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Biochimica et Biophysica Ac

Solubilization, purification and reconstitution of Ca2+-ATPase from bovine

pulmonary artery smooth muscle microsomes by different detergents:

Preservation of native structure and function of the enzyme by DHPC

Amritlal Mandal, Sudip Das, Tapati Chakraborti, Pulak Kar, Biswarup Ghosh, Sajal Chakraborti *

Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India

Received 30 June 2005; received in revised form 21 September 2005; accepted 27 September 2005

Available online 19 October 2005

Abstract

The properties of Ca2+-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with

endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl

ether (C12E8) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca2+-ATPase with

a greater specific activity than solubilization with C12E8 or Triton X-100. DHPC was determined to be superior to C12E8; while that the C12E8 was

determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca2+-ATPase retained the

E1Ca�E1*Ca conformational transition as that observed for native microsomes; whereas the C12E8 and Triton X-100 solubilized preparations did

not fully retain this transition. The coupling of Ca2+ transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was

similar to that of the native micosomes, whereas that the coupling was much lower for the C12E8 and Triton X-100 purified enzyme reconstituted

in liposomes. The specific activity of Ca2+-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and

3-fold greater than that achieved with C12E8 and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in

Ca2+ uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents

solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific

activity. It, therefore, appears that the DHPC purified Ca2+-ATPase retained more organized and native secondary conformation compared to

C12E8 and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light

scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C12E8 and Triton X-100

preparations have to some extent smaller size. These studies suggest that the Ca2+-ATPase solubilized, purified and reconstituted with DHPC is

superior to that obtained with C12E8 and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and

the role of protein– lipid interactions in the function of the membrane-bound enzyme.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Ca2+-ATPase; Endoplasmic reticulum; Bovine pulmonary artery smooth muscle microsome; Liposome; DHPC; C12E8; Triton X-100; DOPC; FCCP

0304-4165/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbagen.2005.09.013

Abbreviations: ER, endoplasmic reticulum; SR, sarcoplasmic reticulam;

DHPC, 1,2-diheptanoyl-sn-phosphatidylcholine; C12E8, poly(oxy-ethylene)8-

lauryl ether; DOPC, dioleoyl-phosphatidylcholine; DTT, D,L-1,4-dithiothreitol;

HBPS, Hank’s buffered physiological saline; MOPS, 3-(N-morpholino)propa-

nesulphonic acid; TBS, tris-buffered saline; EGTA, ethylene glycol bis(2-

aminoethyl ether)-N,N,N V,N V-tetraacetic acid; PMSF, phenylmethylsulfonyl

fluoride; DFBAPTA, 5-5V difluoro derivative of 1,2-bis(o-aminophenox-

y)ethane-N,N,N V,N V,tetraacetic acid; CD, circular dichroism; FCCP, carbonyl-

cyanide-p-trifluoromethoxyphenylhydrazone

* Corresponding author. Tel.: +91 9831228224; fax: +91 33 25828282.

E-mail address: saj_chakra@rediffmail.com (S. Chakraborti).

1. Introduction

Changes in the intracellular Ca2+ concentration [(Ca2+)i]

regulate the contraction–relaxation cycle of smooth muscle [1].

Plasma membrane Ca2+-ATPase and endoplasmic reticular

(ER) Ca2+-ATPase play important roles in counteracting an

increase in [(Ca2+)i] in smooth muscle caused by different

agonists [1]. Recent research provided evidence suggesting that

the ER Ca2+-ATPase is one of the intrinsic Ca2+ transport

membrane protein, which is involved in Ca2+ uptake in the

suborganelle [2]. Three genes of ATP2A1, ATP2A2 and

ATP2A3 encode three types of SERCA protein. SERCA 1,

ta 1760 (2006) 20 – 31

http://www

A. Mandal et al. / Biochimica et Biophysica Acta 1760 (2006) 20–31 21

SERCA 2 and SERCA 3 and alternative splicing of the three

primary transcripts gives rise to at least nine SERCA isoforms

with different functions [3–7]. SERCA2b isoform having mol

mass of ¨115 kDa [8] is the principal form of the Ca2+-ATPase

in smooth muscles and appears to represent a generic ‘‘ER’’

form of the Ca2+-ATPase [9].

Ca2+-ATPase couples the transport of 2 mol of Ca2+ across

the ER membrane with the hydrolysis of 1 mol of ATP and a

conformational change accompanies the reaction cycle [10–

12]. To elucidate fully the structure and function of Ca2+-

ATPase, and particularly the role of lipid�protein interactions

in the conformational changes that accompany ion transloca-

tion, it is necessary to isolate the protein and separate it from

other membrane constituents. The most successful approach so

far involves the use of detergents for solubilization [12–19]

and for reconstitution into defined lipids [20–29]. A major

challenge in the solubilization and reconstitution of membrane

proteins is the choice of a detergent that preserves the native

protein structure and biological function. Nonionic detergents

such as C12E8 and Triton X-100 have been employed for the

solubilization and purification of Ca2+-ATPase from sarcoplas-

mic reticulum (SR) [12,13,16,17]. These procedures have

produced varied results in terms of active enzyme yield,

specific activity and formation of phosphorylated enzyme.

Reconstitution of membrane proteins into liposomes provides a

powerful tool in structural as well as functional areas of

membrane protein research [24,30]. It has been shown that in

some systems, for example, sarcoplasmic reticulum, the nature

of the detergent used for reconstitution is a key factor in

determining the properties of the reconstituted system [30]. In

some membrane preparations, C12E8 and Triton X-100 have

emerged suitable detergents for optimizing the recovery of

membrane bound enzymes [18,24,29–35]. To the best of our

knowledge, reconstitution of pulmonary artery smooth muscle

microsomal (enriched with ER) Ca2+-ATPase into defined

lipids has not been performed with any study based on these

detergents. A short chain phospholipid detergent, DHPC has

been described by Kessi et al. [30] and Shivanna and Rowe

[35] for solubilizing membrane proteins [35]. They showed

that DHPC is a better solubilizing agent for a variety of

membrane proteins, while retaining their maximal native

activity.

