J Mol C;eil Cardiol

24, 989-1002

( 1992)

Cardiomyocytes Doina

Popov,

Express Mirela

Institute of Cellular

Albumin

Binding

Hasu, Nicolae Ghinea, and Maya Simionescu

Nicolae

Proteins Simionescu

Biology and Patholog)l, Bucharest-79691,

(Receiued 19 October 1990; accepted in revised form

Romania

25 March

1992)

D. POPOV, M. HASU, N. GHINEA, N. SIMIONESCU AND M. SIMIONESCU. Cardiomyocytes Express Albumin Binding Proteins. pommel of Ma~~~u~~r and Cellular CuTd~a~~~ (1992) 24, 989-1002.W e mvestigated whether cardiomyocytes express specdic albumin bmding proteins (ABP) which may function in the dissociation offatty acids from their non-covalent complexes with albumin. The experiments were performed on rat neonatal cardicmyocyres (freshly isolated and up to 3 days in culture) and on an enriched sarcolemmaf fraction isolated from adult rabbit ventricular myocardium. Three types of experiments were conducted: (a) identification of ABP on electrohlots of cardiomyocytes and sarcolemmal extracts reacted with [‘*“II-b ovine serum albumin ( [“iI]Alb); !b) kinetic assays of [‘z51]Alb interaction with cardiomyocytes (at 37X), and with a sarcolemmal fraction (at 4’Cj: fci affinity isolation of ABP from solubilized radioiodinated sarcolemmal proteins interacted with an albuminagarose matrix. The investigation showed that: first, two pairs of polypeptides (ABP of M, 18 and 31 kDa I in either cardiomyocytes or sarcolemmal fraction reacted on eiectroblots with [tz51]Alb, second, the binding of the latter to cardiomyocytes was saturable and competed by unlabeled albumin: 50 HUM albumin reduced by -90% the binding of radiolabeled albumin. The sarcolemmal fraction bound [‘251]Afb with a Kd of 3.66x 10 ‘M. Thirdly, among the sarcolemmai proteins retained by the albumin-agarose matrix (18 and 31 kDa), the most prominent was the lower band ( * 16 kDa) of the 18 kDa pair of ABP. The observations revealed that albumin interacts with relatively high affinity with specific binding sites on cardiomyocyte sarcolemma. This interaction may be a recognition step for subsequent fatty acid dissociation and translocation. KEY

WORDS:

assays; Affinity

Albumin binding interactions.

proteins;

Cardiomyocytes;

Introduction While heart myocytes have a poor capacity to synthesize fatty acids, they are provided with efficient systems for the uptake of the longchain non-esteriiied fatty acids (FA) from plasma and for the beta-oxidation of their acyI-CoA esters in the mitochondrial matrix 19, 311. To explain the uptake of FA by myocytes, three mechanisms have been postulated: (i) simple diffusion of plasma FA determined by the rate of metabolic reactions of the cardiomyocytes, and by the concentration of FA in the membranes [S, 231; it has been considered that this passive diffusion of FA is rapid enough for a sufficient supply ofcardiomyocytes with FA 123, 24241; (ii) a carriermediated transport of FA through the myocyte membranes accomplished by a transmembrane fatty acid binding protein (FABP) Please address Street, Rucharest 002’-.2828/92/090989

all correspondence 79691, Romania. + 14 $08.0010

to: Doina

Popov,

Sarcolemmal

proteins;

Ligand-blotting;

Kinetic

[37, 381; (iii) binding to a plasmalemmal albumin receptor which accelerates the dissociation of albumin-FA complexes (Alb-FA) thus facilitating FA uptake into the cardiomyocytes [35, 381. Sorrentino et al. 1351 have shown that at physiologic albumin/oleate concentrations, only unbound oleate participates in the uptake process, while at low nonphysiologic albumin/ligand concentrations there are deviations from the above kinetics that require alternative explanations. Evidence supporting the involvement of an albumin receptor mechanism emerged from the work of Hiitter et al. [16j on the uptake of albumin-bound FA by the myocardial tissue. In addition, Rauch et al. [ZZ] investigating the initial rate kinetics of FA uptake in myocardial single cells suggested that cellular palmitate uptake involves an interaction of

Institute

of Cellular

Biology

and Pathology.

$‘199’2

Academic

8. B.P. Ha&u

Press Lirnitc~tl

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D. Popov et al.

palmitate-albumin complex with more or less specific sarcolemmal sites. The present study was aimed at investigating whether such albumin binding sites are present in the plasma membrane of cardiomyocytes. This inquiry was based on our preliminary observations showing that heart extracts contain two pairs of polypeptides reacting with [‘251]Alb of apparent M, of 18 and 31 kDa. The findings bring evidence that albumin interacts with relatively high affinity to cardiomyocyte sarcolemma.

