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(Received for publication, September 15, 1995; and in revised form, December 7, 1995) From the
DnaA protein is the initiator of chromosomal DNA replication in Escherichia coli. We examined the influence of artificial
mixed membrane composed of synthetic acidic (phosphate) lipid and basic
(ammonium) lipid on the affinity of DnaA protein for ATP. Two sets of
acidic and basic lipids with distinguishable numbers of hydrophobic
alkyl chains were devised. Synthetic membranes made of the sole acidic
lipid but not the basic bilayers inhibited the ATP binding to DnaA
protein and stimulated the release of ATP from the ATP-DnaA complex.
The basic bilayer-forming compounds served as the matrix for the guest
acidic lipids. Acidic lipids dispersed in the basic matrix membrane had
little effect on ATP binding and on ATP release. Conversely, acidic
lipids forming cluster structures in the mixed artificial membranes
inhibited the ATP binding and stimulated the release of ATP. These
observations suggest that in mixed lipid bilayers, a cluster structure
of acidic lipids seems to be an important parameter to decrease the
affinity of DnaA protein for ATP. Chromosomal DNA replication in Escherichia coli initiates at a unique site, oriC, the origin of
chromosomal DNA replication, depending on function of the initiator
protein, DnaA protein(1, 2, 3) . DnaA protein
seems to be a key factor in mechanisms controlling DNA replication. The
replication activity of DnaA protein is likely to be controlled by a
number of regulatory factors functioning in concert. A protein factor
that negatively regulates the activity of DnaA protein has recently
been reported(4, 5) . There is a line of evidence that
suggests that transcription by RNA polymerase facilitates the function
of DnaA protein to open up the oriC region for entrance of
other replication proteins(6, 7, 8) . The
contribution of DNA supercoiling to control the capacity for initiation
of DnaA protein has also been suggested(9, 10) . DnaA protein has a high affinity for ATP (K Lipid interaction of DnaA protein
accompanied by a decrease in the affinity for ATP is an important clue
for investigators examining the initiation of DNA
replication(12, 13, 14) . The initiation of
chromosomal DNA replication in bacterial cells seems to occur on
membranes (15, 16, 17, 18, 19) . To
assume that the activity of DnaA protein is regulated by phospholipids
in biological membrane seems reasonable, and this notion is supported
by the genetic studies of Xia and Dowhan(20) . They showed that
a lethal phenotype of a mutation in the pgsA gene, which is
responsible for the synthesis of phosphatidylglycerol, is suppressed by
a mutation in the rnhA gene. Because the mutation of this gene
induces stable DNA replication, which is independent on DnaA protein
and oriC DNA, they concluded that phosphatidylglycerol is
necessary for the initiation of oriC DNA replication. The
bilayer membrane of E. coli is comprised of various
phospholipids, such as four-chained cardiolipin, double-chained
phosphatidylglycerol, and double-chained phosphatidylethanolamine.
These natural lipids are differentiated by the number of alkyl chains,
not only the charge of the hydrophilic group. Most studies concerning
the interaction between lipids and DnaA protein have focused less on
the unique property of mixed membrane and more on single component
amphiphilic assemblage. When the numbers of hydrophobic chains are
different among the bilayer-forming lipids, there is usually a tendency
to take place a phase separation (cluster formation) in mixed membranes (21, 22, 23, 24) . This phase
separation phenomena is a fundamental and unique property of mixed
membranes. Our objective in this study was to examine the importance
of the phase separation in mixed membrane for a decrease in the
affinity of DnaA protein for ATP. A precise system of regulation for
phase separation in mixed membrane with synthetic lipids is a sine
qua non for such studies. Through synthesis and characterization
of hundreds of non-natural bilayer-forming lipids, we prepared a
molecular design for bilayer assemblage, including the regulation of
phase separation (25, 26, 27, 28, 29, 30) .
A combination of a single-chained lipid containing azobenzene and
glutamate-based double-chained amphiphile makes available a systematic
mixed membrane system in which the cluster formation is
controlled(21, 22, 23, 24) .