We have applied DHPC for solubilization, purification and

reconstitution of Ca2+-ATPase from bovine pulmonary artery

smooth muscle microsomes. In this present communication, we

compared the effectiveness in solubilizing and purifying Ca2+-

ATPase using DHPC with that prepared by using C12E8 and

Triton X-100 by studying simultaneous measurements of Ca2+-

ATPase activity and ATP dependent Ca2+ uptake. Furthermore,

detailed comparison of the DHPC, C12E8 and Triton X-100

purified preparations was made by tryptophan fluorescence and

also by circular dichroism spectroscopy. Reconstitution of

Ca2+-ATPase into dioleoylphosphatidylcholine (DOPC) using

DHPC, C12E8 and Triton X-100 were performed and the

resulting activities were compared. Our results demonstrate that

the DHPC purified and reconstituted Ca2+-ATPase into DOPC

gives a higher activity compared to that of C12E8 and Triton X-

100 purified preparations. To our knowledge, this is the first

report for purification and reconstitution of Ca2+-ATPase of

bovine pulmonary artery smooth muscle microsomal Ca2+-

ATPase into DOPC using the short-chain phospholipid

detergent DHPC.

2. Materials and methods

2.1. Materials

DHPC and DOPC were obtained from Avanti Polar Lipids. C12E8, Triton

X-100, Tris-ATP, PMSF, sucrose, DTT, CaCl2, rotenone, NADPH, AMP, p-

nitrophenylphosphate, p-nitrophenol, cytochrome c, EGTA, Reactive Red 120-

agarose, FCCP and horseradish peroxidase conjugated secondary antibody

were purchased from Sigma Chemical Co., St. Louis, MO, USA. Sephadex G-

100 was procured from Pharmacia, Upsala, Sweden. Bio-Beads SM-2 (average

pore diameter 90A) was purchased from Bio-Rad, California, USA. DFBAPTA

and Fura Red, tetraammonium salt were the products of Molecular Probes, OR,

USA. BCA protein assay kit was obtained from Pierce, Rockford, Illinois,

USA. Control SERCA2b and its polyclonal antibody were supplied by Dr.

Jonathan Lytton, University of Calgary, Calgary, Canada. All other chemicals

used were of analytical grade.

2.2. Methods

2.2.1. Microsome preparationBovine pulmonary artery collected from slaughterhouse was washed

several times with Hank’s buffered physiological saline (HBPS) (137 mM

NaCl, 1.1 mM MgCl2, 4.69 mM KCl, 3.7 mM HEPES buffer, 11 mM

Glucose, pH 7.4) and kept at 4 -C. The washed pulmonary artery was used

for further processing within 1 h after collection. The intimal and serosal

(external) layers were removed and the tunica media, i.e., the smooth muscle

tissue was collected and used for the present studies [36]. The smooth

muscle tissue was characterized by histology with eosin–hematoxylin stain

in a light microscope [37]. Microsomes enriched with the ER from the

smooth muscle tissue were prepared by following the procedure described by

Chakraborti et al. [38].

2.2.2. Characterization of microsomes by the assay of marker enzymesRotenone-insensitive cytochrome c reductase activity was measured as

previously described [39]. Cytochrome c oxidase was assayed by the procedure

of Wharton and Tzagoloff [40]. Acid phosphatase activity was determined at

pH 5.5 using p-nitrophenylphosphate as the substrate [41]. Release of Pi from

5VAMP, an index of 5V-nucleotidase activity, was determined by the method of

Chen et al. [42].

2.2.3. Determination of proteinProtein concentration was estimated by the Pierce Micro BCA protein assay

kit (Pierce, Rockford, Illinois, USA) by following the procedure of Smith et al.

[43] using bovine serum albumin (BSA) as the standard.

2.2.4. Solubilization and Purification of Ca2+-ATPase from the

smooth muscle microsomeAll buffers used contain 1 mM PMSF, 1 AM pepstatin and 10 AM

leupeptin unless stated otherwise. All purification steps were performed at

4 -C.

2.2.4.1. Solubilization of microsomal vesicles. The freshly prepared

microsomes were solubilized on ice with detergents (DHPC, C12E8 and Triton

X-100) as described by Kessi et al. [30] with some modifications. Briefly, 200

mM stock solutions of detergents in solubilization buffer (50 mM Tris–HCl, 1

M KCl, 20% glycerol, 1 mM DTT, pH 7.5) were added to freshly prepared

microsomes to the desired final detergent concentration at a constant

microsomal vesicle protein concentration of 6 mg/ml in the same buffer.

Overall, the detergent /protein ratio appears to be 1:1 for Triton X-100; 1:0.9 for

A. Mandal et al. / Biochimica et Biophysica Acta 1760 (2006) 20–3122

C12E8; and 1.5:1 for DHPC, which have been determined by us to be optimum

for enzyme solubilization and optimum activity.

Unless stated otherwise, microsomal vesicles were solubilized at 0 -C by

drop-wise addition of stock detergent solution with vortex-mixing for 60 s.

Solubilization continued on ice for 30 min with intermittent mixing. Final

concentrations of each of the detergents were varied between 0 and 50 mM.

The solubilization mixture was centrifuged at 105,000�g in an Ultracentrifuge

(Beckman, USA) at 4 -C for 25 min. The enzyme activity was determined in

both the supernatants and the pellets [30].

