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Vol. 59, Issue 4, 886-893, April 2001
-35S]thio-triphosphate Binding through
M1 Muscarinic Receptors in Transfected Chinese Hamster
Ovary Cell Membranes: 2. Testing the "Two-States" Model of Receptor
Activation
Department of Biochemistry and Nutrition, Medical School, Université Libre de Bruxelles, Brussels, Belgium
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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I suggested in the accompanying article [Mol Pharmacol
2001;59:875-885] that muscarinic receptors catalyzed G
protein activation. Acetylcholine or carbamylcholine recognition
facilitated not only the GDP release from receptor-coupled inactive G
proteins but also the release of
G
S binding in the presence of GDP,
but could be studied separately by comparing [35S]GTPS
binding in the absence and presence of GTP. Guanyl nucleotides affected
the efficiency of receptor-G protein coupling. The relative efficacies
of partial agonists in the absence and presence of GTP should remain
nonlinearly correlated if all agonists stabilize (to different extents)
the same active receptor conformation. The correlation between
M1 muscarinic agonists' efficacy in accelerating [35S]GTPS binding in the absence of other nucleotides
and their in vivo efficacy (inositol phosphate accumulation) was in
fact very poor. This probably reflected the presence of GTP in intact cells: pertussis toxin pretreatment (which inactivates the
Gi/o proteins) did not affect the agonists' efficacy
profile (evaluated in the absence of spare receptors), but the addition
of GTP to the [35S]GTPS binding medium did. These
results did not support the allosteric "two states" model of
receptor activation, but suggested that different agonists induced
different receptor conformations ("induced fit").
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Small
ligands are able to induce significant conformational changes in their
binding proteins. Two extreme descriptions of this phenomenon can be
seen by analogy with the hemoglobin and steroid receptors
ligand-induced conformational changes, respectively. Hemoglobin exists
in two dominant conformations: an empty conformation (T) stabilized by
a network of ionic and hydrogen bonds, and another, oxygen-bound
conformation (R). Allosteric modulators, which recognize a single
binding site between the four subunits, do not affect the "R" and
"T" hemoglobin conformations but stabilize one of the two states.
They thereby inhibit or facilitate oxygen binding. The estrogen
receptor conformation depends on the nature of the bound ligand:
different compounds induce different receptor conformations and
therefore produce variable biological effect patterns (Smith and
O'Malley, 1999
).
G-protein coupled receptors (GPCRs) possess seven transmembrane
-helices. Such -helices are typically stable and rigid: they are
stabilized by an extended of hydrogen bonds network. GPCR activation is
thought to involve the disruption of an intramolecular ionic bridge and
coordinated movement of two or more transmembrane helices (Sealfon et
al., 1995; Porter et al., 1996; Gether et al., 1997; Scheer et al.,
1997; Kobilka et al., 1999; Sheikh et al., 1999; Ghanouni et al., 2000;
Porter and Perez 2000; Schulz et al., 2000). In addition, some GPCRs
dimerize, and this affects their ability to activate G proteins (Hebert
et al., 1998; Hebert and Bouvier 1998). It therefore seemed plausible
that
like hemoglobinGPCRs possess two predominant conformations.
Agonists can be defined as ligands that stabilize an active receptor
conformation and thereby increase G protein activity, whereas inverse
agonists, which recognize preferentially a "resting" receptor
conformation, suppress the basal G protein activity (Samama et al.,
1993, 1994; Chidiac et al., 1994).
In response to agonist binding, GPCRs usually activate one or several
trimeric G protein(s), receptor kinases known as GPCR kinases
(Pitcher et al., 1998), and "Velcro" proteins, such as arrestins
(Krupnick and Benovic, 1998) that are involved in the desensitization
of the functional response and receptor internalization, respectively.
The mode of receptor activation (induced fit or stabilization of the
one-and-only active conformation?) might have important repercussions
on the receptor-intracellular protein interaction: if only two receptor
conformations exist, agonists and inverse agonists should activate or
inhibit, respectively, the same set of intracellular proteins with the
same relative efficacy; if each ligand induces a distinct receptor
conformation, one might expect to find drugs with different G protein,
GPCR kinase, and arrestin activation profiles.