Furthermore, the glutamate dialkyl lipids have actually been used for
modifying the nature of aqueous proteins such as myoglobin and
cytochrome c(31, 32, 33) . We
describe here how the phase separation phenomena of mixed bilayer
membrane affects the affinity between DnaA protein and ATP.
EXPERIMENTAL PROCEDURES (
The four non-natural
lipids shown in Fig. 1were synthesized
(1N(
Figure 1:
Molecular
structure of non-natural lipids used in this experiment. Single-chained
alanate and double-chained glutamate compounds are abbreviated as 1 and
2, respectively. Basic and acidic hydrophilic groups are expressed as
capital N and P, respectively.
Previous studies revealed
that anionic residues in lipids are indispensable for a decrease in the
affinity of DnaA protein for ATP, and cationic and zwitter ionic
(neutral) lipids do not influence the
affinity(12, 13, 14) . Therefore, selecting
negatively and positively charged amphiphiles as guest and matrix
membranes, respectively, is a prerequisite for the basic demands (a) and (b). Moreover, opposite charge mixing makes
guest and matrix membranes miscible (requirement (c)). The
requirements (d), (e), and (f) are discussed
in the following sections. On the basis of a heterogeneous
combination, in terms of the number of hydrophobic alkyl chains, we
devised two pairs of amphiphiles; one is single-chained (1N and 1P),
and the other is a double-chained amphiphile (2N and 2P), as shown in Fig. 1. Double-chained 2N and 2P and single-chained 1N and 1P
provide typical non-natural bilayer membranes, both possessing sharp
gel to liquid crystal phase transitions when the hydrophobic force due
to the alkyl chain is strengthened with aromatic stacking
force(37, 38, 39, 40) . The acidic
compounds, 1P and 2P, are comprised of hydrophobic structures similar
to the basic correspondents 1N and 2N, respectively. We previously
reported that phase separation in a mixed membrane is caused by the
heterogeneous combination of hydrophobic alkyl
chains(21, 22, 23, 24) . Hence, when
the acidic lipid is diluted with the basic matrix consisting of a
similar hydrophobic moiety, namely 1P/1N and 2P/2N, the acidic lipid is
likely to disperse monomerically into the basic matrix membrane due to
electrostatic force. On the other hand, in the cross-bred combinations,
1P/2N and 2P/1N, the acidic amphiphile is segregated by the basic
matrix to show phase separation, if the clustering force arising from
the difference of alkyl chains exceeds the electrostatic force. These
postulations are examined in the following sections.
Table 1summarizes phase transition and membrane fluidity for
aqueous dispersions of the four amphiphiles. Even in the presence of
17% glycerol, the dispersions of 1N, 2P, and 2N possessed symmetric and
single endothermic peaks on heating at 80, 61, and 49 °C,
respectively. For the dispersion of 1P, doublet endothermic peaks were
observed around 80 °C, with and without glycerol. In addition to
the peak shape, the presence and the absence of 17% glycerol made
little difference on values for phase transition temperature (T
Fig. 2A shows
DSC thermograms of the double-chained basic membrane, 2N (matrix
bilayer 50 mM) mixed with the acidic membrane, 1P or 2P (guest
bilayer 5 mM). The mixed membrane 2P/2N has a single and broad
endothermic peak at the same temperature as the T
Figure 2:
DSC thermograms of aqueous dispersions of
mixed membranes made of the single-chained basic matrix 1N (A)
and 2N (B). The conditions were: [acidic lipid]
= 5 mM, [basic lipid] = 50
mM, [tris(hydroxymethyl)aminomethane] = 5
mM, aqueous medium containing 17% (v/v) glycerol, and 0.005%
(w/v) Triton X-100.
The guest amphiphile
1P containing the azobenzene chromophore allows one to monitor
aggregation properties through UV-visible spectroscopy (21, 22, 23, 24, 37, 46, 47) .