2.2.4.2. Reactive Red 120-agarose chromatography. Microsomes

were solubilized with the buffer A (50 mM Tris–HCl buffer, pH 7.5, 0.5

mM MgCl2, 2 mM DTT, 20% glycerol, 0.5 mM EGTA and 50 AM CaCl2)

containing the respective detergents (DHPC or C12E8 or Triton X-100). During

the solubilization, the optimal detergent concentrations were used of 20 mM

DHPC or 10 mM C12E8 or 10 mM Triton X-100 on the basis of the

solubilization data obtained from the present study. Protein concentration was

kept at 4.0–4.5 mg/ml and loaded on to a Reactive Red 120-agarose column

(1.5�10 cm) equilibrated with buffer A containing either of the detergents at

a flow rate of 6 ml/h [44]. Proteins were next eluted with step gradient of

NaCl (0.5 and 2 M) in buffer A containing the final concentration of the

detergents, with the flow rate of 12 ml/h. Proteins were monitored

spectrophotometrically at 280 nm and assayed for Ca2+-ATPase activity. The

enzyme was eluted with 0.5 M NaCl. The fractions with maximum Ca2+-

ATPase activity from several runs were pooled and concentrated by Amicon

ultrafiltration cell with YM10 membrane (Mr cut off =10 kDa). The

concentrated pool was adjusted to contain 100 mM KCl and 400 Ag/ml

phosphatidylcholine.

2.2.4.3. Sephadex G-100 chromatography. The concentrated material

(2 ml) obtained after Reactive Red 120-agarose chromatography was

loaded on a Sephadex G-100 column (2.3�92.5 cm) equilibrated with

buffer A containing 100 mM KCl with the final concentration of detergents

20 mM DHPC, 10 mM C12E8 and 10 mM Triton X-100 and eluted at a

flow rate of 9 ml/h. Proteins were monitored spectrophotometrically at 280

nm. All fractions were assayed for Ca2+-ATPase activity and supplemented

with 400 Ag/ml phosphatidylcholine. Fractions with highest Ca2+-ATPase

activity from several runs were pooled and concentrated by Amicon

ultrafiltration cell with YM10 membrane (Mr cut off=10 kDa). The pooled

concentrated fractions with Ca2+-ATPase activity were stored under liquid

nitrogen.

2.2.5. SDS-polyacrylamide gel electrophoresisSodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was

performed according to the procedure of Laemmli [45] using a minigel Protean

II apparatus (Bio-Rad, USA). The protein bands were visualized by silver

staining methods [46].

2.2.6. Identification of purified Ca2+-ATPase by Western immunoblotCa2+-ATPase purified from the smooth muscle microsomal suspension was

identified by western immunoblot, performed by following the method of

Towbin et al. [47] using the polyclonal anti-SERCA2b as the primary antibody

[48]. Horseradish peroxidase conjugated goat anti-rabbit IgG was used as the

secondary antibody. The nitrocellulose membrane was developed with 4-

chloro-1-naphthol (0.2 mM).

2.2.7. Reconstitution of DHPC, C12E8 and Triton X-100 purified

Ca2+-ATPase into DOPCCa2+-ATPase was reconstituted into the exogenous lipid DOPC by using a

combination of methods from the literature [22,24] with some modifications.

Briefly, 50 mg of DOPC in 2 ml of buffer (50 mM MOPS, 0.25 M sucrose, 1

M KCl, 1 mM MgCl2, 0.1 mM CaCl2, 1 mM DTT, 0.025% sodium azide; pH

7.5) containing either of the detergents to give a final lipid : detergent ratio of

1:1.5 (w/w) [35] was vortex-mixed vigorously for approximately 60 s to

disperse the lipid and left at room temperature (23 -C) for 90 min. The

suspension was clarified by sonication for 1�2 min with a Soni Prep model

150 sonifier/cell disrupter. Purified Ca2+-ATPase was solubilized at a ratio of

0.6:1 (w/w; detergent : protein) [35] by vortexing for 60 s and left at room

temperature (23 -C) for 10 min followed by further incubation on ice for an

additional 90 min.

The solubilized Ca2+-ATPase was centrifuged at low speed to remove

insoluble material and aggregates. The clear sample was then mixed with

pre-solubilized exogenous lipid to a desired molar ratio of lipid to Ca2+-

ATPase (1500:1) [35], and vortex-mixed for 30 s and left at room

temperature for 15 min. This mixture was incubated at 4 -C for 90 min.

At the end of the incubation, the detergent was removed by adsorption on

Bio-Beads SM-2 in two batches of 700 mg for 1 h at 4 -C. The reconstituted

vesicles were separated from the Bio-Beads by low-speed centrifugation. The

reconstituted proteoliposomes were purified on a discontinuous sucrose

gradient.

2.2.8. Assay of Ca2+-ATPase activityCa2+-ATPase activity was determined colorimetrically by measuring Ca2+

dependent release of Pi [2].

2.2.9. Simultaneous measurement of Ca2+ uptake and ATP hydrolysisSimultaneous measurements of Ca2+ uptake and ATP hydrolysis were

performed with Fura-Red absorbance continuous spectrophotometric assay as

described by Karon et al. [49]. All assays were performed at 790 nM. Ionized

free calcium was determined from the equation described by Karon et al. [49]

with ¨50 Ag of microsomal protein or ¨7 Ag of reconstituted Ca2+-ATPase

preparations with the detergents.

2.2.10. Characterization of proteoliposomesThe detergent purified Ca2+-ATPase proteoliposomes were treated with

protonophore FCCP (0.25 AM) as described by Levy et al. [50] and the Ca2+

uptake studies were performed in presence and absence of the protonophore,

FCCP by following the method of Karon et al. [49].

