There is no evidence for
irreversi-bility or conformational drift [30] In the presence of bound
divalent cation, the monomers remain native, even after
45 min at 220 MPa
Exposure of a system at equilibrium to increasing
hydrostatic pressure will shift that equilibrium towards
the system that occupies the smaller volume The native
structure of a protein – secondary, tertiary and quaternary
structure – reflects a balance between opposing factors
Conformational entropy disfavors the native structure,
while van der Waals interactions, electrostatic
inter-actions, hydrogen bonds and hydrophobic interactions are
favorable Hydrostatic pressure, by reducing the size of
internal cavities, decreases flexibility of the protein core
At the same time, portions of the protein near the surface
become more flexible since pressure promotes hydration
of the protein [31] Electrostatic interactions are disrupted
by pressure, with a DV of)17 to )35 mLÆmol)1[32]; this
volume change is due to electrostriction of water around
charged groups Hydrogen bond disruption has a small,
positive DV; these bonds are strengthened and diversified
by pressure [33–36] The direction and magnitude of the
volume change for disruption of hydrophobic bonds is
still under debate [3,37] Creating active monomers of an
oligomeric enzyme may require selectively disrupting
those interactions that maintain quaternary structure
without perturbing those that maintain tertiary structure
According to the crystal structures of yeast enolase, there
are two glutamate and two arginine residues per subunit
that form salt bridges with the two arginine and two
glutamates on the other subunit Subunit interactions in
yeast enolase are not very strong; in the presence of
1 mMMn2+, Kdis 4.5· 10)9and DV¼)120 mLÆmol)1
(Fig 5) Given the large negative volume change for
disruption of salt bridges, the pressure-induced
dissoci-ation of yeast enolase may be driven primarily by
disruption of these interactions
changes is complicated by the fact that low pressure may affect the structure of an oligomeric protein without causing dissociation [38]; similarly, pressure can produce changes in spectra and activity of monomeric enzymes in the absence of denaturation [39] We were fortunate to find a spectral change that monitored dissociation of enolase, and to find conditions in which the monomer was stable
Although the observed changes in the UV spectrum of yeast enolase are small, they provide information on the changes that occur during exposure to pressure The first parameter to change D2, shows a clear dependence upon protein concentration, indicating that D2 is monitoring dissociation of the protein into monomers In the 4th derivative of the UV spectrum, the region of D2, 282–
288 nm, contains contributions from both tyrosine and tryptophan residues [20] Simulations of the enolase spectra, using standard spectra of tyrosine and tryptophan in various solvents, show that the changes in D2 that are produced by pressure are due to changes in the environ-ment of tyrosine residues In an earlier study [13], we proposed that the observed decrease in the polarity of the environment of the tyrosine residues was due to two residues which point into a cleft, between the subunits, that
is filled with immobilized water Upon dissociation, the water would no longer be immobilized and its average polarity would decrease As far as we can tell from the UV and fluorescence spectra (not shown), nothing else is happening to the protein at pressures below 150 MPa The enzyme is being dissociated into monomers which maintain their native conformation
A comparison of the results of apo-enolase [12] with those of enolase in the presence of 1 mM Mg2+[12], low (50 lM) and high (1 mM) Mn2+, gives the following picture: (a) The presence of divalent cations stabilizes the dimeric structure of enolase, as has been demonstrated previously [14] This implies that Me2+ binds more tightly to the dimer than to the monomer (b) The dimeric structure stabilizes the conformation of enolase;
we do not observe changes in the spectra until dissoci-ation occurs (c) The presence of divalent cdissoci-ations also stabilizes the monomer of enolase The stabilizing effect
of the divalent cation is not a unique property of Mn2+, but is also observed with Mg2+
We can now begin to explain the role of the dimeric structure of yeast enolase The dimeric structure stabilizes the structure of the monomer and favors the binding of divalent cations, which in turn stabilize the dimer
We find it difficult to believe that there are not other dimeric proteins that could be dissociated by pressure into native monomers Although the same forces are involved in maintaining tertiary and quaternary structure,
in many cases they will not make the same relative
Trang 7contributions to both levels of structure We believe the
key points for success are threefold: (a) finding conditions
that stabilize the monomer without excessively stabilizing
the dimer This may require exploring a range of
temperatures, pH, ion concentrations, etc; (b) examining
with care the pressure range in which major spectral
changes are not occurring and (c) using, under pressure,
techniques such as fluorescence polarization [11] or
dynamic light scattering that are direct measures of the
size of a protein
Acknowledgements
We thank Concordia University for the sabbatical leave during
which time these experiments were performed, and J A Kornblatt
for encouragement Financial support was provided by the Natural
Sciences and Engineering Research Council of Canada and
INSERM.
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