Protein lipid interaction in bile: effects of biliary proteins on the stability of cholesterol–lecithin vesicles1

Biochimica et Biophysica Acta 1390 Ž1998. 282–292

Protein lipid interaction in bile: effects of biliary proteins on the stability of cholesterol–lecithin vesicles 1 Andrew S. Luk a , Eric W. Kaler a , Sum P. Lee a

b,c,)

Department of Chemical Engineering, Center for Molecular and Engineering Thermodynamics, UniÕersity of Delaware, Newark, DE, USA b Department of Medicine, UniÕersity of Washington, Seattle, WA 98108, USA c Department of Veterans Affairs Medical Center, Seattle, WA 98108, USA Received 14 July 1997; accepted 19 August 1997

Abstract The nucleation of cholesterol crystals is an obligatory precursor to cholesterol gallstone formation. Nucleation, in turn, is believed to be preceded by aggregation and fusion of cholesterol-rich vesicles. We have investigated the effects of two putative pro-nucleating proteins, a concanavalin A-binding protein fraction and a calcium-binding protein, on the stability of sonicated small unilamellar cholesterol–lecithin vesicles. Vesicle aggregation is followed by monitoring absorbance, and upon addition of the concanavalin A-binding protein fraction the absorbance of a vesicle dispersion increases continuously with time. Vesicle fusion is probed by a fluorescence contents-mixing assay. Vesicles apparently fuse slowly after the addition of the concanavalin A-binding protein, although inner filter effects confound the quantitative measurement of fusion rates. The rates of change of absorbance and fluorescence increase with the concentration of the protein, and the second-order dimerization rate constant increases with both the protein concentration and the cholesterol content of the vesicles. On the other hand, the calcium-binding protein has no effect on the stability of the vesicle dispersion. This protein may therefore affect cholesterol crystal formation not by promoting the nucleation process, but by enhancing crystal growth and packaging. Our results demonstrate that biliary proteins can destabilize lipid vesicles and that different proteins play different roles in the mechanism of cholesterol gallstone formation. q 1998 Elsevier Science B.V. Keywords: Bile; Gallstone; Kinetics; Scattering

1. Introduction Abbreviations: CH, cholesterol; L, egg lecithin Žphosphodidylcholine.; SUV, small unilamellar vesicle; CBP, calcium-binding protein; ConA, concanavalin A; ConABP, concanavalin A-binding protein fraction; ANTS, 8-aminonaphthalene-1,3,6trisulfonic acid; DPX, p-xylylene-bisŽpyridinium bromide. ) Corresponding author. Address: Gastroenterology Section Ž111GI., Department of Veterans Affairs Medical Center, 1660 South Columbian Way, Seattle, WA 98108, USA. Fax: q1 206 764 2232. Supported in part by the Medical Research Service of the Department of Veterans Affairs. 1 Supported in part by the Whitaker Foundation and by Grant ŽDK-41678. from the National Institutes of Health.

Cholesterol is solubilized in human bile by bile salt–lecithin mixed micelles and lecithin Žphosphodidylcholine. vesicles w1x. Lecithin vesicles are the primary cholesterol carriers in bile supersaturated with cholesterol w2–4x. Aggregation andror fusion of these thermodynamically metastable cholesterol-rich vesicles and subsequent nucleation and crystallization of cholesterol are the key steps to the formation of cholesterol gallstones w5,6x. Both in vivo and in vitro studies demonstrate that cholesterol nucleates pre-