Several results, in fact, suggest that the conformation of some GPCRs
is ligand-dependent. First, different ligands may induce different G
protein activation profiles (Meller et al., 1992; Spengler et al.,
1993; Robb et al., 1994; Perez et al., 1996; Berg et al., 1998; Bonhaus
et al., 1998; Zuscik et al., 1998). Second, the ligands' abilities to
induce receptor phosphorylation, desensitization, and internalization
are not always correlated with their intrinsic activities for G protein
activation (Keith et al., 1996; Blake et al., 1997; Roettger et al.,
1997; Yu et al., 1997; Mhaouty-Kodja et al., 1999). Third, the rate of
activated G protein release by 
2-adrenergic
receptors depends on the bound agonist (Krumins and Barber 1997).
Fourth, the efficacy of ligands stimulating adenylate cyclase through
2-receptor-GS fusion
proteins in the presence of GTP differed considerably from their
efficacies in the presence of XTP or ITP (Seifert et al., 1999).
My goal was to test whether different muscarinic agonists stabilize (to
different extents) the same unique "active"
M1 receptor conformation or induce different
conformational changes. I designed two experimental approaches: 1) G
protein activation is a cyclic reaction (Cassel and Selinger 1978; Hamm
1998). Reaction cycles cannot occur faster than the slowest reaction in
the cycle, known as the "rate-limiting step". It is possible to
switch the rate-limiting step for G protein activation by changing the
guanyl nucleotide composition of the incubation medium. I verified that
on theoretical grounds, if all muscarinic agonists stabilize the same
receptor conformation, the rank order of agonist efficacy should be
independent of the incubation conditions
(Appendix1); then I
investigated the effect of guanyl nucleotides on the rank order of
efficacy of a panel of muscarinic agonists. 2) M1 muscarinic receptors facilitate [35S]GTPS
binding to Gq/11 and Gi/o
proteins (Lazareno and Birdsall 1993; DeLapp et al., 1999). I tested
the effect of pertussis toxin treatment (which inactivates
Gi/o proteins) on the agonists' efficacy profile
in [35S]GTPS binding assays. My results did
not support the two-states model of G protein activation.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Materials.
4-Diphenylacetoxy-1-(2-chloroethyl)piperidine
(4-DAMP) mustard was a generous gift from Dr. R. Barlow (Kirkby
Stephen, Cumbria, UK).
l-[N-methyl-3H]
scopolamine methyl chloride ([3H]NMS; 80 Ci/mmol) and guanosine
5'-[-35S]thio-triphosphate, triethylamine
salt ([35S]GTPS; >1000 Ci/mmol) were
obtained from Amersham Pharmacia Biotech (Bucks, UK). Unlabeled
guanyl nucleotides (as Li+ salts) were obtained
from Roche Molecular Biochemicals (Mannheim, Germany). The
agonists were obtained from the following sources: acetylcholine
chloride, (rac)-acetyl-(-methyl)choline bromide, acetylthiocholine chloride, arecoline hydrobromide, and
(rac)-carbamyl-(-methyl)choline chloride were from Sigma Chemical
Co. (St. Louis, MO), carbamylcholine hydrochloride was obtained from
Merck (Darmstadt, Germany), oxotremorine sesquifumarate was from
Aldrich (Milwaukee, WI), oxotremorine methiodide was from ICN
Biomedicals Inc. (Aurora, OH) and pilocarpine hydrochloride was from
Janssen Chimica (Beerse, Belgium). Pertussis toxin was obtained from
Sigma. All other chemicals were of the highest grade available. Stably
transfected CHO cells expressing the human M1
muscarinic receptor subtype (Hm1 CHO cells) were a generous gift from
Dr. N. Buckley (London, England).
Methods.
The Hm1 CHO cell culture conditions, 4-DAMP mustard
and pertussis toxin treatments, [35S]GTPS
and [3H]NMS binding experiments were performed
as detailed in the accompanying article (Waelbroeck, 2001).
Inositol Phosphates Synthesis and [3H]NMS Binding to Intact Cells. Hm1 CHO cells were subcultured for 48 to 72 h in 24-well plates, in the presence (for IP turnover studies) or absence (for binding studies) of [3H]inositol (1 µCi/ml). Unless otherwise indicated, confluent cells were treated 1 h with 30 nM 4-DAMP mustard before functional studies.