The azobenzene amphiphile 1P in basic matrix 2N represented its
The same
situation of cluster structure formation arises for another matrix
membrane. Fig. 2B shows DSC thermograms for the
single-chained basic membrane 1N (50 mM) (matrix bilayer)
mixed with the acidic membrane, 1P or 2P (5 mM) (guest
bilayer). Like the double-chained matrix membrane, 2N, a system of
different hydrophobic chains represents a set of broad peaks at 74 and
81 °C, whereas another combination for which similar hydrophobic
modules are used led to a single peak at 84 °C (36 kJ/mol). Mixing
the acidic amphiphile possessing a hydrophobic chain similar to that of
the matrix compound with matrix lipids had little effect on the peak
width (4-5 °C), the enthalpic change (31-36 kJ/mol),
and the temperature range (around 80 °C) of the host membrane 1N.
The acidic amphiphile containing azobenzene is miscible with the
chromophoric matrix membrane. As opposed to the 1P, miscibility of the
double-chained 2P is low enough to yield an additional peak below the
main transition. This provides evidence for the presence of cluster 2P
in the single-chained basic matrix membrane.
Figure 3:
Influence of synthetic lipids on the ATP
binding reaction to DnaA protein. Assay of ATP binding to DnaA protein
(2 pmol) was done as described under ``Experimental
Procedures.''
Figure 4:
Kinetic
traces of the ATP release from DnaA-ATP protein representing effects of
the charge in lipids. Dissociation of the DnaA-ATP complex (1.1 pmol)
in the presence of lipids (1P and 2P, 300 µM; 1N and 2N,
30 µM) at 37 °C was examined, as described under
``Experimental Procedures.'' Ct and Co stand for concentrations of ATP-DnaA retained and of initial
ATP-DnaA, respectively.
The
interaction between DnaA protein and lipid was also evaluated through
the ATP release from DnaA protein-ATP complex (Fig. 4). The k
Figure 5:
Influence of mixed bilayer membranes on
ATP binding reaction to DnaA protein. Mixed bilayer membranes of 1P and
2N or 1P and 2N were constructed by sonication, with the proportion of
acidic and basic lipids being 1:10. The amount of ATP bound to DnaA
protein (2 pmol) was determined as described in the legend of Fig. 3. A,
The same
situation as the 1P system holds in case of the double-chained
phospholipid 2P. Mixing with 2N weakened the inhibitory effect of 2P on
the ATP binding to DnaA protein. On the contrary, the mixed membrane of
the heteroalkyl combination (2P/1N) was more inhibitory than that of
the homoalkyl combination (2P/2N). Because the two basic lipids had
little effect on ATP incorporation over the concentration range of
1-100 µM (Fig. 3), the disparity in the
inhibitory behavior between the mixed membranes of the heteroalkyl
combination and of the homoalkyl combination should be considered from
the viewpoint of acidic lipids. Subsequently, ATP dissociation from
the ATP-DnaA complex in the presence of added mixed membrane was
monitored. Fig. 6shows linear relations between time and
logarithms for the amount of the dissociated ATP. k
Figure 6:
A
family of kinetic traces for the ATP release from DnaA-ATP protein
depending on the mixed bilayer membrane. Dissociation of the DnaA-ATP
complex in the presence of lipids was examined as described in the
legend of Fig. 4. The conditions were: [initial
DnaA-ATP] = 1.1 pmol/40 µl, [acidic lipid]
= 30 µM, [basic lipid] = 300
µM, 37 °C. A,
As mentioned in the previous section, mixed membranes
of the heterocombination (1P/2N and 2P/1N) give rise to the phase
separation (cluster formation) of acidic lipids. It seems apparent that
a mixed membrane decreases the affinity of DnaA protein for ATP under
conditions where acidic components form cluster structures. This
study focused on phase separation (cluster formation) in mixed
non-natural membranes and the impact on the interaction between lipid
membrane and DnaA protein was apparent. The heterogeneous combination,
in terms of the number of alkyl chains, for the mixed membrane (1P/2N
and 2P/1N) gave rise to clusters abundant in acidic lipids, whereas
similarity in the hydrophobic structure (1P/1N and 2P/2N) and the
electrostatic interaction among the hydrophilic groups left the acidic
lipids as a monomeric species in the matrix membrane. With this
information one can examine the influence of the acidic cluster in the
mixed membrane on the affinity of DnaA protein for ATP. The affinity
change caused by the acidic lipids did lessen by diluting with matrix
membranes; however, acidic lipids in a cluster state are as effective
as those in the single component membrane in reducing the affinity.
