From the
Phosphatidylinositol 4,5-bisphosphate (PtdIns
(4,5)P
Phospholipases form a large family of lipid-metabolizing enzymes
involved in regulated and unregulated phospholipid hydrolysis (Dennis,
1983). A family of phospholipases C (PLC)
PLCs, like all
lipid-metabolizing enzymes, act on substrates which form a lipid-water
interface, an arrangement which complicates kinetic analyses of enzyme
catalytic activity. However, a detailed kinetic model of the activity
of small molecular weight PLA
On-line formulae not verified for accuracy SCHEME 1
In this model, enzyme E is proposed to bind
non-catalytically to a phospholipid molecule (S), which forms
a stable point of anchorage of the enzyme to the lipid interface.
Subsequent binding of a second phospholipid molecule at the active site
of the enzyme results in the hydrolysis of substrate to product. Thus,
two phospholipid binding events are required for enzyme activity.
Stable attachment of an enzyme at the interface is an idea which has
been extended further in kinetic analyses of the scooting behavior of
phospholipases (Berg et al., 1991; Jain and Gelb, 1991), where
multiple catalytic cycles can be performed by the enzyme before it
detaches from the interface and redissolves in the aqueous environment.
More recent studies have suggested that the dual substrate/scooting
models may be applicable to a wide range of lipid-metabolizing enzymes
including secretory PLA
Using the
Binding was performed in buffer C in 100 µl
volumes, with 50 ng of TEPLC/tube. Calcium was omitted to eliminate
PLC-catalyzed PtdIns (4,5)P
Data from all three
cases were fitted to the Hill equation (Equation 1) using the curve
fitting programme Ultrafit:
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
Data from case III were sigmoidal and were fitted to a logarithmic
form of the Hill equation:
On-line formulae not verified for accuracy
An equilibrium association constant (K
On-line formulae not verified for accuracy
Enzyme activity was
measured using PtdIns (4,5)P
Several other
non-ionic detergents were tested as diluents in PtdIns (4,5)P
The equilibrium dissociation constant for interface binding by
PLC
Binding of PtdIns
(4, 5) P
The principle behind
experiments using BIA is to measure intermolecular interactions by
immobilizing one molecule (the ``ligand'') followed by
introduction of the second molecule (the ``analyte'') in the
continuous flow-though over the ligand (Fagerstam et al.,
1992). Specific interactions are detected optically due to changes in
the resonance of the plasmon electrons located at the site of
immobilisation of the ligand. Thus, phospholipase C (the ligand) was
immobilized to the sensorchip as described under ``Experimental
Procedures'' and binding of
PtdIns
(4, 5) P
To
confirm that the preincubation of PLC with vesicles had not simply
oligomerized enzyme prior to injection into the biosensor, vesicle-PLC
mixtures were treated with dimethylsuberimidate for 90 min at 25 °C
to cross-link any adjacent protein molecules and separated by
SDS-polyacrylamide gel electrophoresis. A single protein band at 150
kDa was seen in silver-stained gels with no higher molecular mass
forms, confirming that PLC did not self-oligomerize in the presence of
a lipid interface (data not shown). These results were confirmed
several times, including by immobilizing PLC to biosensorchips using
thiol coupling which is likely to reduce the heterogeneity of coupling
at the chip surface due to the low abundance of cysteine resudues in
the enzyme. In light of these two experimental protocols, we suggest
that oligomerization of PLC
The qualitative nature of each of the relationships
established in cases I-III is very similar to that found for secretory
PLA
The Hill equation reduces to the Henri Michaelis-Menten
equation when n = 1, which can describe a unireactant
enzyme or a multireactant one where substrate binding sites behave
independently. These data therefore imply that association of PLC
Three kinetic constants which were
strikingly similar for all three isozymes were derived from this series
of experiments. K
The binding of a PtdIns
(4, 5) P
Complex multireactant lipase kinetics were first shown
for secretory forms of PLA
An effect of PtdSer on lipase activity is well established;
it is thought to be essential for penetration of lipid interfaces by
secretory PLA
The present results, together
with previous studies using monolayer substrates (James et
al., 1994; Boguslavsky et al., 1994; Rebecchi et
al., 1992a) in which PLC activity was inhibited as the surface
pressure increased, suggest that PLCs must penetrate lipid aggregates
in order to bind and hydrolyze substrates. Such a model may seem
unnecessary given that the phosphodiester bond in PtdIns(4,5)P
Our results show that PLC
PLC assays were performed for all three PLC
We are grateful to Ian Batty, Peter Thomason, and
Richard Currie for helpful discussions of the data. We thank Gary Waldo
for the gift of TEPLC, Carol Lyon for scanning electron microscopy, and
Sheila Smith for technical assistance.
) hydrolysis by three different
-isoforms of
phospholipase C (PLC) was examined to investigate the catalytic action
of these extracellular signal-regulated enzymes. Depletion of
phospholipase C from solution by incubation with sucrose-loaded
vesicles of differing compositions followed by ultracentrifugation
demonstrated stable attachment of PLC to the vesicles from which an
equilibrium association constant of PLC with
PtdIns
(4, 5) P
could be determined. A mixed
micellar system was established to assay PLC activity using dodecyl
maltoside, which behaved as an essentially inert diluent of
PtdIns
(4, 5) P
with respect to PLC
activity. Kinetic analyses were performed to test whether PLC
activity was dependent on both bulk PtdIns
(4, 5) P
concentration and surface concentration in the micelles as has
been shown for other lipid metabolising enzymes. Each of the PLC
isoforms behaved similarly in these analyses, which indicated the
involvement of at least two binding events. Interfacial Michaelis
constants were calculated to be between 0.1-0.2 mol fraction for
all three enzymes, and K
(the equilibrium
dissociation constant of PLC for lipid) ranged between 100-200
µM. The apparent multiple interfacial binding events did
not appear to result from lipid-induced PLC
oligomerization
implying that PLC
monomers possess more than one lipid-binding
site. Surface dilution of PLC-catalyzed
PtdIns
(4, 5) P
hydrolysis was assessed in
the presence of increasing concentrations of various nonsubstrate
phospholipids, which profoundly reduced PLC activity, suggesting that
these lipids may inhibit enzyme action. The data indicate that G
protein-regulated isoforms of PLC operate with separate lipid binding
and catalytic steps and imply that under physiological conditions,
PLC
isoforms operate under first-order conditions. These findings
may have implications for the mechanisms of regulation of PLC
s by
G protein subunits.