0005-2760r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 1 6 1 - 6

A.S. Luk et al.r Biochimica et Biophysica Acta 1390 (1998) 282–292

dominantly from vesicles w4,7x. Cholesterol supersaturation, however, is a necessary but not sufficient driving force to accelerate nucleation w8x. Factors other than the cholesterol content must therefore contribute to the prolonged stability of cholesterol-rich vesicles in normal bile w9x. Protein concentrations in human bile are often elevated at the early stages of cholesterol gallstone formation w10,11x. Based on cholesterol nucleation studies under a polarizing microscope w12x, various proteins isolated from human bile have been shown to either shorten or prolong the nucleation time in both human and model bile. Proteins that shorten cholesterol nucleation time are termed pro-nucleating proteins. Pro-nucleating proteins include both mucous glycoproteins or mucin w13–15x and non-mucous glycoproteins w16x. When mucin-free human gall bladder bile is subject to a concanavalin A ŽCon A.-affinity column, the resultant ConA-binding protein fraction Ž ConABP. 1 promotes cholesterol nucleation in a dose-dependent manner w17,18x. A number of pro-nucleating proteins have since been identified in this ConABP fraction such as phospholipase C w19x, immunoglobulins w20,21x, fibronectin w22x, a 1acid glycoprotein w23x, and aminopeptidase N w24,25x. Furthermore, a low molecular weight anionic peptide fraction or calcium-binding protein Ž CBP. has recently been proposed to interact with vesicles and accelerate cholesterol crystallization w26x. In addition to the library of existing pro-nucleating proteins, anti-nucleating proteins such as phospholipase A 2 w27x, apolipoprotein A-I and A-II w28,29x, and other glycoproteins w30x prolong cholesterol nucleation time. Despite the increasing number of putative proand anti-nucleating proteins being reported, there has yet been no mechanistic study of the interactions between vesicles and pro-nucleating proteins. The kinetics of cholesterol gallstone formation have been described qualitatively in terms of a nucleation time w12x. Since this method does not measure the actual nucleation process, this nucleation time is often termed ‘‘crystal appearance time’’. Other techniques focus on measuring the overall rate of cholesterol crystal growth w31x. These times and rates change with the total lipid concentration employed in the experiment and provide no information about the stability of the initial vesicle dispersion and the early events associated with nucleation. Since the metasta-

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bility of bile depends likely on a balance of activities between various pro-nucleating and anti-nucleating proteins, it is of fundamental importance to quantify the kinetics of aggregation and fusion of cholesterol–lecithin vesicles as induced by these pro-nucleating proteins. We report here the effects of two putative pro-nucleating protein fractions, the ConA-binding protein and the calcium-binding protein, on the stability of sonicated small unilamellar vesicles containing cholesterol ŽCH. and lecithin ŽL.. The use of these sonicated CH–L vesicles as a model of biliary vesicles is already well-established w32,33x. In addition, a parallel kinetic study of sonicated vesicles has been reported with phospholipase C as the pro-nucleator w34x. Nonetheless, there is evidence that some proteins may interact differently with vesicle or bilayers prepared in different ways w35x. The observed kinetics of aggregation and fusion of CH–L vesicles can be predicted by a mass-action model w36x, and the aggregation rate constant obtained from the kinetic analysis describes the ‘‘nucleating potency’’ of putative pro-nucleators. 2. Materials and methods 8-Aminonaphthalene-1,3,6-trisulfonic acid Ž ANTS. and p-xylylene-bisŽpyridinium bromide. ŽDPX. were purchased from Molecular Probes ŽEugene, OR. . Cholesterol ŽCH ) 99% pure. and egg yolk lecithin ŽL, type V-E from egg yolk, approx. 99% pure. were obtained from Sigma Ž St. Louis, MO. and used without further purification. Water was distilled and deionized, and all glassware were acid-washed. 2.1. Preparation and purification of biliary proteins ConABP was isolated using the method described by Groen et al. w17x. Briefly, gall bladder bile was obtained from patients undergoing cholecystectomies by using needle aspiration, and frozen at y208C until used. After thawing, the bile was centrifuged at 5000 = g for 15 min and the supernatant was loaded to a concanavalin A-Sepharose column. The column was initially washed with 20 column volumes of buffer ŽpH 7.4. containing 50 mM Tris–HCl, 0.2 M NaCl, 1 mM CaCl 2 , 1 mM MnCl 2 , and 1 mM MgCl 2 . The bound fraction was subsequently eluted with 10 column volumes of buffer Ž pH 7.4. containing 50 mM