Diversity in size of the cluster and its purity might provoke a
difference in the affinity change. Therefore, cluster formation of
phospholipids in a mixed membrane may be one factor regulating the
interaction between DnaA protein and lipids. The heterogeneity in
hydrophobic moiety between the component lipids is a key factor for
formation of cluster domains in mixed artificial
membranes(21, 22, 23, 24) . Partial
hydrolysis and/or ester exchange for acyl chain of phospholipid by
phospholipases could provide biological lipid membranes with
heterogeneity in hydrophobic domains and might be a candidate offering
a membrane signal to the DnaA protein. In fact, the rapid turnover of
acidic phospholipids in E. coli has been
reported(48, 49) . Moreover, Ohki reported that
breakdown of acidic phospholipids in E. coli is apparently
coupled with cycles of cell proliferation(50) . All these
results taken together lead to the thesis that the cluster structure of
acidic phospholipids is controlled in concert with the cell cycle and
that such a process may regulate DnaA protein activity.
Volume 271,
Number 7,
Issue of February 16, 1996 pp. 3633-3638
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES (**)
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
= 0.03 µM) and ADP (K
= 0.1 µM)(11) . ATP bound to
DnaA protein hydrolyzes slowly to ADP in the presence of
DNA(4) . ADP also tightly binds to DnaA protein and is slowly
replaced by ATP, and the ATP-bound form is active in an oriC replication system reconstituted with purified proteins, whereas
the ADP-bound form is inactive(11) . Thus, adenine nucleotide
binding is likely to be a primary process involved in the regulation of
the activity of DnaA protein.
)
Materials
DnaA protein was purified by the
method described previously(34) , except that a newly
constructed overproducer was used. (
)Specific activity of
the protein was 0.7 
10
unit/mg. Purity of the
fraction used exceeded 90%, as determined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis.
)(24, 35) , 2N(36) ,
1P(37) , and 2P (38, 39, 40) ).
Powdery lipid was sonicated in a Branson Sonifier 185 (sonic power 30,
2 min, 1 ml) in distilled water to yield a translucent dispersion
(10-20 mM). When acidic (
)lipids were used,
equimolar tris(hydroxymethyl)aminomethane was added for purposes of
neutralization. Sonicating the powdery basic and acidic lipids (sonic
power 40, for 3 min, 1 ml) in water led to mixed membrane dispersions
(10-20 mM). This co-sonication method was used to avoid
a nonfusion state where an aggregate of the acidic lipid and of the
basic compound independently co-exist. These aqueous dispersions were
used as stock solutions for the following assays.
[
-P]ATP (5 mCi/mmol) was obtained from
Amersham Corp.
Differential Scanning Calorimetry
Differential
scanning calorimetry (DSC) was conducted on a Seiko SSC-5200 instrument
equipped with the DSC-120 thermal balance. The lipid dispersions
(10-55 mM, 52 µl in buffer G (50 mM HEPES-KOH (pH 8.0 at 1 M), 0.5 mM magnesium
acetate, 0.3 mM EDTA, 5 mM dithiothreitol, 10 mM ammonium sulfate, 17% (v/v) glycerol, and 0.005% (v/v) Triton
X-100)), which were prepared by the co-sonication method, were sealed
in silver sample pans. The buffer mentioned below was mounted on the
thermal balance as a control sample. All DSC thermograms were scanned
at a rate of +1 °C/min from 0 to 100 °C. The thermograms
were reproducible for the cooled samples.Fluorescence Polarization (P value)
A stock
solution of 1,6-diphenyl-1,3,5-hexatriene (1 mM) was prepared
in tetrahydrofurane. An aliquot of the tetrahydrofurane solution (10
µl) was, after solvent removal, mixed with aqueous glycerol
dispersions of lipids (1 mM, 1 ml), and the mixture was
lightly sonicated. The fluorescence intensity was monitored using a
Hitachi F-4500 under conditions of excitation at 363 nm and emission at
434 nm. The P value was calculated with the common
equation(21) .Assay for Influence of Lipids on ATP Binding to DnaA
Protein
DnaA protein (2 pmol) and lipids were mixed at 0 °C
for 15 min in 40 µl of buffer G.