(
)
has
been identified which is involved in hormone and growth
factor-stimulated signal transduction in mammalian cells (Rhee et
al., 1989; Rhee and Choi, 1992). This family is subdivided into
three groups of PLC isoenzymes, distinguished by their modes of
receptor-dependent regulation. The
-isoforms are regulated by G
protein
and
subunits (Taylor et al., 1991;
Smrcka et al., 1991; Camps et al., 1992; Blank et
al., 1992; Boyer et al., 1992) and the
-isoforms by
changes in enzyme tyrosine phosphorylation (Margolis et al.,
1989; Wahl et al., 1992). Possible regulatory mechanisms of
PLC
isoforms have not been identified.
s has been developed (Deems
et al., 1975; Eaton and Dennis, 1976; Roberts et al.,
1977; Hendrickson and Dennis, 1984) according to :
(Hendrickson and Dennis, 1984;
cytosolic PLA
(Hanel et al., 1993), secretory PLC
(Volwerk et al., 1994), PtdIns 4-kinase (Buxeda et
al., 1991), PLC
(Cifuentes et al., 1993), and
PLC
(Wahl et al., 1992) but similar experiments using G
protein-regulated PLCs have not been reported.
-isoform of PLC from turkey erythrocyte cytosol assayed with lipid
monolayers, we have established that enzyme activity is influenced by
factors such as monolayer lipid composition and interfacial surface
pressure (James et al., 1994). We now report kinetic analyses
and vesicle-binding studies for turkey erythrocyte PLC (TEPLC) and
recombinant forms of mammalian PLC
1 and PLC
2 in which
separate lipid binding events can be discerned. Analysis of data
according to the Hill equation indicates that PLC
isoforms may
operate with an ordered sequential mechanism in which
PtdIns
(4, 5) P
occupancy of the active site
is secondary to binding at the interface. The experiments reported here
were performed in the absence of the physiological G protein activators
of PLC so that a clear idea of the enzyme-lipid interactions could be
established as a prelude to investigations into the molecular basis of
G protein-mediated activation of the PLC
s.
Materials
Phosphatidylcholine (PtdCho) and phosphatidylethanolamine
(PtdEtn) were from Sigma. Phosphatidylserine (PtdSer) was from
Boehringer Mannheim. Polyphosphoinositides were purified from Folch
fraction type I (Sigma) as described previously (James et al.,
1994). Non-ionic detergents were of the highest grade available and
were from Calbiochem (octyl glucoside), Boehringer Mannheim (Triton
X-100 purified for membrane research), Fluka (dodecylmaltoside),
Bio-Rad (Tween 20), and Merck (Brij-35 as a 30% solution by mass). All
other chemicals were AnalaR grade or better.
[H]PtdIns
(4, 5) P
was
prepared by labeling inositol-depleted 1321N1 astrocytoma cells with 1
mCi of [
H]inositol (Amersham Corp.) for 2 days
and subsequent high performance liquid chromatography purification of
labeled lipids (James et al., 1994). 30 µCi of
[
H]PtdIns (4,5)P
was recovered at a
specific radioactivity of 7-15.5 Ci/mmol. Q-Sepharose FF,
Sephacryl S-300 SF, heparin-Sepharose CL-6B, and the Mono Q HR 5/5 FPLC
column were purchased from Pharmacia LKB Biotechnol. The Bio-Gel HPHT
(100
7.8 mm) column, Bio-Gel HPHT (4
50 mm) precolumn
and Bio-Gel HPT hydroxylapatite media were from Bio-Rad. Biosensor
coupling reagents and CM5 sensorchips were from Pharmacia Biosensor,
Uppsala, Sweden.
Methods
Purification of TEPLC
TEPLC was
purified from the cytosol of turkey erythrocytes by the method detailed
by Waldo et al.(1990). Washed turkey erythrocytes were
disrupted by nitrogen cavitation and the supernatant collected after
centrifugation (36,000
g, 20 min, 4 °C). Ammonium
sulfate was added to a final concentration of 226 g/liter and the
precipitate collected by centrifugation (36,000
g, 10
min, 4 °C). The washed precipitate was resuspended in 20
mM Tris, pH 7.4, 1 mM EDTA, 1 mM
dithiothreitol, 0.1 mM benzamidine, 0.1 mM
phenylmethylsulfonyl fluoride, the solution clarified by centrifugation
(36,000
g, 30 min, 4 °C) and the TEPLC
purified by sequential chromatography of the clarified solution on
Q-Sepharose FF, Bio-Gel HPT (hydroxylapatite), heparin-Sepharose CL-6B,
Sephacryl S-300 SF, and Mono Q ion-exchange.