Materials

and

Methods

Materials Special reagents were obtained from the following sources: collagenase Worthington, 154 U/mg from Seromed (Germany) ; carrierfree Na[‘251] from the Institute of Atomic Energy (Otwock-Swierk, Poland); iodogen from Pierce Chemical Co. (Rockford, IL, USA); hyaluronidase type II, albumin bovine, fraction V (9699% albumin), mouse albumin fraction V, albumin-agarose and hemoglobin, type III (sheep erythrocytes) from Sigma (St Louis, MO, USA); nitrocellulose membrane filters BA-85, 0.45 pm from Schleicher and Schuell (Dassel, Germany); glass microfiber paper GF/C from Whatman (England); urea and sucrose from Merck (Darmstadt, Germany); culture medium 199 from Gibco (Grand Island, NY, USA); X-ray films from Eastman-Kodak (Rochester, NY, USA) and Azo-Mures (Tg. Mures, Romania). All other chemicals used were of analytical grade. Radioiodinated albumins Bovine serum albumin was radioiodinated for 15 min at room temperature using iodogen ( 100 pg iodogen/l50 ,ug protein) in the presence of 2 mCi Na[lz51]. The unreacted iodine was removed by extensive dialysis against PBS. The same procedure was used for radioiodination of formaldehyde-treated and of anionized bovine serum albumins as well as for mouse serum albumin. Isolation of neonatal cardiomyocytes Pregnant

rats (strain

R) were

given

a stan-

dard pellet diet and water ad libitum and checked daily for offspring. Within 24 h of birth, 10 neonates from each litter were collected from the same mother. At birth, the pups’ weight ranged from 5 to 6 g, and in the conditions of our laboratory the rat colony produced a mean of 12 pups per litter at birth. Each newborn rat (under mild ether anesthesia) had its thorax opened by a mid-line incision, the heart aseptically excised and quickly processed for the isolation of cardiomyocytes using the digestion protocol of McDonagh et al. [20], except that collagenase concentration was 1 mg/ml and supplemented with 1 mg/ml hyaluronidase. Briefly, the heart tissue was minced in K-free balanced salt solution containing 1.32 mM CaCl, and the enzymatic digestion was performed for 20 min in a medium devoid of Ca*+ ions. Following grathis step, Ca*+ ions were reintroduced dually up to 1.25 mM in the cell preparation. All handling procedures were performed in a laminar flow hood to reduce contamination. The following experimental steps appeared to be essential for obtaining viable cardiomyocytes: first, continuous oxygenation of the solutions used, keeping their temperature above 25°C [2]; second, a gentle dissociation procedure of the cells from the heart by simply pipetting the small pieces of tissue in the enzymatic dissociation medium at 37°C [IO]; and third, the ability to obtain isolated myocytes within 20-30 min from the heart’s excision. The release of cardiomyocytes into the dissociating medium was continuously monitored microscopically and the cells were pelleted by centrifugation at 850 g for 10 min at 4°C. To allow cardiomyocytes to recover from the possible adverse effects of the isolation procedure, cells were rewashed from the degradative enzymes and suspended in co!d Dulbecco’s modified essential medium (DMEM) containing 10% calf serum, 0.1 mg/ml streptomycin and 100 U/ml penicillin. Primary cultures of cardiomyocytes Enzymatically dissociated cardiomyocytes were seeded on plastic culture dishes (35 mm diameter), covered with a thin film of 1% gelatin and incubated at 37°C under 95% air and 5% CO,. The myocytes adhered in a few

Albumin

Binding

Proteins

hours and the medium was changed at 48 h. After 3 days, the myocyte culture was invaded by non-myocardic cells whose growth was promoted by the serum mitogens [39]. The biochemical assays were performed on cardiomyocytes up to 3 days in culture. Radioiodination

oJ cardiomyocyte sarcolemmal proteins

Suspensions of isolated myocytes were reacted in 50 mM phosphate buffer pH 7.4 with 175 PCi [‘251]Na in a test tube coated with 1OOpg iodogen for 5 min at 4°C. The radioiodinated cells were removed and the proteins solubilized either directly in the electrophoresis sample buffer (1% SDS, 1% beta-mercaptoethanol, 6 M urea in 0.1 M phosphate buffer pH 7.0) or concentrated by precipitation in 10% trichloroacetic acid (TCA) containing 0.015% sodium deoxycholate. In both cases, the solubilized proteins were subjected to 5-15% polyacrylamide gel electrophoresis in the presence of 0.1% SDS (SDS PAGE) followed by autoradiography of the gels. For cultured cardiomyocytes, the mediumexposed sarcolemmal proteins were radioiodinated selectively, using the method previously described for endothelial cells [12]. To each culture dish 20 mg or iodogen-coated Sephadex beads (5 pug iodogen/mg Sephadex) and 175,&i [‘251]Na were used to radioiodinate the adherent myocytes. After 5 min the beads were removed with PBS and the cells were scrapped and processed as above. Mation

oJ‘a membrane preparation from neonatal cardiom-yocytes

Freshly isolated cardiomyocytes (from 20 pups’ hearts) or up to 3-day cultured cells (originating from 15 pups’ hearts) were used for these experiments. The cells were thoroughly washed by repeated centrifugations in 50 mM Tris-HCl buffer pH 7.4 containing 0.15 M NaCl, 1 mM phenyl methyl sulphonyl fluoride (PMSF) and 1 mM benzamidine. IAfter homogenization in the above buffer in Ultra Turrax (Janke and Kunkel KG, Staufen i Breisgau, Germany) at 4°C (at maximum sperd) for 3 min. the homogenates were sonicatrd for 3 min in an Ultrasonic Cell Disrup-