[-P]ATP (5 mCi/mmol) was then added to give
the final concentration of 2 µM and incubated at 0 °C
for 15 min. The binding of ATP to DnaA protein reached equilibrium
within 5 min (data not shown). The solution was passed through a
membrane filter (Millipore HA 0.45 µm pore) presoaked in buffer G.
The filter was washed with 6 ml of ice-cold buffer G and dried under an
infrared lamp. The retained radioactivity was measured in a liquid
scintillation counter.
Assay for Influence of Lipids on Release of ATP from DnaA
Protein
DnaA protein (2 pmol) was preincubated with 2 µM [-P]ATP (5 mCi/mmol) in 40 µl of
buffer G at 4 °C for 15 min. Lipids were added, and the mixture was
incubated at 37 °C, followed by passage through a nitrocellulose
membrane filter. Radioactivity on the filter was then measured.
Design of Mixed Membranes to Control Phase
Separation
To examine the importance of a cluster of acidic
lipids in mixed membrane on the affinity of DnaA protein for ATP, we
first selected appropriate synthetic lipids that satisfied the
following criteria: (a) a guest amphiphile should be capable
of interacting with DnaA protein and reducing the affinity of DnaA
protein for ATP, (b) a matrix membrane should not affect the
affinity, (c) both the guest and matrix membranes should be
miscible, (d) a system by which both states, phase separation
and monomeric dispersion, should be controllable, (e)
amphiphiles that met these criteria should make available a bilayer
membrane in aqueous glycerol, which is essential for maintenance of
ATP-binding activity of DnaA protein, and (f) the membrane
should represent a clear gel to liquid crystal phase transition so as
to be detectable with thermal analysis.Aggregation Properties of Non-natural Amphiphiles in
Aqueous Glycerol
In the following experiments, we usually added
17% (v/v) glycerol to the buffer to protect DnaA protein from
denaturation. Mixing distilled water with protic organic solvents,
however, generally results in destabilization of
membrane(41, 42, 43, 44, 45) .
Thus, we first determined the stability of the bilayer membrane in the
buffer containing glycerol. Aqueous bilayers can undergo the gel to
liquid crystal phase transition. This macroscopic phenomenon is one of
the most fundamental membrane properties and causes microscopic
physicochemical changes in membrane fluidity(24, 25) .
To estimate damage of bilayer structures by the addition of glycerol,
DSC and fluorescence polarization (P value) of
1,6-diphenyl-1,3,5-hexatriene were used in characterizing phase
transition and membrane fluidity, respectively. 
), enthalpic change associated with the
transition (
H), and peak width, without regard for
amphiphile structures, except for peak width of 2N. P values for the
dispersions of 2N and 2P lessened by 0.06-0.08 with 17% glycerol.
The microscopic environment of the double-chained membrane interior is
affected by glycerol, but this change is not so extensive as to
conclude the amphiphiles exist in nonbilayer state such as micellar or
monomeric dispersion. We actually confirmed the presence of vesicular
(2N, diameter 40-400 nm) and tube-shaped (1N, length 500-3000 nm,
width 150 nm) membranes even in the presence of 17% glycerol by
transmittance electron microscopy for the dispersions stained with
uranyl acetate (data not shown).