The inositol phosphates accumulation was measured as follows. Each well was incubated 30 min at 37°C with 250 µl of DMEM enriched with myo-[3H]inositol (1 µCi/ml), 20 µl of 150 mM LiCl (final concentration, 10 mM), and 30 µl of agonist solution in the HEPES/NaCl-MgCl2 binding buffer. The medium was then aspirated, the incubation stopped by 0.5 ml of ice-cold methanol, and the cells scraped. Each well was rinsed by a second 0.5-ml methanol addition. The pooled fractions were extracted by 2 ml of chloroform in the presence of 1 ml of water. A 1.5-ml fraction of the aqueous phase was deposited on ion exchange column to separate [3H]inositol from the [3H]inositol phosphates (IP, IP2, IP3, and IP4) as described previously (Van-Rampelbergh et al., 1997). The total radioactivity was measured by liquid
scintillation counting.
[3H]NMS binding was measured in a larger
incubation volume to minimize tracer depletion by muscarinic receptors.
Each well was incubated for 30 min at 37°C with 1.25 ml of DMEM
enriched with [3H]NMS (0.8 nM), 100 µl of 150 mM LiCl (final concentration, 10 mM) and 150 µl of agonist solution
in the HEPES-NaCl-MgCl2 binding buffer. The
medium was then aspirated, the wells rinsed with ice-cold DMEM, and the
cells harvested in 1 ml of ice-cold PBS (phosphate-buffered saline)
enriched with 1 mM EDTA. Each well was rinsed with 1 ml PBS/EDTA, the
two fractions were pooled, and the bound radioactivity measured by
liquid scintillation counting. The tracer's affinity (KD, 0.30 ± 0.06 nM) was determined
in the same incubation conditions by saturation curves (0.03 to 2.0 nM
[3H]NMS). I verified the validity of the Cheng
and Prusoff (1973) equation by analysis of
[3H]NMS/agonist competition curves at different
tracer concentrations.
Data Analysis. Non linear curve fitting was performed with a computer assisted curve fitting program (Prism; GraphPAD Software, San Diego, CA).
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Agonist Binding and Functional Properties in CHO Cell
Membranes.
I verified that agonists achieved equilibrium binding
to CHO cell membranes within 10 min [accompanying article (Waelbroeck, 2001)]. I then compared the agonist binding properties in the absence
and presence of guanyl nucleotides with their functional properties in
the same membranes (see Tables 1 and
2). In the absence of guanyl nucleotides,
the full and "almost full" agonists (efficacy > 80% of
acetylcholine, see Table 1) recognized 25 to 30% of the binding sites
with a high affinity and had a significantly lower affinity for the
remaining [3H]NMS binding sites (Table 2). The
partial agonist competition curve fitting was not significantly
improved by using a two-sites model: I report a single "corrected
IC50 " value (using a log scale) in Table 1.
These values are identical to the agonists' KD values only if they indeed recognize a
single site. I observed, however, that all agonists had a lower
affinity in the presence of GTPS (
1 µM), GTP, ( 1 µM) or
GDP ( 3 µM) than in their absence (Table 1): it is likely that,
like full agonists, partial agonists discriminated two receptor states
(HR and HRG) in the absence of guanyl nucleotides. It is indeed known
that the existence of two receptor states is very difficult to
demonstrate if the proportion of high- or low-affinity sites or their
affinity ratio is too low (De Lean et al., 1982).
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S binding in the absence and in the
presence of GDP. Their potencies (pEC50 values) and efficacies (Emax, expressed in
percentage of the maximal response to acetylcholine) are summarized in
Table 1. In the absence of unlabeled guanyl nucleotides, the agonist
concentrations necessary for half-maximal
[35S]GTPS binding stimulation
(EC50 values) were lower than the concentrations
necessary for occupancy of half of the receptors (corrected
IC50 values). As shown in the accompanying
article (Waelbroeck, 2001), this was not because of spare receptors:
the high-affinity agonist-receptor complexes were more efficient than the low-affinity complexes for [35S]GTPS
binding stimulation in the absence of competing nucleotides. The
agonists' EC50 and binding
KD values were well correlated in the
presence of 3 µM GDP (Table 1).
Agonists Binding and Functional Properties in Intact Cells.