Purification of rPLC
Both
rPLC1 and rPLC
2
1 and rPLC
2 were purified after expression in Sf9 cells,
as described in more detail elsewhere (Paterson and Harden, 1994). In
brief, monolayers of 3
10
Sf9 cells were infected
with 3
10
plaque-forming units recombinant
baculovirus encoding either rat PLC
1 or human PLC
2 and the
cells incubated at 27 °C in Graces medium supplemented with 3.3
g/liter yeastolate, 3.3 g/liter lactalbumin hydrolysate, 10% (v/v)
fetal bovine serum, 2 mM glutamine, 100 µg/ml kanamycin,
and 50 µg/ml gentamycin. Forty-eight h after infection, the cells
were collected, resuspended in hypotonic lysis buffer, and homogenized
in a loose-fitting Dounce homogenizer. Either recombinant PLC
isoform was then purified from the high speed supernatant prepared by
centrifugation (105,000
g, 65 min, 4 °C) of the
whole cell lysate. rPLC
1 was isolated by sequential chromatography
of the high speed supernatant on Q-Sepharose FF and heparin-Sepharose
CL-6B, while rPLC
2 required chromatography on Q-Sepharose FF,
heparin-Sepharose CL-6B, and Bio-Gel HPHT.
PLC
Assays
[H]PtdIns
(4, 5) P
enzyme assay stock solutions were prepared using unlabeled and
labeled lipid (sufficient for 15,000 disintegrations/min/assay) which
were dried to a film from chloroform stock solutions under a stream of
nitrogen and resuspended in 10 mM HEPES, pH 7.4, by
sonication. Dodecylmaltoside was added from stock solutions to
individual assay tubes at concentrations giving the desired mole
fraction calculated as
(PtdIns
(4, 5) P
)/((PtdIns
(4,5)P
) + (detergent)). Assays were performed in
buffer A (comprising 10 mM HEPES, pH 7.4, 120 mM KCl,
10 mM NaCl, 2 mM EGTA, 1 mM
MgCl
), and sufficient calcium to give a free ionic
concentration of 1 µM (calculated using a
K
of EGTA for calcium of 5.17
10
M
). Assays (100 µl
volume) were started by addition of enzyme (10-25 ng) to tubes
containing all other constituents prewarmed to 37 °C. Assays
proceeded for up to 15 min (under which conditions <20%
PtdIns
(4, 5) P
was utilized and assays were
linear) and were stopped by addition of 750 µl
chloroform/methanol/concentrated HCl (40:80, 1 v/v), 100 µl of
H
O, 250 µl of chloroform, and 250 µl of 0.1
M HCl, followed by centrifugation.
[
H]InsP
was measured by liquid
scintillation counting of 500 µl upper phase.
Vesicle Binding
Large unilamellar vesicles (LUV)
were produced by extrusion through 100-nm polycarbonate membranes using
a phospholipid extruder (Lipex Biomembranes Inc.) according to the
manufacturer's instructions. Briefly, 15 mg of lipid of various
compositions (PtdCho/PtdSer/PtdIns
(4, 5) P (70:27:3 by molarity); PtdCho/PtdIns
(4, 5) P
(97:3); PtdCho alone; PtdCho with increasing masses of PtdSer)
were dried to a film, resuspended by vortexing in buffer B comprising
10 mM HEPES, pH 7.4, 200 mM sucrose, 3.4 mM
EDTA, 20 mM KCl, followed by repeated freeze-thawing in liquid
nitrogen/40 °C water bath. Lipids were extruded with >10 passes
through the polycarbonate filters, and LUVs were stored at 4 °C.
For PLC-binding studies, vesicles were diluted to 100 µM
with respect to PtdIns
(4, 5) P
in buffer C
(10 mM HEPES, pH 7.4, 3.4 mM EDTA, 150 mM
NaCl) and used as a stock for all lower concentrations required in the
binding studies.
hydrolysis. Enzyme was
incubated with vesicles for 10 min on ice (better reproducibility of
data was observed at this temperature), followed by ultracentrifugation
(60,000 rpm, 30 min at 4 °C, TLA 100 rotor, Beckman TL100
centrifuge), and PLC activity remaining in the supernatant was assayed
against 25 µM
[
H]PtdIns
(4, 5) P
as
above.
Measurements of PLC Oligomerization by BIA
Real
time intermolecular interaction between PLC molecules and lipids were
determined using the BIALite system (Pharmacia Biosensor, Uppsala,
Sweden). PLC was immobilized to a CM5 biosensor chip by amine coupling,
using the manufacturer's recommended procedures with certain
modifications. The hydrogel matrix on the sensor chip was activated by
a 6-min injection (5 µl/min flow rate) of a 1:1 mixture of
N-hydroxysuccinimide and
N-ethyl-N`-[3-(diethylamino)propyl]
carbodiimide. PLC was immobilized by a 7-min injection of between
5-25 µg/ml protein at either pH 4.2 (in sodium acetate
buffer) or pH 7.2 (in buffer C which was also used as the flow-through
buffer) and any free unreacted sites on the matrix were blocked with 1
M ethanolamine chloride. Immobilization at pH 4.2 captured
between 9-11 ng of PLC/mm sensorchip surface
(9,000-11,000 RUs increase) whereas only 0.15-0.2
ng/mm
PLC (150-200 RUs) was immobilized at neutral
pH, approximated according to the manufacturer's recommendations.
Subsequent assessments of PLC and/or lipid vesicle binding to
immobilized enzyme were performed at a flow rate of 5 µl/min.
Experiments were performed at pH 7.2 in buffer C and some measurements
were made at pH 4.2 in sodium acetate buffer. Analytes were injected
for various times (at least 4 min), and dissociation was allowed to
progress for at least 450 s. After injections of PLC, the chip surface
was regenerated by injection of a 2-µl pulse of 1 M NaOH;
a pulse of 0.12% (w/v) SDS was used to regenerate the chip after
injections of phospholipid vesicles. Data were analyzed using the
BIAevaluation software version 2.0 supplied by the manufacturer and are
presented without subtraction of control bulk refractive index changes
because of the markedly different optical properties of vesicle
preparations compared to enzyme dilutions.