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Tris–HCl, 0.2 M NaCl and 0.1 M a-D-methylmannopyranoside. The final eluate was dialyzed for 48 h at 48C, lyophilized, and subjected to SDS-PAGE. The 130 kDa band was cut and electroeluted, and the material electroluted from this band is referred to as ConABP. There were no cholesterol microcrystals in the ConABP preparation as examined using polarizing microscopy. CBP was prepared using the method previously described w37x. Hepatic bile was collected from gallstone patients through surgically placed T-tubes and frozen at y208C. After thawing, bile was centrifuged at 5000 = g for 15 min, and CaCl 2 was added to give a final concentration of 0.5 M in the supernatant. This solution was refrigerated at 48C for 48 h, during which time a precipitate formed. The precipitate was collected by centrifugation at 10 000 = g for 20 min at 48C and dissolved by stirring in 0.5 M EDTA ŽpH 8.2. for 24 h. Insoluble material was removed by a final centrifugation. The supernatant was exhaustively dialyzed, lyophilized, and subjected to polyacrylamide gel electrophoresis. The sample was first dissolved in distilled water to a concentration of 10–15 mgrml and mixed with an equal volume of reducing sample buffer Ž 0.1 M Tris–HCl, 8% 2-mercaptoethanol, 12% sucrose, and 0.005% bromophenol blue, pH 6.8.. The molecular weight was approximated with low-molecular weight standards ŽBio-Rad, Richmond, CA. . A 12% native gel was used in a Mini-Protean II unit Ž Bio-Rad. at a constant voltage of 175 V until the dye front was within 5 mm of the bottom of the gel. The pigmented band at the gel front was harvested without staining and electroeluted with 50 mM NH 4 HCO 3 elution buffer in the absence of SDS ŽBio-Rad electroeluter. . The subsequently lyophilized material is referred to as CBP. 2.2. Vesicle preparation Cholesterol and lecithin mixtures were coprecipitated from chloroform in various molar ratios Žapproximately 1:1, 1:3, and pure lecithin.. The lipid composition was chosen to be within the range of human hepatic and gall bladder bile. We used 50 mol CH as an upper limit because above this level the vesicle become metastable. The solvent was evaporated by a stream of nitrogen in a rotary evaporator, and the residual solvent was eliminated under vac-

uum. The lipid film was resuspended in the appropriate buffer solution. Small unilamellar vesicles were made by directly sonicating the hydrated lipid solution in a container flushed with nitrogen in an icewater bath for 90 min ŽHeat Systems Ultrasonics Model W-225.. The resulting dispersion was centrifuged at 258C for 2 h at 32 000 = g, similar to the method described by Barenholtz et al. w38x, and the supernatant was incubated overnight at 258C. At this temperature, our vesicles remain stable over several days when repeatedly monitored with absorbance or optical density. The vesicles we prepared were monodisperse with a standard deviation of the hydrodynamic radii consistently less than 5% of the mean. The mean hydrodynamic radius of vesicles with vary˚ Ž0% ing chemical composition are as follows: 110 A; ˚ Ž20% CH. CHrL 3.3–260 A˚ Ž50% CH.. CH. 150 A; Since vesicles of the same compositions were monodisperse, the minor variation in size with different composition should not affect the experimental observations. Experimental procedures have been described previously w34x and are summarized here in brief. Vesicle fusion was probed by a contents-mixing assay using ANTS and DPX as the fluorescence probes. Vesicles were prepared in the appropriate buffer solutions Ž pH 7. containing either Ž a. 25 mM ANTS, 80 mM NaCl, 5 mM CaCl 2 and 5 mM HEPES, Ž b. 90 mM DPX, 40 mM NaCl, 5 mM CaCl 2 , and 5 mM HEPES, or Žc. 12.5 mM ANTS, 45 mM DPX, 60 mM NaCl, 5 mM CaCl 2 , and 5 mM HEPES. Vesicles were separated from unencapsulated materials by gel filtration ŽSephadex G-75. with the elution buffer containing 0.15 M NaCl, 5 mM CaCl 2 , and 5 mM HEPES. All of the above buffer solutions were treated with 0.02 wt% of NaN3 to inhibit bacterial growth and checked to be isosmotic ŽOsmotte A, Precision Instruments.. The final lecithin concentration was 0.5 mM, and vesicles prepared with the elution buffer were used for absorbance experiments. Lipid phosphorus was determined by the method of Bartlett w39x, and the cholesterol concentration was measured by a cholesterol oxidase assay w40x. 2.3. Absorbance and fluorescence measurements Vesicle aggregation and fusion were monitored by recording absorbance or turbidity at 350 nm Ž 300 nm