2.2.11. Fluorescence spectroscopyFluorescence measurements were made by diluting an aliquot of native

microsomes (¨50 Ag/ml of protein) or purified enzyme with the detergents (¨7

Ag protein) into 2.5 ml of buffer {50 mM MOPS, 2 mM EGTA, pH 7.5}. The

intrinsic fluorescence of tryptophan was measured with a Perkin Elmer

spectrofluorimeter model LS50B. Fluorescence spectra were measured with

kex=295 nm and kem=340 nm and band widths of 4 and 16 nm for excitation

and emission, respectively. Fluorescence emission spectra of the E1, E1*Ca and

E.Mg states of Ca2+-ATPase in native microsomes and in the purified enzyme

preparations were recorded at room temperature (23 -C). Ca2+ and Mg2+

transitions were induced by adding aliquots of 1 M of CaCl2 and 5 M of MgCl2stock solutions to give final concentrations of 1 mM CaCl2 and 10 mM MgCl2,

respectively. The sequential additions were followed at a constant emission

wavelength of 340 nm.

2.2.12. Circular dichroismCD measurements were made at room temperature with a Jasco J600

spectropolarimeter with a band width of 2 nm and a scan speed of 2 nm/min.

Each sample was scanned four times; the scans were automatically averaged.

The possible effects of light scattering in the CD measurements were

minimized by using a CD cell with a narrow path length (0.1 cm) and by

using identical concentrations of protein and other experimental parameters for

each sample in these comparative studies.

2.2.13. Quasi-elastic light scatteringQuasi-elastic light scattering was performed with the Nicomp particle sizer

from the Pacific Scientific (Silver Spring, MD, USA) with temperature

controlled Peltier heating and cooling as described previously [51] to determine

the vesicle size of the native microsomes and the other reconstituted

proteoliposomes.

2.2.14. Statistical analysisData were analyzed by unpaired t test and analysis of variance,

followed by the test of least significant differences for comparison

Table 1

Specific activities of different marker enzymes at different steps in the preparation of microsomes from bovine pulmonary artery smooth muscle tissue

Fraction Cytochrome c

oxidase

Acid

phosphatase

Rotenone insensitive

NADPH-cytochrome

c reductase

5V-Nucleotidase

600–15,000 g pellet 3.34T0.21 3.40T0.23 0.16T0.02 0.18T0.0315,000–100,000 g pellet 0.24T0.04 (7) 0.33T0.05 (10) 1.68T0.12 (1050) 2.25T0.16 (1250)

Microsomes 0.09T0.01 (3) 0.13T0.02 (4) 2.20T0.16 (1375) 0.07T0.01 (39)

Plasma membrane 0.08T0.01 (2) 0.14T0.02 (4) 0.10T0.01 (63) 3.12T0.20 (1733)

Cytochrome c oxidase activity is expressed as Amol of cytochrome c utilized/30 min/mg protein, acid phosphatase activity as Amol of p-nitrophenol/30 min/mg

protein, NADPH cytochrome c reductase activity as reduction of absorbance of cytochrome c at 550 nm/30 min/mg protein and 5V-nucleotidase activity as Amol of

Pi/30 min/mg protein. Results are meanTSE (n =4). Values in the parentheses indicate the activity as percentage of that of the 600–15,000 g pellet (values of 600–

15,000 g pellet are set at 100%).

A. Mandal et al. / Biochimica et Biophysica Acta 1760 (2006) 20–31 23

within and between the groups. P <0.05 was considered as significant

[52].

3. Results and discussion

3.1. Characterization of microsomes

We characterized the microsomal fraction at different steps

in the preparation process by measuring the activities of

cytochrome c oxidase (a mitochondrial marker) [53], acid

phosphatase (a lysosomal marker) [54], rotenone insensitive

NADPH-cytochrome c reductase (a microsomal marker) [55]

and 5V-nucleotidase (a plasma membrane marker) [54].

Microsomal fraction showed, respectively, 37-fold decrease

in sp. activity of cytochrome c oxidase and 26-fold decrease in

the sp. activity of acid phosphatase activity compared with

600–15,000�g pellet. It also showed 45-fold decrease in sp.

activity of 5V-nucleotidase, compared with plasma membrane

fraction. Furthermore, the microsomal fraction showed, respec-

tively, 14-fold and 22-fold increase in the sp. activity of

rotenone insensitive NADPH-cytochrome c reductase, com-

pared with the 600–15,000�g pellet and the plasma membrane

fraction (Table 1).

Fig. 1. Ca2+-ATPase activity of the supernatant obtained after solubilization of micro

100 and expressed as percentage of starting microsomal Ca2+-ATPase activity. Total

100 at various detergent concentration as percentage of starting microsomal Ca2+-A

3.2. Comparison of solubilization of microsomes by DHPC,

C12E8 and Triton X-100

Solubilization of membrane proteins by suitable detergents

with preservation of native structure and function is important

for its detailed biochemical and biophysical studies. The

purpose of our present research was to investigate the efficacy

of the detergents: Triton X-100, C12E8 and the short chain

phospholipid DHPC to solubilize the microsomes isolated from

bovine pulmonary artery smooth muscle tissue for structural

and functional studies of Ca2+-ATPase. DHPC is structurally a

phospholipid but its short fatty acid chains of seven carbon

atoms endow it with detergent like properties [30]. It forms

micelles [56] rather than bilayers when dispersed in water with

a relatively high c.m.c. of 1.4 mM and shows a broad size

distribution dependent on its concentration and on the NaCl

concentration of the suspension [57]. DHPC has the advantage

that it is available in pure form, it has no net charge, it is stable

over a wide pH range of 4–10, and it does not interfere with

spectrophotometric measurements [30,35].