[3H]NMS recognized a single binding site
population with a KD = 300 pM at 37°C, in
the Dulbecco's minimal essential medium. I did not observe biphasic
agonist competition curves in intact cells: all the agonists studied
recognized a single receptor state (not shown). The agonist
KD values in intact cells at 37°C
(summarized in Table 3) were, as a rule,
similar to the "low-affinity" KD values
observed in membranes at 30°C.
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Effect of the Incubation Conditions on the Agonists'
Efficacies.
The agonists' efficacies in intact cells were poorly
correlated with their efficacies on membranes in the absence of
unlabeled nucleotides (Fig. 2A). I can
provide two explanations for this discrepancy: 1) GTP is present in
very large concentrations in intact cells, but absent from the
[35S]GTPS binding buffer: this affects the
rate-limiting step of the G protein activation cycle [accompanying
article (Waelbroeck, 2001)]. Partial agonists might differ in their
relative abilities to stimulate the ternary complex formation as
opposed to activated G protein release. 2) It has been demonstrated
that muscarinic agonists stimulate [35S]GTPS
binding to Gq/11 as well as to
Gi/o proteins (Lazareno and Birdsall 1993; DeLapp
et al., 1999). Gi/o proteins poorly stimulate the
phospholipase C: should agonists differ in their abilities to stimulate
the different G protein subtypes, those that activate preferentially
Gq/11 proteins must be relatively more efficient
in the inositol phosphate turnover assay. To test these two hypotheses,
I 1) compared [35S]GTPS binding in the
absence and presence of unlabeled nucleotides (Fig. 2B), and 2) studied
the impact of pertussis toxin treatment on
[35S]GTPS binding in the absence and
presence of muscarinic agonists (not shown).
|
S binding after 1 h incubation
in the presence of large GTP (3 µM) concentrations. The results are
summarized in Table 3 and Fig. 2, B and C. The partial agonists'
efficacy profile in the presence of GTP was very similar to their
profile in intact cells (Fig. 2C), not with their profile in the
absence of nucleotides (Fig. 2B). In the presence of GDP (Fig.
3, D-F), an intermediate efficacy profile
was observed. It was reasonably correlated with their efficacies in
intact cells (Fig. 2E) as well as their efficacies in membranes in the
absence (Fig. 2D) or presence (Fig. 2F) of GTP.
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S binding stimulation increased
significantly after pertussis toxin treatment. In addition, all partial
agonists achieved at least 80% of the maximal effect of acetylcholine
in toxin-treated cell membranes. These results suggested that, because
of the very low G protein density available to muscarinic receptors
after pertussis toxin treatment, activation of a few of the muscarinic receptors was sufficient to rapidly saturate the few residual G
proteins by GTPS (i.e., that pertussis-toxin treated membranes possessed spare receptors). I compared acetylcholine dose effect curves
at different receptor concentrations (Table
4) to verify this hypothesis. It was
necessary to inactivate more than 60% of the muscarinic binding sites
by 4-DAMP mustard before decreasing the maximal acetylcholine response
(Table 4). The partial agonists' efficacies were identical in
membranes from control cells and from cells pretreated with both
pertussis toxin and 4-DAMP mustard (not shown). Pertussis toxin
treatment decreased the density of G proteins responding to muscarinic
receptor activation, but did not affect the agonists' efficacy profile
(not shown). Taken together, these results suggested that the
agonists' efficacy profile at M1 muscarinic
receptors depended on the guanyl nucleotides' concentration, but not
on the G protein composition.
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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My goal in this work was to test the hypothesis that muscarinic
M1 receptors exist in only two conformations in
equilibrium (R and R*) and that all agonists stabilize (with different
efficiencies) the one and only "active" receptor conformation, R*.
I assumed in addition (for the sake of simplicity) that once the
receptor has been activated, it completes at least one G protein
activation cycle before deactivating. This model (Appendix) is much
more restrictive than the "allosteric model" analyzed by the groups of Lefkowitz and Kenakin (Samama et al., 1993, and Weiss et al., 1995,
respectively): these authors indeed included a parameter describing the
effect of agonists on G protein recognition by HR*. If the activated
receptor's affinity for the G protein depends on the nature of the
agonist, this implies that different agonists induce different active
receptor conformations.