A.S. Luk et al.r Biochimica et Biophysica Acta 1390 (1998) 282–292

for pure lecithin vesicles. in a Perkin-Elmer Lambda 2 spectrophotometer at room temperature. The scattering from a dilute solution of small polydisperse particles illuminated with an unpolarized light source can be described in the Rayleigh–Gans–Debye approximation w42x and is related to the measured absorbance w34x as Is Ž t . Io

s 1 y 10y t Ž t .Pl M

Ý i2P

sAP

Vi Ž t . P f i Ž u . ,

Ž1.

is1

where fi Ž u . s

2p

H0

Pi Ž u . sin u Ž 1 q cos 2 u . d u

and IsŽ t .rIo is the total scattered intensity, t Ž t . denotes the absorbance or optical density at time t, A is a constant that depends on experimental parameters, i is the degree of aggregation, M is the largest aggregate considered, w Vi Ž t .x represents the number density of the ith aggregate Ž i-mer., and Pi Ž u . is the particle form factor that depends on the size and shape of the ith aggregate and the scattering angle u . Since the form factor of a vesicle is known 2 w42x, it is assumed that all higher-order aggregates are vesicular in shape. For the early stages of dimer formation Ž M s 2., Eq. Ž1. reduces to 1 y 10yt Ž t . s A

½

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˚ 2 for pure lecithin and between the values of 70 A ˚ 2 for an equimolar CH–L mixture w43x. 96 A Vesicle fusion and leakage experiments were performed with a Perkin-Elmer MPF-66 spectrofluorometer. Excitation and emission wavelengths were set to 355 and 515 nm, respectively. In order to eliminate scattered light, a 430 nm filter was placed at the emission pathway. The protein solution was injected into a vesicle dispersion at concentrations of 1, 3, and 5 mgrml with a final volume of 2.5 ml. Vesicle fusion was registered as a decrease in ANTS fluorescence by collisional quenching with DPX. The 100% fluorescence intensity Žor 0% fusion. was set by using a 1:1 mixture of ANTS and DPX vesicles. The 100% fusion intensity Žor 0% leakage. was set by the fluorescence intensity of vesicles coencapsulating ANTS and DPX at the same lecithin concentration. Vesicles do not leak in the presence of either ConABP or CBP. A theoretical analysis of the kinetics of vesicle aggregation and fusion as determined from this assay has been developed w36,41x. 2.4. Mass-action model Vesicle aggregation is described by the following mass-action model w41x

V1Ž t . P f 1Ž u .

q4 P

Ž3.

ž

V1Ž 0 . y V1Ž t . 2

/

5

P f2 Žu . .

Ž2. The initial number density of vesicles was calculated from the mean hydrodynamic radius of the vesicles as measured by quasi-elastic light scattering ˚ and w42x, with the bilayer width assumed to be 35 A, the lecithin headgroup area obtained by interpolation

2

The form factor for vesicles, PÕ Ž u ., is given by the following expression: PÕ Ž u . s w3rŽ u 3 y Õ 3 .4Žsin uy u cos uysin Õ y Õ cos Õ .x 2 , where us qR o , Õ s qR i , q is the scattering vector given as Ž4p n r l.sinŽ u r2., and R o and R i denote the outer and the inner radii of vesicles, respectively.

The rate of each step is determined by the aggregation rate constants K i j . In order to simplify the model, all rate constants are assumed identical; hence the fitted rate constant K represents an average rate of all aggregation steps. In the early stage of aggregation, vesicle dimerization should follow second-order kinetics, so 1

1 y

V1Ž t .

V1Ž 0 .

s 2 K dim t.

Ž4.

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The dimerization rate constant K dim Ž K 11 . can be obtained from the absorbance data via a plot of w V1Ž t .xy1 against time. In principle, a more elaborate mass-action model can be used to characterize the kinetics of both aggregation and fusion of vesicles w34x. Unfortunately, due to the interference by both the light absorption by the protein and sample turbidity, the measured fluorescence signal could not be quantitatively corrected for inner-filter effects.