Microsomes isolated from bovine pulmonary artery smooth

muscle tissue were solubilized with DHPC, C12E8 and Triton

X-100 at several concentrations, each ranging from 0 to 50

somes with several concentrations (0–50 mM) of DHPC, C12E8 and Triton X-

activity in the supernatant: ( ), DHPC; ( ), C12E8; ( ), Triton X-

TPase activity.

Fig. 2. Ca2+-ATPase activity of the pellets obtained after solubilization of microsomes with several concentrations (0–50 mM) of DHPC, C12E8 and Triton X-100

and expressed as percentage of starting microsomal Ca2+-ATPase activity. Total activity in the pellets: ( ), DHPC; ( ), C12E8; ( ), Triton X-100 at

various detergent concentration as percentage of starting microsomal Ca2+-ATPase activity.

A. Mandal et al. / Biochimica et Biophysica Acta 1760 (2006) 20–3124

mM. To compare the microsome-solubilizing ability of these

detergents and Ca2+-ATPase activity, a percentage of the

starting amount of native microsomes was determined in the

supernatant of the solubilized membranes after pelleting. The

supernatant and the resuspended pelleted materials were treated

with Bio-Beads SM-2 to remove the detergents present prior to

being assayed for the enzyme activity. Phosphatidylcholine

(400 Ag/ml) was added to the resulting sample preparation

finally in order to avoid aggregation and denaturation of the

enzyme in the assay mixture.

Fig. 1 shows the Ca2+-ATPase activity recovered from the

supernatant of the respective detergent-treated samples. In Fig.

1, it appears that significantly more activity is observed upon

Fig. 3. Specific activity of Ca2+-ATPase in supernatant obtained after solubilization o

( ) and Triton X-100 ( ).

solubilization by the DHPC over a broad range of DHPC

concentration than by the C12E8 and Triton X-100. Fig. 2

shows the activity associated with the pelleted fractions of each

detergent-treated sample.

The sp. activities of these preparations in the soluble

fractions are shown in Fig. 3 as a function of detergent

concentration. At very low detergent concentration (5 mM), the

C12E8 and Triton X-100 solubilized enzyme had a higher sp.

activity than that solubilized by DHPC. Above 5 mM

detergent, however, the sp. activity of the DHPC solubilized

Ca2+-ATPase was significantly greater than that solubilized by

any of the other two detergents. Additionally, the C12E8 and

Triton X-100 solubilized enzyme rapidly lost their activities

f microsomes with several concentrations (0–50 mM) of DHPC ( ), C12E8

Fig. 4. (A) 7.5% SDS-PAGE profile of Ca2+-ATPase purification with DHPC.

Lane 1, microsome; lane 2, DHPC solubilized microsome; lane 3, Reactive Red

120-agarose flow through; lane 4, 0.5 M NaCl eluate of Reactive Red 120-

agarose; lane 5, Sephadex G-100 eluate; lane 6, standard mol. wt. markers. (B)

7.5% SDS-PAGE profile of Ca2+-ATPase purification with C12E8. Lane 1,

microsome; lane 2, C12E8 solubilized microsome; lane 3, Reactive Red 120-

agarose flow through; lane 4, 0.5 M NaCl eluate of Reactive Red 120-agarose;

lane 5, Sephadex G-100 eluate; lane 6, standard mol. wt. markers. (C) 7.5%

SDS-PAGE profile of Ca2+-ATPase purification with Triton X-100. Lane 1,

microsome; lane 2, Triton X-100 solubilized microsome; lane 3, Reactive Red

120-agarose flow through; lane 4, 0.5 M NaCl eluate of Reactive Red 120-

agarose; lane 5, Sephadex G-100 eluate; lane 6, standard mol. wt. markers. (D)

Western blot profile of purified Ca2+-ATPase. Lane 1, control; lane 2, DHPC

purified Ca2+-ATPase; lane 3, C12E8 purified Ca2+-ATPase; lane 4, Triton X-

100 purified Ca2+-ATPase; lane 5, standard mol. wt. markers.

A. Mandal et al. / Biochimica et Biophysica Acta 1760 (2006) 20–31 25

with increasing detergent concentration and the optimum range

for these detergents is, therefore, very narrow (Fig. 3). In

contrast, the optimum detergent concentration range for DHPC

is relatively broad (Fig. 3).

The above data for DHPC and C12E8 support the findings of

Shivanna and Rowe [35]. It appears that the maximum

solubilization as well as the Ca2+-ATPase activity of the

microsomes were obtained with 20 mM DHPC, 10 mM C12E8

and 10 mM Triton X-100 (Figs. 1–3).

Table 2

Purification of Ca2+-ATPase from bovine pulmonary artery smooth muscle microso

Purification steps Detergents

used

Total protein

(mg)

Microsomes – 507

Detergent solubilized microsomes DHPC 53.88

C12E8 47.55

Triton X-100 42.46

Reactive Red 120-agarose

chromatography

DHPC 3.49

C12E8 2.96

Triton X-100 2.63

Sephadex G-100 chromatography DHPC 1.382

C12E8 1.320

Triton X-100 1.269

a Unit=Amol Pi/min at 37 -C.

3.3. Purification of detergent-solubilized Ca2+-ATPase

The detergent-solubilized preparations were purified by

Reactive Red 120-agarose chromatography followed by

Sephadex G-100 gel filtration. The optimum detergent con-

centrations for solubilization of the microsomes were chosen as

20 mM for DHPC, 10 mM for C12E8 and 10 mM for Triton X-

100 (Figs. 1–3). The Ca2+-ATPase activity-containing frac-

tions of the Reactive Red 120-agarose column obtained with

0.5 M NaCl were pulled, concentrated and loaded on a

Sephadex G-100 gel filtration column. The ATPase activity

containing fractions of the Sephadex G-100 chromatography

were eluted between 100 and 160 ml. The Sephadex G-100

purified Ca2+-ATPase showed a single band ¨115 kDa (Fig.