Analysis of Kinetic
Data
PtdIns
(4, 5) P hydrolysis by all
three PLC
isoforms was determined under three conditions
previously described for secretory PLA
(Hendrickson and
Dennis, 1984). Case I involves determination of PLC activity with
increasing bulk concentrations of PtdIns (4,5)P
and a
single substrate mole fraction. Since initial binding to the interface
is thought to be dependent on the bulk substrate concentration, this
allows the determination of K
, the
equilibrium dissociation constant in bulk concentration terms. Case II
allows determination of the interfacial Michaelis constant
(K
) for PLC by determining PLC activity
with a single bulk concentration of PtdIns
(4, 5) P
and increasing mole fractions. Case III involves measuring PLC
activity when bulk PtdIns
(4, 5) P
concentration and surface PtdIns (4,5)P
concentrations are varied simultaneously.
Determination of K
For
case I experiments, Equation 1 reduced to the Henri Michaelis-Menten
equation (Equation 2), from which values for the equilibrium
dissociation constant (K) were derived:
Determination of Interfacial
K
Hill coefficients from case II data were
similar to those derived from logarithmic transformations of case III
experiments (). Subsequently, double-reciprocal plots were
made from case II velocity curves, where the ordinate was raised to the
power of the Hill number. This yields a straight line (Segel, 1976) and
the ordinate intercept is equal to
(-1/K)
.
)
for PLC
binding to vesicles was determined according to the method
of Rebecchi et al. (1992b). Assuming 1:1 complex formation
between PLC and PtdIns
(4, 5) P
,
K
was determined as the slope of the plot
of PLC bound/PLC free versus the concentration of
PtdIns
(4, 5) P
according to Equation 5:
Data Presentation
Representative experiments
which have been repeated multiple times with similar results are shown
in all figures. Data points are the means of duplicates with a range of
10%.
Assay Conditions to Examine PLC
PtdIns (4,5)P
Activity
hydrolysis by PLC
was
analyzed according to a bireactant model to test whether interface
binding and PtdIns (4,5)P
hydrolysis represent independent
steps required for catalysis as has been proposed for PLC
(Rebecchi et al., 1993; Cifuentes et al., 1993) and
PLC
(Wahl et al., 1992). The activity of three PLC
isoforms was examined using a three case kinetic analysis in which
PtdIns
(4, 5) P
bulk and surface
concentrations were varied independently and concurrently, as
established by Hendrickson and Dennis(1984).
/dodecylmaltoside mixed
micelles as substrate. These conditions induce submaximal PLC activity
due to the absence of cholate which facilitates
PtdIns
(4, 5) P
hydrolysis and would
compromise the kinetic analyses undertaken. Under the conditions used,
dodecylmaltoside appeared to behave as a true neutral diluent of PtdIns
(4,5)P
and did not appear to bind to the enzyme or inhibit
PLC activity. This was judged by assaying PLC activity using a fixed
mole fraction of PtdIns (4.5)P
in the presence of
increasing concentrations of dodecylmaltoside. Activity was linear with
respect to increasing bulk lipid concentration despite increases in
dodecylmaltoside (data not shown). Thus, dodecylmaltoside did not
appear to inhibit PLC activity. The effects of dodecylmaltoside on
phospholipid vesicle structure was assessed by scanning electron
microscopy. 100 µM PtdIns
(4, 5) P
sonicated into 10 mM HEPES, pH 7.2 (stock assay lipid),
resulted in heterogeneous unilamellar vesicles whose diameters varied
between 40-280 nm. Addition of 150 µM
dodecylmaltoside (to give a mole fraction of
PtdIns
(4, 5) P
of 0.4) did not affect
vesicle lamellarity, but reduced the size heterogeneity, with vesicles
between 30-120 nm diameter (data not shown).
mixed micelles including octyl glucoside, Triton X-100, Brij 35,
and Tween 20. Triton X-100 profoundly inhibited PLC activity at
concentrations above 25 µM, and all other detergents
exerted inconsistent effects on PLC activity such that the relationship
between PLC activity and increasing detergent concentration was not
linear (data not shown). These effects were independent of the critical
micellar concentrations for each detergent. Because dodecylmaltoside
was the only detergent which did not inhibit PLC, it was used to dilute
surface concentrations of PtdIns (4,5)P
.
PLC
To
investigate whether PLC Activity Is Dependent on Bulk and Surface
Concentrations of PtdIns
(4, 5) P
activity involves multiple binding events
due to interactions with the bulk lipid interface and subsequent
substrate binding within the interface, the bulk concentration and mole
fraction of PtdIns (4,5)P
were increased simultaneously and
InsP
production was measured. This was achieved by assays
using a single concentration of dodecylmaltoside and increasing
concentrations of PtdIns
(4, 5) P
(case III
in the protocols described under ``Experimental Procedures'')
The relationship betwen PLC
activity and bulk and surface
concentrations of substrate was sigmoid (Fig. 1, showing results
for PLC
). Similar results were obtained for
PLC
and TEPLC. The solid line on
Fig. 1
is a fit of the data to Equation 1. The sigmoidicity of
this relationship is strong evidence that PLC-mediated
PtdIns
(4, 5) P
hydrolysis involves at least
two distinct binding events (Hendrickson and Dennis, 1984). A
logarithmic Hill plot of the data gave a straight line, from which the
slope (equal to the Hill coefficient, n) was determined and
was found to range between 1.4-1.8 for the three enzymes
(). These values of n confirmed that the
interactions of PLC
isoforms with lipid surfaces involves at least
two lipid-binding sites.