3. Results In our experimental system, the addition of the protein samples to the elution buffer, and the equibration of the vesicles by themselves did not contribute to a change in turbidity. After addition of ConABP to dispersions of vesicles containing various amounts of cholesterol, absorbance increases continuously with time ŽFig. 1.. Since the absorbance increase begins immediately after the addition of protein, vesicle aggregation is not rate-limited by the binding of proteins to vesicles. Both the rate of absorbance increase and the extent of vesicle aggregation Ž as shown by the absorbance plateau. scale with the ConABP concentration. ConABP also appears to mediate vesicle fusion in a concentration-dependent manner ŽFig. 2.. Since protein binding is not the rate limiting step, a single parameter mass-action model can be employed to describe vesicle aggregation. The solid lines in Fig. 1 represent the theoretical fits generated by such a model, and the fitted values of the aggregation rate constant K are in Table 1. This simple model gives an excellent fit to the experimental absorbance data. As depicted in Fig. 1, the model fits slightly underestimate the early part of the absorbance increase. In other words, the rate constant associated with each step is likely to decrease as aggregation proceeds. In order to better describe the earliest stages of vesicle aggregation, the early data are analyzed according to Eq. Ž4. ŽFig. 3.. The dimerization rate constants determined from the slope of the fitted line correlate with both the protein concentration and the cholesterol content of vesicles ŽFig. 4.. Likewise, the aggregation rate constant in other agents known to induce vesicular aggregation

Fig. 1. Vesicle stability upon the addition of pro-nucleating proteins is shown over time by monitoring absorbance at the indicated wavelengths. Elution buffer containing no vesicles is used as the reference. Cholesterol content of vesicles Žin mol% CH. is as follows: Ža. 50; Žb. 28; and Žc. 0 Žpure lecithin.. Three ConA-binding protein concentrations are used: Žv . 1 mgrml; Ž`. 3 mgrml; and Ž=. 5 mgrml. The ConA-binding protein induces vesicle aggregation at a concentration-dependent fashion, but the calcium-binding protein at a concentration of 1 mgrml Ž^. does not affect vesicle stability. The lecithin concentration used was 0.5 mM, and the solid lines represent the fits predicted by the mass-action model.

and fusion such as phospholipase C also correlates with the cholesterol content in the vesicles w34x. This is most probably due to the steric repulsion of the phospholipid headgroups Žsee Section 4. . As shown in Table 1, the early dimerization rate constant is generally larger than the rate constant obtained from

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Table 1 Rate constants for ConA-induced aggregation of cholesterol– lecithin vesicles Sample

Second-order aggregation rate constants from the mass action model and the dimerization rate constant a K mass-action

Fig. 2. Stability of CH–L vesicles Ž28 mol% CH. upon the addition of ConABP and CBP is shown over time by the ANTSrDPX fluorescence fusion assay. Due to the coupled interferences of protein absorption and sample turbidity, the measured fluorescence data cannot be corrected for inner-filter effects. The fluorescence intensities are expressed on a qualitative scale in terms of percent fluorescence quenched as discussed in Section 2. The lecithin concentration used was 0.5 mM. Symbols for protein concentrations are the same as in Fig. 1.

b K dim ŽMy1 sy1 .

Pure lecithin 1 mgrml ConABP 3 mgrml ConABP 5 mgrml ConABP

2=10 3 2=10 4 4=10 4

4=10 3 3=10 4 1=10 5

28 mol% CH 1 mgrml ConABP 3 mgrml ConABP 5 mgrml ConABP

2=10 4 5=10 4 1=10 5

2=10 4 1=10 5 5=10 5

50 mol% CH 1 mgrml ConABP 3 mgrml ConABP 5 mgrml ConABP

9=10 3 1=10 5 3=10 5

2=10 4 3=10 5 7=10 5

c Smoluchowski upperlimit k sm : ;10 9 Ž258C.

the overall mass-action model, showing that the rate of vesicle aggregation decreases with increasing size of aggregates. With the availability of both absorbance and artifact-free fluorescence experiments, a complete mass-

Fig. 3. A dimerization plot Žreciprocal number density of vesicles versus time. for the early stages of ConABP-induced aggregation of CH–L Ž28 mol% CH.. The straight line fits for each protein concentration show that dimerization follows second-order kinetics. The slope of the line is twice the dimerization rate constant. Symbols for protein concentrations are the same as in Fig. 1.