4A–C). Furthermore, the immunoblot profile of the purified

Ca2+-ATPase with the detergents indicate that the enzyme

migrates with a single band at an apparent molecular mass of

¨115 kDa (Fig. 4D). The sp. activity of the purified enzyme

from the DHPC solubilization was significantly greater than

that obtained from the preparation solubilized with C12E8 and

Triton X-100 (Table 2).

The results of the purification scheme are summarized in

Table 2. The sp. activity of the DHPC purified Ca2+-ATPase is

1.9 times greater than that obtained from C12E8 and 2.2 times

greater than that obtained with Triton X-100 solubilized

preparations. These data demonstrate that the DHPC solubili-

zation results in a significantly greater quantity of high sp.

activity of Ca2+-ATPase than the other two detergents.

The present comparative studies of the result from

solubilization and purification of microsomal Ca2+-ATPase

demonstrate the inherent variations exhibited by various

detergent groups when interacting with the microsomes. The

three detergents employed in this study: DHPC, C12E8 and

Triton X-100 represent well-differentiated groups of soluble

amphiphiles, i.e., the phospholipid and non-ionic detergents.

The DHPC solubilization consistently provides greater yields,

with specific activities significantly greater over a wide

concentration range of the detergent than that achieved either

by C12E8 or the Triton X-100 (Table 2 and Fig. 3). The yield of

total units of activity at the optimal solubilization concentration

for DHPC is 32% and 56% higher than that obtained with

C12E8 and Triton X-100, respectively (Fig. 1). Combining the

mes

Total activity

(Unitsa)

Specific activity

(Units/mg)

Fold

purification

Recovery

%

45.60 0.09 – 100

30.55 0.567 6 67

20.97 0.441 5 46

10.69 0.252 3 23

18.56 5.318 59 41

9.07 3.064 34 20

7.31 2.779 31 16

12.37 8.951 99 27

6.21 4.704 52 14

5.17 4.074 45 11

Table 3

Reconstitution of the purified Ca2+-ATPase from bovine pulmonary artery

smooth muscle microsomes into liposomes

Detergents

used

Specific activity

of the purified

Ca2+-ATPase

(Amol Pi/min/

mg protein)

Specific activity

of the purified

Ca2+-ATPase in

reconstituted

vesicles

(Amol Pi/min/

mg protein)

% change of

specific activity

(reconstituted

vesicle with

purified enzyme)

DHPC 8.951T0.76 13.758T0.81a +54

C12E8 4.704T0.37 5.612T0.42 +19

Triton X-100 4.074T0.33 4.518T0.38 +11

a P <0.01 compared to purified Ca2+-ATPase.

Table 5

Ca2+-uptake and Ca2+-ATPase activities of microsomes and purified recon

stituted enzyme

Sample Simultaneous

Ca2+-uptake

(Amol Ca2+/min/

mg protein)

Simultaneous

Ca2+-ATPase

activity

(Amol ATP/min/

mg protein)

Coupling ratio

(Amol Ca2+/Amol

ATP/min/mg

protein)

Microsomes 2.42T0.1 1.25T0.05 1.94

DOPC-DHPC

reconstituted

enzyme

3.73T0.2 2.05T0.12 1.82

DOPC-C12E8

reconstituted

enzyme

3.11T0.2 1.82T0.06 1.71

DOPC-Triton

X-100

reconstituted

enzyme

2.68T0.1 1.65T0.05 1.62

Ca2+-uptake and Ca2+-ATPase activities were measured simultaneously as

described in Materials and methods at 790 nM free ionized Ca2+ at 25 -C. The

coupling ratio is the ratio of Ca2+-uptake to Ca2+-ATPase activity (measured by

the simultaneous assay in both cases). Values given are meanTSE (n =4).

Table 6

Ca2+-uptake of Ca2+-ATPase reconstituted proteoliposomes in absence and

presence of protonophore FCCP

Sample Ca2+-uptake

(Amol Ca2+/min/mg protein)

% change of Ca2+

uptake in the

reconstituted

FCCP treated

proteoliposomes

compared to

FCCP untreated

proteoliposome

�FCCP +FCCP (0.25 AM)

DOPC-DHPC

reconstituted

3.73T0.2 7.83T0.6a +110

A. Mandal et al. / Biochimica et Biophysica Acta 1760 (2006) 20–3126

relative yields through solubilization and affinity purification

followed by final purification by gel filtration, the total yield of

activity units with DHPC was 99% and 139% more than that

obtained with C12E8 and Triton X-100, respectively (Table 2).

3.4. Reconstitution of purified Ca2+-ATPase into DOPC

The DHPC, C12E8 and Triton X-100 purified Ca2+-ATPase

were reconstituted into DOPC with the procedure described in

Materials and methods. DOPC was chosen because it has

previously been shown to maximize the recovery of activity

upon reconstitution [22,58]. To maximize the replacement of

endogenous lipid, a high lipid-to-Ca2+-ATPase ratio of 1500:1

was used. The sp. activity of Ca2+-ATPase reconstituted into

DOPC vesicles with DHPC was 2.5-fold and 3-fold greater

than that achieved with C12E8 and Triton X-100, respectively

(Table 3). The sp. activity recovered for the DHPC-reconsti-

tuted vesicles was 54% higher than that DHPC purified

enzyme, whereas the sp. activity recovered for the C12E8 and

Triton X-100 reconstituted vesicles were, respectively, 19%

and 11% higher than the starting activity for the C12E8 and

Triton X-100 purified enzyme (Table 3).