Distribution of Acidic Amphiphiles in Basic Matrix
Membrane
Phase separation in the mixed membranes was examined
using DSC and UV-visible absorption spectroscopy. It is important for
the DSC study that phase transition parameters of a cluster domain
differ from those of a matrix membrane. As shown in Table 1, the
single-chained bilayers represented a phase transition temperature
higher by 20-40 °C over the double-chained bilayers. Also
phase transition temperatures of the acidic bilayers being at pH 8.0
are 5-10 °C higher than those of the corresponding basic
membranes. The four assemblies can be readily distinguished as
different peaks on DSC thermograms. (49 °C) of the matrix bilayer 2N. The width of the peak of
the dispersion 2P/2N is 5-6 times larger than that of the single
component bilayer of 2N (5 °C, Table 1). On the other
hand, the 1P/2N thermogram has two additional peaks below (
35
°C, 15% [ratio of area]) and above (62 °C, 5%) the
major transition (51 °C, 80%). The width of the major peak
(
2.2 °C) is nearly twice that of the bilayer 2N but about half
as large as that of the mixed membrane 2P/2N.
at 336 nm at 37 °C, thereby indicating the
presence of the face to face stacking (H-aggregate) (47) . When
raising the temperature to 70 °C, the shifted
to 360 nm and reverted to the original wavelength by cooling to room
temperature. This reversible spectral change is a typical instance of
aggregation and disintegration of azobenzene amphiphiles associated
with fluidity change of the matrix
membrane(21, 22, 23) . Despite the presence
of electrostatic force, the single-chained anionic amphiphile forms a
cluster in the double-chained cationic matrix bilayer, as represented
by small additional peaks. The spectral shift seen with cluster
formation would be relevant to the multiendothermic peaks.Cluster Size
The presence of multiple endothermic
peaks does not necessarily mean that only the guest lipids converge on
the acidic domains. This situation can be readily confirmed in the
2P/1N mixed membrane. Assuming that a phase-separated cluster is made
up of only the 2P molecules, H values for the additional
peak (73 °C) and the main transition (81 °C) can be calculated
to be 36 and 26 kJ/mol, respectively. The former value is nearly 30%
more and the latter is almost 20% less than the H values
for 2P and 1N single-component bilayers, respectively (Table 1).
The excess of the additional peak over the hypothetical value can be
offset by a decrease in the main transition. In other words, the acidic
cluster domain of 2P contains matrix component 1N as minor ingredient.
This situation can be confirmed also from the peak position in the DSC
thermogram. In the case of the phase-separated cluster exists as the
same assembly as the single-component 2P bilayer, the additional peak
should appear close at the T of the 2P bilayer, 61
°C. The fact of showing the peak (73 °C) between the T values for the 2P (61 °C) and for 1N (80
°C) bilayers indicates that the phase transition property of the
acidic cluster is affected by the mixing with matrix lipids.Effect of Single Component Membrane on the Affinity of
DnaA Protein for ATP
We explored the single component membrane
from the viewpoint of requirements (a) and (b) in the
initial section. We used filter binding assay for examination of ATP
binding to DnaA protein(11) . Fig. 3shows the influence
of the bilayer-forming lipid on ATP binding to DnaA protein. The two
basic lipids, 1N and 2N, had little effect on the ATP binding to DnaA
protein over the range of the lipid concentration from 1 to 1000
µM. On the contrary, in case of acidic lipids, 1P and 2P,
the amount of bound ATP decreased with increase in the lipid
concentration. The curve shifted toward the direction of low
concentration of lipid when 2P replaced 1P, thereby suggesting the
superior aggregation stability of the double-chained lipid, as compared
with the single-chained compound. Because the addition of acidic lipids
to the DnaA-ATP complex without incubation at 37 °C did not affect
the amount of ATP on the filter (Fig. 4), the inhibitory effect
of acidic lipids on ATP binding to DnaA protein cannot be explained by
the release of DnaA protein from the filter by acidic lipids.
, 1P; 
, 1N; 
, 2P; ,
2N.
, 1P; , 1N; , 2P; ,
2N.
(apparent rate constants) values for 2P, 1P,
2N, and 1N were calculated to be 1.1
10, 8.5
10, 9.6
10 (s
), and 9.6
10 (s
), respectively. The two acidic lipids (30
µM) accelerate the release of ATP 1 order of magnitude
faster than does the basic amphiphile (300 µM). Moreover,
the finding that the k
value of 2P is slightly
larger than that of 1P is consistent with the observation of ATP
binding behavior. ATP release was nonexistent in the absence of lipid.