a

The number density of vesicles was calculated from the mean hydrodynamic radius of the vesicles as measured by quasi-elastic ˚ and a headlight scattering, assuming a bilayer width of 35 A ˚ 2 for pure group area by interpolating between the values of 70 A 2 ˚ lecithin and 96 A for an equimolar CH–L mixture. The lecithin concentration was 0.5 mM, and the measured radii for pure lecithin vesicles were 11.0 nm, for CHrL s1:4 – 16.0 nm, and for CHrL s1:1 – 26.5 nm. Under these conditions, the vesicle number densities are 1.57=10y7 M Žmoles of vesicles per liter. for pure lecithin, 8.20=10y8 M for CHrL s1:4, and 3.1= 10y8 M for CHrL s1:1. b In this calculation, only the early part of the absorbance curve was used for dimerization analysis. c The Smoluchowski value is given by k sm s8 kT r3h , where h is the viscosity of the medium, k is the Boltzmann constant, and T is the temperature, and gives the upper diffusion-limited rate of aggregation.

action model can be employed to determine the fusion rate constants w34x. However, due to the innerfilter effects caused by both protein absorption and sample turbidity, the fluorescence data Ž Fig. 2. overestimate the extent of vesicle fusion. As a result, only an upper limit for the first-order fusion rate constants can be estimated from the fluorescence experiments. This upper limit is approximately 1 = 10y3 sy1 for all cases.

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Fig. 4. The fitted second-order dimerization rate constant K dim for the ConA-binding protein experiments versus protein concentration for vesicles containing different mol% CH: Ž`. 50; Žv . 28; and Ž^. 0 Žpure lecithin..

In sharp contrast, the addition of up to 5 mgrml of CBP to a vesicle dispersion causes no changes in either turbidity or fluorescence intensity. Thus the candidate pro-nucleator CBP does not affect vesicle stability. We also performed leakage measurements for both ConABP and CBP and found no change in fluorescence. 4. Discussion Cholesterol gallstone formation is a highly complex and dynamic process. Biliary proteins, though present in small amounts in bile, are believed to play a major contributory role in gallstone disease, with proteins modifying different stages of cholesterol crystallization. Pro-nucleating proteins can promote cholesterol crystallization by accelerating the aggregation and fusion of biliary vesicles. Two pronucleating proteins, phospholipase C and gall bladder mucin, do induce both vesicle aggregation and fusion w34,44,45x. In addition, proteins may facilitate the nucleation of cholesterol from vesicles, modify crystal morphology w46–48x, or function as cholesteroltransfer agents to enhance crystal growth. The protein concentrations examined here correspond to those of physiological importance for human gall bladder bile. In normal bile, the mean concentration of ConA binding protein is approximately 1 mgrml; and for biliary anionic polypeptide

fraction, which is similar to the CBP studied here, the mean value is estimated to be approximately 0.8 mgrml w49,50x. However, gall bladder bile that contains gallstones is usually more concentrated and has a much higher protein content than does normal bile w51x. Thus protein concentrations from 1 to 5 mgrml span the range from those of normal bile to that in abnormal bile in which gallstones are formed. Protein-induced fusion of lipid vesicles or liposomes is common in cellular processes w52x. Since liposome fusion requires the close contact of apposing membranes and substantial bilayer destabilization, proteins can facilitate the fusion process by enhancing the rate of these key intermediate steps w53x. For example, surface dehydration, hydrophobic bridging, and charge neutralization drive vesicle adhesion, while lateral phase separation and inverted phase formation disorganize lipid packing and enhance lipid intermixing between the apposed membranes. For the pro-nucleating phospholipase C, vesicle aggregation and fusion are the result of the membrane-destabilizing effects of the hydrolysis product diacylglycerol w34,54x. Diacylglycerol is proposed to induce vesicle fusion through the transient formation of non-lamellar lipid intermediates w55x. Most previous work has focused on the fusion of cholesterol-free lipid vesicles and thus provides no insight into the nucleation of cholesterol from vesicles. Thus it is important to focus here primarily on the effects of cholesterol content on the aggregation and fusion of CH–L vesicles. Bile contains a mixture of glycoproteins, each with a different proportion and distribution of monosaccharides w56x. On the basis of lectin-affinity chromatography, these crude non-mucous glycoproteins have been further purified, and the isolated ConABP promotes cholesterol crystallization in a dose-dependent manner w17,18x. Upon the addition of ConABP, vesicle aggregation begins instantaneously Ž Fig. 1. and may be accompanied by slow vesicle fusion Ž Fig. 2.. ConABP presumably promotes cholesterol crystallization by stimulating the aggregation and fusion of biliary vesicles, possibly through hydrophobic bridging between the different monosaccharide or polysaccharide moieties of the bound glycoproteins. We have used a lipid mixing assay w57x, employing NRDrPE-rRho-PE lipid dequenching and shown lipid mixing of vesicles on treatment with phospholi-