The size distributions of the reconstituted liposomes were

measured by quasi-elastic light scattering. The results are

shown in Table 4 along with the data for microsomal vesicles

and the reconstituted enzyme preparations from each detergent.

The results show a particle size of 187 nm for the most active

reconstituted vesicles with DHPC. The results presented in

Table 4 shows that each reconstituted vesicles have different

diameter, with DHPC>C12E8>Triton X-100.

The Ca2+ transport rate and the ATPase activity of the

reconstituted preparations were measured by the simultaneous

assay (Table 5). The coupling efficiencies of the DHPC

Table 4

Quasi-elastic light scattering particle size analysis of native microsomal

vesicles and DOPC reconstituted liposomal preparations of Ca2+-ATPase

Sample Vesicle size (nm)

Native microsomal vesicle 190T17

DOPC-DHPC reconstituted enzyme 187T16

DOPC-C12E8 reconstituted enzyme 139T12

DOPC-Triton X-100 reconstituted enzyme 134T11

Each result is the meanTSE (n =4). Results obtained are from the best fitting

volume weight Nicomp distribution analysis.

enzyme

DOPC-C12E8

reconstituted

enzyme

3.11T0.2 5.59T0.5b +80

DOPC-Triton

X-100

reconstituted

enzyme

2.68T0.1 4.52T0.4b +69

Ca2+-uptake was measured as described in Materials and methods at 790 nM

free ionized Ca2+ at 25 -C. Values given are meanTS.E. (n =4).a P <0.001 compared to FCCP untreated proteoliposomes.b P <0.01 compared to FCCP untreated proteoliposomes.

-

reconstituted vesicles appeared to be close to that of the native

microsomal vesicles; while that of the C12E8 and Triton X-100

purified vesicles were somewhat less than that of the native

microsomes.

It has been established that Ca2+-ATPase are in fact Ca2+/H+

counter transporter [50]. In the Ca2+-ATPase proteoliposomes,

a relatively low Ca2+ accumulation is observed in the absence

of FCCP (Table 6). In contrast, adding FCCP led to a marked

increase in Ca2+ accumulation (Table 6), which discriminates

between efficacy of reconstitution and residual permeability of

different reconstitutions.

A. Mandal et al. / Biochimica et Biophysica Acta 1760 (2006) 20–31 27

Our results indicate that the method with DHPC resulted in

reconstituted vesicles with a 1.5-fold greater activity than

the purified enzyme and for the C12E8 purified ATPase

preparation it is 1.2-fold only (Table 3). In case of Triton X-

100 purified ATPase, the reconstituted vesicles show 1.1-

fold increased ATPase activity (Table 3). In addition, the

range of detergent concentration over which enzyme with

high sp. activity was recovered in the solubilization step is

considerably broader for DHPC than for C12E8 and Triton

X-100 (Fig. 3). This is in accordance with the observation

of Shivanna and Rowe [35] who studied the efficacy of

DHPC over C12E8 for purification and reconstitution studies

of sarcoplasmic reticular Ca2+-ATPase isolated from rabbit

skeletal muscle. These factors alone make the DHPC

Fig. 5. (A) Emission fluorescence spectra of the E1Ca to E1*Ca transition for native m

kem=340 nm, and bandwidths of 4 and 16 nm, respectively, for excitation and emi

microsomal vesicles and to purified Ca2+-ATPase preparations with 20 mM DHPC,

Ca2+ are 10 mM and 1 mM, respectively.

preparation superior to the C12E8 and Triton X-100

preparation for obtaining material for biophysical and

biochemical studies such as calorimetry, NMR and ion

transport studies.

3.5. Conformational characterization of purified Ca2+ -ATPase

Tryptophan fluorescence was used to determine the extent to

which the native conformational properties that have been

demonstrated in native microsomal vesicles are preserved in

the purified preparations from the three detergents of this study.

Many studies have shown that changes in tryptophan fluores-

cence parallel Ca2+ binding to the ATPase. Binding of Ca2+

ions to the high affinity Ca2+ binding site of Ca2+-ATPase

icrosomal vesicles. Fluorescence spectra were measured with kex=295 nm and

ssion. (B) Fluorescence changes on the addition of MgCl2 and CaCl2 to native

10 mM C12E8 and 10 mM Triton X-100. The final concentrations of Mg2+ and

A. Mandal et al. / Biochimica et Biophysica Acta 1760 (2006) 20–3128

results in an increase in the intrinsic tryptophan fluorescence

intensity [59,60]. The change in tryptophan fluorescence was

suggested to be indicative of the E1Ca to E1*Ca transition of

the protein [59–62].