These results indicate that the acidic lipid is much superior to the
basic compound in stimulating ATP release from the DnaA-ATP complex and
in inhibiting ATP binding to DnaA protein. These results are consistent
with previous studies showing the importance of acidic residues in
lipids for interactions with DnaA
protein(12, 13, 14) .
Decrease in Affinity of DnaA Protein for ATP by Acidic
Lipids Forming Cluster Structures in Mixed Bilayer
Membranes
From the viewpoint of phase separation, we examined
the interaction between mixed membrane and DnaA protein through the
release and incorporation of ATP. Fig. 5shows the inhibition
for ATP incorporation by various mixed membranes. In the case of the
single-chained phospholipid 1P, mixed with 1N possessing the same
hydrophobic moiety, the inhibition curve shifted from that of sole 1P
by 1 order of magnitude toward the direction of higher concentrations.
The curve for the mixed membrane of the heteroalkyl combination
(1P/2N), however, is coincident with that for the single acidic
component membrane of 1P, within experimental error.
, 1P; , 1P/2N; , 1P/1N. B, , 2P; , 2P/1N; ,
2P/2N.
can be calculated from the slope. The k
values for membranes 1P, 1P/1N, 1P/2N, 2P, 2P/2N, 2P/1N were
determined to be 8.5
10, 5.7
10, 7.6
10, 1.1
10, 6.6
10, and 9.2
10(s
), respectively. The
ATP release varies with the combination of the number of alkyl chains.
The mixed membranes of heteroalkyl combination (1P/2N and 2P/1N)
facilitate ATP release more rapidly than do those of the homoalkyl
combination (1P/1N and 2P/2N). These observations on ATP release are
consistent with results of its inhibitory effects on ATP binding to
DnaA protein.
, 1P; , 1P/2N;
, 1P/1N. B, , 2P; , 2P/1N; ,
2P/2N.
)))
We thank M. Ohara for comments on the manuscript.
Hideaki Horiuchi (Kyushu University) kindly developed home-designed
glass equipment for synthesis of the non-natural lipids.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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T. Mizushima, T. Takaki, T. Kubota, T. Tsuchiya, T. Miki, T. Katayama, and K. Sekimizu Site-directed Mutational Analysis for the ATP Binding of DnaA Protein. FUNCTIONS OF TWO CONSERVED AMINO ACIDS (LYS-178 AND ASP-235) LOCATED IN THE ATP-BINDING DOMAIN OF DnaA PROTEIN IN VITRO AND IN VIVO J. Biol. Chem., August 14, 1998; 273(33): 20847 - 20851. [Abstract] [Full Text] [PDF]
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J. Garner, P. Durrer, J. Kitchen, J. Brunner, and E. Crooke Membrane-mediated Release of Nucleotide from an Initiator of Chromosomal Replication, Escherichia coli DnaA, Occurs with Insertion of a Distinct Region of the Protein into the Lipid Bilayer J. Biol. Chem., February 27, 1998; 273(9): 5167 - 5173. [Abstract] [Full Text] [PDF]
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T. Mizushima, K. Yokoyama, S. Mima, T. Tsuchiya, and K. Sekimizu Inhibition of Thymidine Transport in dnaA Mutants of Escherichia coli J. Biol. Chem., August 22, 1997; 272(34): 21195 - 21200. [Abstract] [Full Text] [PDF]
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T. Mizushima, S. Sasaki, H. Ohishi, M. Kobayashi, T. Katayama, T. Miki, M. Maeda, and K. Sekimizu Molecular Design of Inhibitors of in Vitro oriC DNA Replication Based on the Potential to Block the ATP Binding of DnaA Protein J. Biol. Chem., October 11, 1996; 271(41): 25178 - 25183. [Abstract] [Full Text] [PDF]
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M. Makise, S. Mima, T. Tsuchiya, and T. Mizushima Molecular Mechanism for Functional Interaction between DnaA Protein and Acidic Phospholipids. IDENTIFICATION OF IMPORTANT AMINO ACIDS J. Biol. Chem., March 2, 2001; 276(10): 7450 - 7456. [Abstract] [Full Text] [PDF]
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