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pase C, ConABP Ž data not shown. and bile salts w58x. This further suggests that vesicular fusion could have taken place. The convincing proof, however, can only come from physical evidence such as obtained by ultra-rapid cryo-TEM. The kinetics of ConABP-induced vesicle aggregation can be predicted by the single parameter massaction model, which assumes an identical aggregation rate constant for each step ŽFig. 1. . The theoretical fits, however, slightly underestimate the early stage of the absorbance increase, suggesting that the aggregation rate constant may decrease with increasing aggregate size. ConABP consists of a mixture of pro-nucleating proteins such as immunoglobulins, a 1-acid glycoprotein, aminopeptidase N, phospholipase C, and fibronectin. The observed absorbance increase is thus a summation of absorbance curves created by each individual pro-nucleating protein. At the same ConABP concentration, the dimerization rate constant correlates with cholesterol content in vesicles. Similarly the aggregation rate constant in phospholipase C-induced vesicle aggregation scales with the cholesterol content w34x. This rate enhancement can be attributed to the effects of cholesterol on the physical properties of lipid membranes. Cholesterol lowers the short range steric repulsion in lecithin bilayers by spreading apart the phospholipid headgroups, and also increases the long-range van der Waals attractions between membranes w59x. Both effects mean that cholesterol-loaded vesicles are less stable to colloidal aggregation than are pure lecithin vesicles. The pro-nucleating ability of CBP in bile has been reported w26x. CBP is a low molecular weight Ž10 kDa., highly acidic anionic peptide fraction isolated from the pigment-lipoprotein complex in bile w37,60,61x. Upon the addition of CBP to a vesicle dispersion, no change in turbidity and fluorescence intensity occurs, indicating that CBP does not affect vesicle stability. Moreover, CBP strongly inhibits the precipitation of calcium carbonate from supersaturated bile w61x. If CBP truly enhances cholesterol crystallization, possible mechanisms include promoting cholesterol crystal growth or effective crystal packaging. Since CBP is often present in the center of cholesterol gallstones, calcium-rich complexes of aggregated CBP can also perhaps serve as nuclei for cholesterol nucleation w62,63x. Similar calcium bind-

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ing proteins found biologically also play an important role in crystal growth and packaging in dentine and bone mineralization, and in the growth of the shells of shellfish and sea urchins w64x. The complete characterization of cholesterol gallstone formation requires a systematic study of the kinetics of each intermediate step. As an initial approach, we have quantitatively examined the kinetics of the proposed early stages of gallstone formation, namely the aggregation and fusion of CH–L vesicles at physiological ConABP concentrations. Considering the gradual nature of cholesterol gallstone formation, the significance of ConABP on the stability of biliary vesicles can be appreciable. Nonetheless, the second-order dimerization rate constant serves as an excellent index of the potency of putative pronucleating proteins. The results of this study indicate that different proteins may have different mechanisms of action in accelerating cholesterol crystallization in bile. We also suggest that the method of quantifying the aggregation and fusion of lipid vesicles, in the presence of physiological concentrations of similar pro-nucleating proteins, can be a comparative measurement of their purported pro-nucleating potency or activity, thus distinguishing in vitro artifacts from physiological relevance. Using a similar but not identical experimental design, de-Bruijn et al. w66x concluded that vesicular disruption may be the predominant mechanism of its nucleation promoting action. In support of our observations, Afdhal et al. w65x have recently used a fluorescent assay in conjunction with light scattering and morphology to characterize the quantity and kinetics of mucin-induced vesicular aggregation and fusion. Finally, the application of this protocol in the study of gallstone formation may afford an opportunity to understand the molecular events of cholesterol crystallization and provide mechanistic insights into the protein–lipid interactions that ultimately lead to cholesterol nucleation in bile.

Acknowledgements We are grateful for the use of Dr. A.M. Lenhoff’s spectrofluorometer, and for the experimental assistance of Dr. H. Park in the preparation and purification of biliary proteins from human bile.

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