Figure 1:
PtdIns
(4,5)P hydrolysis by PLC
as a function of
both bulk concentration and mole fraction of substrate (case III). The
concentration of dodecylmaltoside was held constant at 196
µM and PtdIns (4,5)P
bulk concentration was
increased up to 100 µM. Assays were as otherwise stated
under ``Experimental Procedures.'' Inset, Hill plot
of the data fitted to Equation 3 under ``Experimental
Procedures.'' Solid lines were fitted using the Ultrafit
Programme. Data are representative of three experiments and were
repeated three times for each of PLC
and TEPLC with
similar results (Table I).
Dissection of Separate Lipid Binding Events in the
Catalytic Cycle of PLC
Lipid-metabolizing enzymes are
considered to bind to and sometimes penetrate lipid interfaces with
subsequent further substrate binding within the interface as component
parts of their catalytic mechanism. The initial interaction with the
interface is dependent on the bulk lipid concentration while binding
within the interface is dependent on the substrate mole fraction. The
separate steps can be examined independently of each other by variation
of bulk lipid concentration at a fixed mole fraction and vice versa.
was determined by assaying PtdIns
(4, 5) P
hydrolysis as a function of bulk concentration at a fixed mole
fraction of PtdIns (4,5)P
of 0.2 (case I). This was
achieved in practice by varying both bulk
PtdIns
(4, 5) P
and dodecylmaltoside
concentrations proportionately, maintaining the mole fraction constant.
This value was chosen because at mole fractions greater than
0.25-0.3, the kinetics of PtdIns
(4, 5) P
hydrolysis deviated from linearity whereas below 0.25, reactions
were linear, a phenomenon which is not understood at present. PLC
activity showed a hyperbolic relationship with
PtdIns
(4, 5) P
bulk concentration
(Fig. 2). The data shown are for TEPLC and were similar to those
obtained for both recombinant PLC
and
PLC
. The solid line on Fig. 2is the
curve obtained by fitting the data to Equation 1. The Hill number
obtained was close to 1, indicating that binding of PLC to the lipid
interface likely involved a single site of interaction. Case I data
therefore fitted Equation 2 from which values for the equilibrium
dissociation constant (K
) were obtained
().
Figure 2:
TEPLC activity toward PtdIns
(4,5)P/dodecylmaltoside mixed micelles as a function of
bulk PtdIns (4,5)P
concentration (case I). PtdIns
(4,5)P
mole fraction was 0.2. Assays were performed as
described under ``Experimental Procedures.'' Solid lines were fitted using the computer programme Ultrafit. Data are
representative of four experiments and were repeated several times for
PLC
and PLC
2.
This relationship between substrate bulk
concentration and PLC binding was further investigated using a
non-catalytic vesicle binding assay, as described previously for
PLC
(Rebecchi et al., 1992b). TEPLC was incubated with
sucrose-loaded vesicles of differing phospholipid compositions, as
described under ``Experimental Procedures,'' and the amount
of TEPLC in the supernatant was determined by assay after
ultracentrifugation. When incubated with vesicles composed of
PtdCho/PtdSer/PtdIns
(4, 5) P
(70:27:3
mol/mol), TEPLC was depleted from the supernatant and bound to the LUV
pellet in a manner which depended on the total vesicle concentration
(Fig. 3A). The relationship appeared hyperbolic in
agreement with that found for case I experiments and the solid line
resulted from fitting the data to a single site binding model from
which K
values of between 2.5 and 5
µM were calculated. An apparent equilibrium association
constant (K
) was determined using the
model proposed by Rebecchi et al. (1992b, Equation 4),
assuming 1:1 interaction between PLC
and vesicles in these
conditions (Fig. 3A, inset). The
K
value for TEPLC was 5 ± 1
10
M
with respect to
bulk PtdIns (4,5)P
concentration (mean ± S.D., five
experiments).
Figure 3:
A, binding of turkey erythrocyte PLC to
sucrose-loaded vesicles. 50 ng of TEPLC was incubated with a range of
concentrations of PtdIns (4,5)P-containing vesicles (PtdIns
(4,5)P
/PtdCho/PtdSer, 3:70:27 mol/mol) followed by
ultracentrifugation, as described under ``Experimental
Procedures.'' PLC activity remaining in the supernatant was
assayed against PtdIns (4,5)P
and compared with no vesicle
controls. PLC activity in no vesicle controls was 11.7 ± 0.8
pmol IP
/min. Data are shown as PLC bound versus PtdIns (4,5)P
concentration in the vesicles fitted to
a single site binding model. Inset, plot of the ratio of bound
PLC to free PLC versus the PtdIns (4,5)P
concentration, from which K was determined. The
experiment shown is representative of four which gave similar results.
B, effects of vesicle lipid composition on TEPLC binding.
TEPLC was incubated with vesicles lacking PtdIns (4,5)P
but
containing PtdCho and increasing molar proportions of PtdSer (9:1, 3:1,
1:1 PtdCho/PtdSer by molarity) and treated as for A. Top
panel shows enzyme activity remaining in the supernatant after
centrifugation, and results for PtdCho/PtdSer/PtdIns (4,5)P
(3:1:0.1) are shown for comparison. PLC activity in no-vesicle
controls was 19.0 ± 2.2 pmol IP
/min. Lower
panel, amounts of PLC in nanograms bound to the different
vesicles.