Fig. 5A shows about 9% increase in the fluorescence

intensity of native microsomal Ca2+-ATPase in the presence of

Ca2+. This is in accordance with the observation of Dupont

[59] who studied sarcoplasmic reticular Ca2+-ATPase. The

effect of Mg2+ and Ca2+ binding on the conformational

transition was monitored by following the fluorescence

change at constant wavelength on the sequential addition of

Mg2+ and Ca2+. Fig. 5B shows the successive response of the

three purified Ca2+-ATPase preparations to the addition of

Mg2+ and Ca2+ compared with native microsomes. Ca2+

binding to the DHPC-solubilized and purified Ca2+-ATPase

resulted in a net 8% increase in tryptophan fluorescence,

which is in accordance with that observed for native

microsomes. In contrast, the enzyme from the C12E8 and

Triton X-100 purified preparation showed 3% and 2% Ca2+-

induced conformational transition as compared to that

obtained for native microsomes (Fig. 5B). The microsomes

showed typical Mg2+ and additive Ca2+-induced transitions

when MgCl2 was added to a final concentration of 10 mM

followed by CaCl2 to a final concentration of 1 mM. The

DHPC, C12E8 and Triton X-100 purified enzyme did not

exhibit the Mg2+ induced change (Fig. 5B). Taken together,

these results show that DHPC purified enzyme retained more

native characteristics than that of the other detergent purified

preparations. This is consistent with the low coupling ratio for

the C12E8 and Triton X-100 preparations compared with

DHPC preparation (Table 5). The reason for this difference in

the properties is probably related to the mechanism of

Fig. 6. CD spectra of DHPC ( ), C12E8 ( ) and Triton X-100 (—) purified Ca2

averages of four accumulations. The purified samples contained 20 mM DHPC, 10

solubilization of the detergents. In the DHPC preparation, it

seems conceivable that the immediate surroundings of the

protein might retain more endogenous lipids than that

obtained after treatment with C12E8 or Triton X-100 even

though all the preparations have approximately the same lipid-

to-protein ratio [30]. This is in accordance with the

observation of de Foresta et al. [63] who suggested, for

example, that C12E8 directly interact with the protein,

replacing lipids in contact with the protein, so that the

C12E8 purified Ca2+-ATPase retains significant C12E8 in the

interfacial region of the protein. The relatively low activities

of C12E8 and Triton-X-100 purified enzyme could be due to

the fact that both the detergents remain bound with the

enzyme even after Bio Beads SM-2 adsorption.

The CD spectra of DHPC, C12E8 and Triton X-100 purified

preparations were measured to detect any secondary structural

changes. Fig. 6 shows the CD spectra of the DHPC, C12E8 and

Triton X-100 solubilized and purified Ca2+-ATPase prepara-

tions. The CD spectra shows a positive single peak at ¨192 nm

and a double lobed negative at ¨208 nm and a minimum at

¨223 nm, which is actually the signature of the high a-helix

content of the purified protein. None of the spectra showed any

significant effect of added Ca2+ (data not shown). The negative

ellipticity of the purified enzyme preparation increases from

DHPC to C12E8 to Triton X-100 (DHPC<C12E8<Triton X-

100). These results together with the sp. activity of the

respective preparations indicate that the increased negative

ellipticity at 223 nm is well correlated with decreased sp.

activity. It appears conceivable that the activities of the C12E8

and Triton X-100 purified enzyme could be due to some

denaturation of the protein (Fig. 6). Thus, DHPC purified Ca2+-

ATPase retained more organized and native secondary confor-

+-ATPase. Path length 0.1 cm; protein concentration 0.1 mg/ml. The spectra are

mM C12E8 and 10 mM Triton X-100.

A. Mandal et al. / Biochimica et Biophysica Acta 1760 (2006) 20–31 29

mation compared to that of C12E8 and Triton X-100 solubilized

and purified preparations.

3.6. Mechanism of action of DHPC

It has previously been demonstrated that short-chain

phospholipids are able to disperse bilayers of long-chain

phospholipids [35,64]. It was subsequently shown by Kessi

et al. [30] and Shivanna and Rowe [35] that DHPC could be

used as an effective solubilizing agent for biological mem-

branes. Herein, we have presented the study of purification of

the Ca2+-ATPase by DHPC and reconstitution with DOPC,

while preserving the native characteristics of the membrane

proteins in terms of structure and function. As suggested by

Shivanna and Rowe [35], the efficacy of DHPC could be due to

a mechanism of solubilization that involves disruption of the

lipid portion of the membrane rather than by direct interactions

with the membrane proteins. DHPC, a natural phospholipid,

has negligible interactions with membrane protein interface. It

could be excluded from the domain by the chain length

incompatibility. This is in contrast with the probable mecha-

nism of solubilization of C12E8, which has been shown to

displace lipids from the interfacial region of Ca2+-ATPase

[35,63]. Our results in comparing the Ca2+-ATPase prepared

with the use of solubilization and reconstitution by DHPC,

C12E8 and Triton X-100 support this mechanism of solubili-

zation for DHPC. In the detergent-solubilized and purified

preparations, the DHPC enzyme retains its ability to undergo

the Ca2+-induced conformational change. The C12E8 and Triton

X-100 preparations appear to lost these native properties. The

retention of these native properties by the DHPC preparation is

consistent with a mechanism of solubilization of Ca2+-ATPase

by DHPC in which the detergent either does not interact

directly with the protein or because it is in itself a phospholipid,

can replace these lipids, or it would contribute to diluting the

endogenous lipids during solubilization and allow replacement

by exogenous lipid without affecting appreciably the protein

conformation and activity [35].

In summary, we have demonstrated that the pulmonary

artery smooth muscle microsomal Ca2+-ATPase solubilized,

purified and reconstituted with the short-chain phospholipid

detergent DHPC is superior to that obtained from C12E8 and

Triton X-100 purified preparations in terms of specific activity;

and retained more native properties. This provides a model

membrane protein–lipid system that can be used for detailed

biochemical and biophysical studies such as the mechanism of

ion transport and lipid–protein interactions, which requires

relatively large amount of the purified enzyme.

Acknowledgements

Thanks are due to the Life Science Research Board

(Ministry of Defence, Govt. of India), Department of Biotech-

nology (Govt. of India) and the Indian Council of Medical

Research (New Delhi) for partly financing the work. Thanks

are also due to the Department of Science and Technology

(DST), Govt. of India for supporting our Department by DST-

FIST program. The authors are grateful to Dr. Jonathan Lytton

(University of Calgary, Calgary, Canada) and Dr. Nirmalendu

Das (Scientist, Membrane Biology Division, Indian Institute of

Chemical Biology, Kolkata, India) for their help and cooper-

ation in this work.

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