©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Kinetic Analysis of Phospholipase C Isoforms Using Phospholipid-Detergent Mixed Micelles
EVIDENCE FOR INTERFACIAL CATALYSIS INVOLVING DISTINCT MICELLE BINDING AND CATALYTIC STEPS (*)

Stephen R. James (§) , Andrew Paterson (1)(¶), T. Kendall Harden (1), C. Peter Downes

From the (1) Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee, Tayside DD1 4HN, Scotland, United Kingdom Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7365

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5)P) 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 PLCs by G protein subunits.


INTRODUCTION

Phospholipases form a large family of lipid-metabolizing enzymes involved in regulated and unregulated phospholipid hydrolysis (Dennis, 1983). A family of phospholipases C (PLC)() 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.

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 PLAs has been developed (Deems et al., 1975; Eaton and Dennis, 1976; Roberts et al., 1977; Hendrickson and Dennis, 1984) according to :

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 (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.

Using the -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 PLC1 and PLC2 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 PLCs.


EXPERIMENTAL PROCEDURES

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 rPLC1 and rPLC2

Both rPLC1 and rPLC2 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 PLC1 or human PLC2 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. rPLC1 was isolated by sequential chromatography of the high speed supernatant on Q-Sepharose FF and heparin-Sepharose CL-6B, while rPLC2 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 Kof EGTA for calcium of 5.17 10M). 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 HO, 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.

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 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.

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

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:

On-line formulae not verified for accuracy

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).

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) 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, Kwas determined as the slope of the plot of PLC bound/PLC free versus the concentration of PtdIns (4, 5) P according to Equation 5:

On-line formulae not verified for accuracy

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%.


RESULTS

Assay Conditions to Examine PLC Activity

PtdIns (4,5)P 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).

Enzyme activity was measured using PtdIns (4,5)P/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).

Several other non-ionic detergents were tested as diluents in PtdIns (4,5)P 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 Activity Is Dependent on Bulk and Surface Concentrations of PtdIns (4, 5) P

To investigate whether PLC 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.

The equilibrium dissociation constant for interface binding by PLC 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 PLC2.



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 Kvalues 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 Kvalue for TEPLC was 5 ± 1 10M 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 10M (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.

Binding of PtdIns (4, 5) P 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 Kwas determined from the intercept on the ordinate as described under ``Experimental Procedures.'' Kvalues are shown in . However, because the PLC reaction appears to become non-linear at mole fractions greater than 0.3, these values of Kshould 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.''

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-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 PLC1 and PLC2 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.

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 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.

The qualitative nature of each of the relationships established in cases I-III is very similar to that found for secretory PLA (Hendrickson and Dennis, 1984), which suggests the PLCs 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.

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 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.

Three kinetic constants which were strikingly similar for all three isozymes were derived from this series of experiments. Kvalues 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 Kis the reciprocal of the association constant (K). However, the value of Kderived from catalytic assays was greater than the reciprocal of the Kcalculated 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 Kfor 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 Kdetermined for the PLCs suggests that under physiological conditions these enzymes are likely to operate under first-order conditions.

The binding of a PtdIns (4, 5) P 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 PLCs 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.

Complex multireactant lipase kinetics were first shown for secretory forms of PLA (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 PLCs 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).

An effect of PtdSer on lipase activity is well established; it is thought to be essential for penetration of lipid interfaces by secretory PLA (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 Kof 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.

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 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.

Our results show that PLC 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 PLCs 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

PLC assays were performed for all three PLC 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.



FOOTNOTES

*
This work was supported by Agriculture and Food Research Council of Great Britain Grant AG94-516 (to C. P. D.) and by United States Public Health Service Grant GM 29536. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee, Tayside DD1 4HN, Scotland, United Kingdom. Tel.: +44-1382-344729; Fax: +44-1382-201063.

Recipient of a Wellcome Trust Travel Grant.

The abbreviations used are: PLC, polyphosphoinositide-specific phospholipase C; BIA, biospecific interaction analysis; LUV, large unilamellar vesicles; PLA, 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.


ACKNOWLEDGEMENTS

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.


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