I analyze in the Appendix A strictly two-states model of G protein
activation. Three parameters were necessary to describe G protein
activation in the absence and presence of agonists. First, the constant
J = [R*] / [R] is related to
the free energy difference (
G°) between the (empty,
uncoupled) resting and activated receptor conformations
(J = e
(
G°/RT)).
Second, the allosteric parameter (defined by J = [HR*] / [HR]) is related to the effect of
agonist recognition on the free energies of the resting and activated
receptor conformations. Agonists are characterized by 
1, neutral
antagonists by = 1, and inverse agonists by 
1. Third,
the variable 
, defined by the equation
![&dgr;J=<FR><NU>[R*]+[R*G<SUB><UP>GDP</UP></SUB>]+[R*G]+[R*G<SUB><UP>GTP</UP></SUB>]</NU><DE>[R]+[RG<SUB><UP>GDP</UP></SUB>]+[RG]+[RG<SUB><UP>GTP</UP></SUB>]</DE></FR>,](/content/vol59/issue4/fulltext/886/img002.gif)
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, is not a
thermodynamic constant: it depends not only on the G protein subtypes
encountered by the receptor but also on the nucleotides (GDP, GTP...
) concentrations. Pertussis and cholera toxins (which inactivate
Gi/o and persistently activate
Gs proteins, respectively), guanyl nucleotides
(which affect the G proteins' conformations), and the tissue or cell
lines' G protein expression pattern may profoundly affect the value of
.
The agonists' efficacy is usually defined as the maximal over-basal
effect (Emax) induced by the ligand of
interest, relative to the maximal over-basal effect of the ligand that
induced the largest Emax. I had no
information about the extent of receptor activation in the absence of
agonist (constitutive receptor activity, J), or on the ability of
acetylcholine to switch the receptors in the active state (). I
therefore used an unusual scale to describe the agonist effects in
Figs. 3 and 4: "0" represented the G
protein activation rate by resting receptors (R), and "1" represented G protein activation by fully activated receptors, R*.
|
The basal rate of G protein activation in the absence of agonist is
proportional to e0 = J
/(I + J), and the G protein activation rate observed in the presence of a saturating ligand concentration is
proportional to eligand = J
/(1 + J). As shown in Fig. 3 (dotted
and dashed lines), if the proportion of empty but nevertheless activated receptors is very low (J 
0), the
constitutive activity of the receptors will be negligible
(e0 J) and the efficacy of most agonists, proportional to their ability to stabilize the active
receptor conformation (eligand J). Very efficient agonists (1) are necessary to
fully activate the receptors, and inverse agonists cannot significantly
decrease the (already negligible) receptor activity. Should G proteins
interact significantly and preferentially with activate receptors (Fig.
3, full line), J will become large. The rate of
receptor-induced G protein activation in the absence of agonists
(e0) will then become significant, and the
effect of inverse agonists will become readily detectable. Because of
the cooperation of G proteins in maintaining the receptors in the
active state, agonists that have a rather small effect on the active
receptor conformation's stability ( near 1) may nevertheless fully
activate the receptors (Fig. 3, full line).
The agonists' potencies (EC50 value) do not
merely reflect their affinities for either uncoupled (R) or precoupled
receptors (RG), but also depends on the G protein's ability to
"preactivate" the receptors, . Let us assume that there are no
spare receptors in the membranes and that the
G
Nucleotides may affect the potency and efficacy of GPCR-G protein
coupling (Meller et al., 1992; Spengler et al., 1993; Robb et al.,
1994; Berg et al., 1998; Bonhaus et al., 1998; Breivogel et al., 1998;
Seifert et al., 1999). GDP, like GTP, markedly inhibited the binding of
muscarinic agonists (Tables 1 and 2): they prevented G proteins from
stabilizing agonist-bound activated receptors and decreased
J. I therefore expected guanyl nucleotides to
decrease the agonists' efficacies (eago)
and potencies (Kact) (Fig. 4). If full and
partial agonists differ only in their ability to stabilize the same
(one and only) activated receptor conformation, their rank order of
efficacies in the absence and presence of nucleotides should remain
(nonlinearly) correlated (Fig. 5).