Stable binding of PLC to vesicles was influenced by
the lipid composition of the vesicles. Thus, when PtdSer was omitted
and PtdCho was increased to 97% of the lipid, Kwas decreased by one order of magnitude to 0.4 ± 0.1
10
M
(mean ±
S.D., three experiments). In addition, binding was absolutely dependent
upon the presence of PtdIns
(4, 5) P
in the
vesicles under the chosen lipid proportions. When TEPLC was incubated
with vesicles lacking PtdIns
(4, 5) P
, but
containing 1.2 mM PtdCho, 0.45 mM PtdSer (the maximum
concentrations examined) very little depletion of PLC from the
supernatant was observed (Fig. 3B, top panel)
and binding to the vesicles was measurable only when the vesicular
PtdIns (4,5) P
concentration was significant
(Fig. 3B, lower panel). Thus,
PtdIns
(4, 5) P
molecules appear to form
anchorage sites for TEPLC to the lipid interface, and in their absence,
the enzyme is unable to interact stably with the lipid aggregate.
molecules
within the interface was assessed in case II experiments, in which
PLC
catalytic activity was measured at a constant bulk
concentration of PtdIns (4,5)P
and varied mole fraction.
This was achieved by varying the concentration of dodecylmaltoside
alone. Experiments were performed at a bulk concentration of 200
µM PtdIns
(4, 5) P
which ensured
that all dodecylmaltoside concentrations added were above the critical
micelle concentration of this detergent (100 µM in these
ionic conditions). Experiments at higher bulk concentrations of PtdIns
(4,5)P
gave inconsistent data, possibly due to non-uniform
mixing of phospholipid with detergent. The relationship between enzyme
velocity and substrate mole fraction appeared sigmoidal (Fig. 4,
data for PLC
are shown), and fitted Equation 1 with
Hill coefficients of between 1-2. The Lineweaver-Burk
double-reciprocal plot of the data was not linear, but curved upward
and was linearized by raising the ordinate to the power of the Hill
number (Fig. 4, inset), and the interfacial
K
was determined from the intercept on
the ordinate as described under ``Experimental Procedures.''
K
values are shown in .
However, because the PLC
reaction appears to become non-linear at
mole fractions greater than 0.3, these values of
K
should be regarded as minimum values
and true values may be somewhat higher, as have been reported for
PLC
(Wahl et al., 1991; Carpenter et al., 1992).
Figure 4:
PLC activity toward
PtdIns (4,5)P
/dodecylmaltoside mixed micelles as a function
of PtdIns (4,5)P
surface concentration (case II). PtdIns
(4,5)P
bulk concentration was 200 µM. Assays
were as described under ``Experimental Procedures.''
Inset, Lineweaver-Burk transformation of case II data. The
sigmoid line was fitted using the Ultrafit programme. Data presentation
are as for Fig. 2.
The experiments described above were performed using a free calcium
concentration of 1 µM. To examine the effects of
Ca concentration on PLC catalysis, case I and II
experiments were repeated in 100 µM calcium for
PLC
, representing a supraphysiological concentration
of calcium unlikely to be evoked physiologically. Data showed that
there was no alteration in the three kinetic constants. However, in the
presence of 50 nM free calcium, we have found that
V
was reduced by almost half (data not shown).
Thus, calcium concentrations likely to occur in the cytoplasm appear to
affect enzyme velocity rather than association of the enzyme with the
lipid.
Measurements of PLC Oligomerization by BIA
The
data above indicated that separate PtdIns
(4, 5) P binding and catalytic steps occur in the catalytic cycle of
PLC
. This might suggest that PLC
monomers contain multiple
PtdIns (4,5)P
-binding sites, with one region of the
molecule acting as an anchor, locating the enzyme at the interface.
Alternatively, PLC
could oligomerize at a lipid interface thus
presenting multiple substrate binding sites which might serve both as
anchors and catalytic sites, as proposed for PLA
(Roberts
et al., 1977). We examined the possibility of lipid-dependent
and independent PLC
oligomerization using Biospecific Interaction
Analysis with the BIALite biosensor, as desribed under
``Experimental Procedures.''
-containing vesicles (the
analyte; composition 70% PtdCho, 27% PtdSer, 3% PtdIns (4,5)P
by molarity) was measured (Fig. 5), by injection of
vesicles over the ligand PLC (Fig. 5, upper trace). The
trace shows an initial instantaneous increase in response due to
changes in the bulk optical density contributed by the LUV followed by
specific interaction and binding of vesicles to PLC. At the end of the
injection, the signal decreased initially due to reduction in the
refractive index of the flow-through buffer followed by complex
dissociation of the vesicles from PLC.
Figure 5:
Lipid-dependent and -independent TEPLC
oligomerization measured using BIA. TEPLC was amine coupled to a
sensorchip and interactions with phospholipid vesicles (composition 70%
PtdCho, 27% PtdSer, 3% PtdInsP by molarity, upper
trace), 2.5 µg/ml TEPLC (pH 7.2, bottom trace), and
TEPLC-phospholipid vesicles mixture (middle trace) were
measured. Injections commenced at the upward arrow and
dissociation started at the end of the analyte injection (downward
arrow). Data are representative of two experiments with TEPLC,
which have been repeated with both PLC
1 and PLC
2 with similar
results.
Binding of PLC to immobilized
enzyme in the absence of lipid was also determined. At neutral pH, no
PLC-PLC association could be detected (the signal increased by
approximately 120 RUs which was wholly attributable to nonspecific
interactions with the chip surface as judged using a non-coupled
sensorchip as control; Fig. 5, bottom trace). PLC
binding at the sensorchip was subsequently determined in the presence
of phospholipid vesicles to see if lipid enhanced PLC oligomerization.
PLC and vesicles were preincubated together and injected as a combined
analyte mixture over immobilized PLC. Binding to the sensorchip was
reduced relative to binding of vesicles alone (Fig. 5, middle
trace), which suggested that the preincubation step simply
saturated PLC and vesicles such that binding at the chip was abrogated.
The slightly elevated base-line signal after the injection stopped was
totally due to a small amount of vesicle association with immobilized
PLC and was not attributable to PLC oligomerization. Thus, no evidence
for oligomerization of PLC was detectable using this approach.
isoforms is unlikely to explain the
observed multisite PtdIns
(4, 5) P
binding
kinetics.
PLC Hydrolysis Using Nonsubstrate Phospholipids as
Diluents
PtdIns
(4, 5) P hydrolysis by
PLC was measured as a function of mole fraction in mixed phospholipid
vesicles, using PtdCho, PtdSer, or PtdEtn as diluents of PtdIns
(4,5)P
. When the mole fraction of PtdIns (4,5)P
was reduced to 0.67 with nonsubstrate lipid (equal to 12.5
µM ``background'' lipid), PLC activity was
reduced to less than 30% of the maximal velocity with all three lipids
(Fig. 6). PtdIns
(4, 5) P
hydrolysis
was reduced further when the PtdIns
(4, 5) P
mole fraction was reduced to 0.5 (Fig. 6) after which
further reductions in enzyme activity were not measurable. Thus,
diluting the mole fraction of PtdIns
(4, 5) P
with phospholipid reduced PLC activity by an amount that was
greater than that seen with dodecylmaltoside. The reduction in PLC
activity was disproportionately large with respect to the mole fraction
of nonsubstrate lipid added, suggesting that these nonsubstrate lipids
exert an additional inhibitory effect on PLC activity. Thus, the
kinetic analysis performed with PtdIns
(4,5)P
-dodecylmaltoside mixed micelles cannot be achieved
using nonsubstrate lipids as diluents. The mechanism of this inhibitory
effect is not known, and the effects of such lipids on PLC
activity will be examined in greater detail elsewhere.
Figure 6:
PLC
activity as a function of PtdIns (4,5)P mole fraction (case
II) using nonsubstrate lipids as diluents. PLC activity was assayed in
the absence of any detergent using 25 µM PtdIns
(4,5)P
and in the presence of increasing concentrations of
nonsubstrate lipids (PtdCho (circles), PtdSer
(diamonds), or PtdEtn (squares)) such that the PtdIns
(4,5)P
mole fraction was reduced to 0.2. Data shown were
obtained using PLC
and were repeated for each of
PLC
and TEPLC with similar
results.
DISCUSSION
Phospholipases C which are involved in signal transduction
responses to cellular stimuli are members of a diverse family of
enzymes whose mode of interaction with lipid substrates is complex and
only partly defined. In this study, we have examined the kinetic
characteristics of PtdIns
(4, 5) P hydrolysis
by the
-isoforms of PLC in the absence of their physiological
activators. The data help to establish a basic understanding of how
these enzymes behave toward lipid-water interfaces from which
physiologically relevant mechanisms of regulation may eventually be
discernible.
(Hendrickson and Dennis, 1984), which suggests the
PLC
s may interact with lipid surfaces in a similar manner. The
sigmoidicity of case III is likely due to simultaneous alterations in
both the bulk and surface concentrations of
PtdIns
(4, 5) P
and indicates that PLC
behaves as a multireactant enzyme. Calculation of Hill coefficients
suggested at least two binding sites are involved. Fitting case I and
case II data to the Hill equation yielded hyperbolic and sigmoidal
velocity curves with Hill coefficients of 1 and
2, respectively.
The hyperbolic relationship between bulk substrate concentration and
PLC
binding to the interface was confirmed in a vesicle binding
assay.
isoforms with membrane interfaces and binding
PtdIns
(4, 5) P
at the active site are
separate events. For efficient PLC-catalyzed production of second
messengers, PLC
may bind to membrane interfaces in a
PtdIns
(4, 5) P
-specific manner, and
subsequent secondary binding within the interface may help form stable
anchorage of PLC at the membrane. This mechanism may also lead to
processive catalysis whereby PLC could hydrolyze multiple
PtdIns
(4, 5) P
molecules before detaching
from the interface. As similar behavior was observed with each of the
PLC isoforms used, the proposed mechanism may be generally applicable
to members of the PLC
family. The sigmoidicity of case II may be
due to several factors including alterations in the architecture of the
mixed micelles as mole fraction of phospholipid increases or positive
cooperativity between multiple substrate binding sites within the
PLC
molecule. Nevertheless, the dependence of PLC
catalytic
activity on both bulk and surface concentrations of
PtdIns
(4, 5) P
clearly indicates that
binding to the lipid interface and within the interface are component
parts of the catalytic mechanism.
values were between
100-200 µM, slightly higher than that found for
PLC
(Cifuentes et al., 1993) and lower than that for
PLC
(Wahl et al., 1991). In theory, the
K
is the reciprocal of the association
constant (K
). However, the value of
K
derived from catalytic assays was
greater than the reciprocal of the K
calculated from vesicle-binding assays (Fig. 3). These data
lend support to the idea that the characteristics of lipid interfaces
will affect the affinity of PLC interactions since the disparity is
likely to be due to the differences in the conditions of the two
assays. Experiments designed to determine the interfacial
K
for each PLC isoenzyme showed that over
10% of the interface must comprise PtdIns
(4, 5) P
for half V
to be attained. This value is
lower than that calculated for PLC
(Wahl et al., 1991;
Carpenter et al., 1992) although it is possible that it is an
underestimate of the true value for the reasons stated earlier. Under
physiological circumstances, PtdIns forms approximately 5-10% of
plasma membrane phospholipid content (Vance, 1985) and
polyphosphoinositides are approximately 1-3% of the PtdIns (Creba
et al., 1983). Although these values probably underestimate
the proportions of polyphosphoinositides at sites where they are
localized in the cell, such as the inner leaflet of the plasma
membrane, the high interfacial K
determined for the PLC
s suggests that under physiological
conditions these enzymes are likely to operate under first-order
conditions.