|
The agonists' efficacies for [35S]GTPS
binding stimulation in the absence of unlabeled nucleotides were "on
average" equivalent to their efficacies in intact cells or in the
presence of GTP (Fig. 2, A and B). The correlation, however, was poor
(Fig. 2A): there was a 6.5% chance of randomly observing such a
substantial incubation condition effect in an experiment of this size
(two-way ANOVA, n = 4 experiments). The agonists'
efficacies for [35S]GTPS binding stimulation
in the presence of GTP were well correlated with their effect on
inositol phosphates' synthesis activation (Fig. 2C), and pertussis
toxin pretreatment did not affect the agonists' efficacy profile in
the absence of spare receptors (not shown). These two results suggested
that the guanyl nucleotide (but not G protein) composition of the
incubation system was an important determinant of the agonists'
efficacy profile.
According to the "Cassel and Selinger", G protein activation cycle
[accompanying article (Waelbroeck, 2001)], the rate-limiting step for
G protein activation depends on the nucleotide concentration. M1 muscarinic agonists accelerated G protein
activation in the presence of high GTP concentrations, by facilitating
the GDP release, and facilitated binding of
[35S]GTPS (at very low concentrations) to
empty G proteins by accelerating the
GS binding in the
presence of GDP: this probably explains why the agonists' effect in
the presence of GDP was reasonably correlated with their effect in both
other incubation conditions (Fig. 2, D-F).
Taken together, my results did not support the "two conformations"
model of G protein activation. There are two possible interpretations for the discrepancies:
To simplify the equations, I assumed in the "two-states"
model (Appendix of this and the accompanying article) that the
activated receptor does not deactivate before completing at least one G protein activation cycle. If some agonists are released faster than the
activated G protein by the HRG HR*G H + R*G H + RG H + RG + GTP HR*G + GTP,
etc.).
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| If different agonists stabilize different receptor conformations,
the variable might depend not only on the G protein and guanyl
nucleotide composition of the incubation medium but also on the agonist itself.
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In conclusion: my results did not support the two-states model of G protein activation.
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Acknowledgments |
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I thank P. Poloczeck for his outstanding technical help.
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Footnotes |
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Received August 8, 2000; Accepted January 9, 2001
1 Because of the length of the Appendix, it is not printed herein. It can be found in its entirety in the online version of this article.
Supported by Grant 3.4504.99 from the Fonds de la Recherche Scientifique Médicale, by an "Action de Recherche Concertée" from the "Communauté Française de Belgique" and by a "Interuniversity Poles of Attraction Program - Belgian State, Prime Minister's Office - Federal Office for Scientific, Technical and Cultural Affairs".
Send reprint requests to: Dr. Waelbroeck, Laboratoire de Chimie Biologique et de la Nutrition, Faculté de Médecine de l'Université Libre de Bruxelles, Bât. G/E, CP 611, 808 Route de Lennik, B-1070 Brussels, Belgium. E-mail: mawaelbr{at}ulb.ac.be
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Abbreviations |
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GPCR, G protein-coupled receptors;
GTPS, guanosine 5'-thio-triphosphate;
4-DAMP mustard, [4-diphenylacetoxy-1-(2-chloroethyl) piperidine];
[3H]NMS, L-[N-methyl-3H]scopolamine
methyl chloride;
CHO, Chinese hamster ovary cells;
IP, inositol
phosphate;
DMEM, Dulbecco's minimum essential medium;
GS-bound G proteins.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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3401-3407This article has been cited by other articles:
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M. Waelbroeck Activation of Guanosine 5'-[{gamma}-35S]thio-triphosphate Binding through M1 Muscarinic Receptors in Transfected Chinese Hamster Ovary Cell Membranes: 1. Mathematical Analysis of Catalytic G Protein Activation Mol. Pharmacol., April 1, 2001; 59(4): 875 - 885. [Abstract] [Full Text]
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) and
pilocarpine (
) dose-effect curves were obtained in control cells and
in cells pretreated for 1 h with 30 nM 4-DAMP mustard before
extensive washing. The carbamylcholine and pilocarpine EC50
values decreased in this experiment from 6.16 ± 0.07 and
4.80 ± 0.06 to 5.02 ± 0.09 and 3.75 ± 0.08, respectively. These data are representative of three experiments in
duplicate.