molecule to at least one site in PLC other than the active site
is inherent in the above proposal. The data presented in cases I-III do
not allow a distinction to be made between whether the PLC
s
interact specifically with the lipid-water interface by binding to an
individual phospholipid molecule or by binding nonspecifically to the
interface. The latter possibility would be in line with an earlier
surface dilution model of phospholipase activity (Deems et
al., 1975). This model predicts that binding of phospholipase will
occur to an interface composed solely of nonsubstrate surface diluents.
However, when TEPLC was incubated with sucrose-loaded PtdCho or
PtdCho/PtdSer vesicles lacking
PtdIns
(4, 5) P
, no measurable binding of the
enzyme to vesicles was seen. These data strongly support a
multisubstrate mechansism in which binding at the interface is a
specific process requiring the presence of substrate lipid. Thus,
PLC
isozymes apear to possess at least two lipid-binding sites,
each of which confers specificity toward PtdIns(4,5)P
,
which are responsible for interfacial binding and catalysis,
respectively.
(Deems et al., 1975).
To account for such behavior in a small protein, it was proposed that
PLA
dimerized in the presence of a lipid interface (Roberts
et al., 1977) where one enzyme monomer acted as the anchor for
a second catalytic monomer. This mechanism remains an open question
with evidence supporting both monomeric (Jain et al., 1991)
and multimeric (Fremont et al., 1993) behavior. While there is
no need to invoke oligomerization as the multireactant mechanism for a
protein as large as PLC
, we examined this possibility and were
unable to detect any evidence of lipid-dependent PLC self-association.
Experiments using BIA showed that very little oligomerization of PLC
occurred, either alone or in the presence of phospholipid vesicles.
Therefore, oligomerization appears an unlikely mechanism to explain the
kinetic behavior of PLC
isozymes presented in this study. By
analogy with the similar proposal for PLC
(Cifuentes et
al., 1993), we propose that PLC
s may contain a noncatalytic
PtdIns
(4, 5) P
-binding pocket. In keeping
with this, all PLC isoforms contain pleckstrin homology domains in
their amino termini (Parker et al., 1994), a disparate region
of primary sequence which has PtdIns (4,5)P
binding
capacity (Harlan et al., 1994). In line with this suggestion,
it is becoming plain that both amino and carboxyl termini of PLC
play important functions in G protein regulation of activity and
association with membranes (Lee et al., 1993; Park et
al., 1993; Schnabel et al., 1993; Wu et al.,
1993).
(Berg et al., 1991) and enhances the
binding of PLC
to lipid vesicles (Rebecchi et al.,
1992b). The latter authors suggested that PLC
might bind PtdSer in
the interface or that the electrostatic potential of the interface in
the presence of PtdSer favors PLC
association. In this respect,
phos-phoinositide hydrolysis is enhanced by the presence of another
anionic phospholipid in lipid interfaces, phosphatidic acid (Hirasawa
et al., 1981), an effect that may be due to it acting as an
allosteric regulator of the enzyme (Jones and Carpenter, 1993). Here we
have shown that PtdSer increases PLC
association with vesicles,
seen as an increase in the K
of the
enzyme, but does not appear to bind the enzyme itself, supporting the
electrostatic potential model discussed above. However, although PtdSer
facilitates stable penetration of lipid aggregates by PLC
, it does
not increase the catalytic rate of the enzyme. PtdSer acted as a
diluent of PtdIns (4,5)P
and inhibited
PtdIns
(4, 5) P
hydrolysis in an identical
manner to the zwitterionic lipid PtdCho. Therefore, we suggest that
PtdSer may help localize PLC
to cell membranes but is not a
physiological activator of the enzyme.
is likely to be exposed in the aqueous environment at the surface
of the membrane. We propose this mode of action may facilitate
catalysis by restricting diffusion of PLCs to the two-dimensional
membrane. Indeed, this may be why a dual substrate mechanism is
apparently conserved among a wide range of lipid metabolizing enzymes,
an extreme example being PtdIns 4-kinase (Buxeda et al., 1991)
in which the reaction catalyzed appears to involve the most hydrophilic
region of the substrate lipid.
enzymes are fully competent to interact with lipid substrates in the
absence of activation by receptor-regulated G proteins and that,
similar to other enzymes such as protein kinase C and other
phospholipases, this interaction is influenced by substrate
concentration and diluent lipid composition of the interface. The
kinetic characteristics of PLC
s established here in the absence of
physiological regulation will allow future investigations into the
mechanisms of activation by G protein subunits, calcium ion
concentration, and membrane composition.
Table: Kinetic constants derived for three isoforms
of PLC from case I and case II catalytic assays
isoenzymes
under the three case kinetic analysis described under
``Experimental Procedures'' and the equilibrium dissociation
constant (K
), interfacial
Michaelis-Menten constant (K
), and
V
were calculated. Data are means ± S.D.
for multiple independent experiments for each enzyme which were
performed in duplicate.
,
phospholipase A
; PtdCho, phosphatidylcholine; PtdEtn,
phosphatidylethanolamine; PtdIns, phosphatidylinositol; PtdIns
(4,5)P
, phosphatidylinositol (4,5)-bisphosphate; PtdSer,
phosphatidylserine; RUs, resonance units; TEPLC, turkey erythrocyte
phospholipase C.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.