of Cardiomyocytes

991

tor (Heat Systems-Ultrasonics Inc. Plainview, NY, USA) using the microtip with the amplitude at setting 6. The homogenates were subsequently centrifuged for 60 min at 32 K in a 60Ti rotor. The sedimented pellet consisted of membrane materials. Isolation of enriched cardiac sarcolemma fractions The ventricles of adult rabbit hearts, which provide a larger amount of starting material than the neonatal rat heart, were processed as described by van Alstyne et al. [41]. In addition, we had supplemented the solutions used with protease inhibitors: 1 mM PMSF and 5mM benzamidine. The final membrane preparation was collected at sucrose density 1.1 g/ ml, and used immediately or stored at - 70°C. Purity of the preparation was assessed by marker enzyme determinations and by transmission electron microscopy. Enzyme assays of the enriched cardiac sarcolrmmal fraction Na+/K’-ATPase and K’-p-nitrophenyl phosphatase were determined as described by Bers [3], and 5’-nucleotidase was assayed according to Evans [S]. Na+/K’-ATPase was determined after unmasking the latent activity by incubation with SDS (0.6 mg/mg protein) [13]. To establish the contamination of sarcolemmal fraction with internal membrane materials originating from sarcoplasmic reticulum and mitochondria, we assaved the activity of Ca’+/K’-ATPase and azide-sensitive ATPase [17, 18. Zs]. The angiotensin converting enzyme activity of sarcolemmal preparation was measured as a test of tontamination with endothelial cell membranes 1.51. Electron microscopy of the enriched cardiac .sarcolemmal fraction Electron microscopy of the enriched cardiac sarcolemmal fraction was performed using the protocol described by Simionescu et al. [32]. Briefly, equal volumes of sarcolemmal preparation and “triple fixative” mixture were incubated for 1 h on ice, and sedimented for 5 min at 13 500 g. The pellet was washed 2 x 5 min in 0.14 M cacodylate-HCI buffer pH 7.4, en bloc stained for 7.5 min in 0.5’&

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many1 acetate, dehydrated in graded ethanols, and embedded in Epon 812. Thin sections were stained with many1 acetate and lead citrate before examination with Philips 400HM or Philips 201C electron microscopes.

Ligand-blotting

identijication proteins

of albumin binding

Proteins obtained either from freshly isolated or cultured myocytes, or proteins solubilized from whole membrane preparations solubilized in 1% SDS in PBS were subjected to SDS-PAGE (in the conditions described above). The separated proteins were electrotransferred in a semi-dry system by the method of Andersen [I]. Previous experiments showed that SDS does not interfere with albumin binding to ABP [II]. Blotted proteins were quenched with 1% hemoglobin in PBS or with 2 ,ug/pl bovine IgG in PBS. To identify the albumin binding proteins (ABP) on blots, the following ligands were used: bovine serum albumin; formaldehyde-treated BSA prepared in the laboratory using the technique of Horiuchi et al. [15]; anionized BSA obtained by conventional methods using succinic anhydride; and mouse serum albumin. Blots were incubated for 1 h at room temperature with 2 ,&i/ml of radioiodinated ligands in the presence of the quencher. Unreacted albumin and the free iodine were removed by repeated washings of the blots. Finally, blots were exposed to X-ray films at - 70°C and the autoradiographs developed.

Kinetics of [‘251]-a1bumin

myocyte interaction

Neonatal cardiomyocytes were incubated with increasing concentrations of [‘251]A1 b (from 8 nM to 2.5~~) for 30 min at 37°C. After 3 quick washes in PBS, the cell layer was lysed in 2 M NaOH and the cell associated radioactivity was counted. To evaluate the non-specific binding, the incubation mixtures were supplemented with 50 ,UM cold BSA. The specific binding was estimated by subtracting the non-specific binding from the value of total binding. Competition experiments were performed in the presence of one- to lOOO-fold excess of cold albumin.

Kinetics of [1251]-albumin binding to cardiac sarcolemmal fractions Cardiac sarcolemmal fractions (20 ,ug protein) were incubated for 30 min at 4°C with increasing concentrations of [‘251]Alb (24.9397.8 nM). The reaction mixture had a final volume of 150~1 and consisted of PBS pH 7.4 supplemented with 2 pg/pl bovine IgG (as quencher for non-specific binding) and protease inhibitors (to avoid any degradation during experiments). [‘251]Alb binding to the sarcolemmal membranes was determined by vacuum filtration assay. Thus, 100~1 of the incubation mixtures were quickly filtered on Whatman GFjC glass filters, rapidly washed with 16 ml ice-cold PBS containing 2pg/@ bovine IgG, and the filter-associated radioactivity (total binding) counted. To establish the non-specific [‘251]Alb binding, similar experiments were conducted in the presence of 79.6,u~ cold albumin. Specific binding of [‘251]Alb was obtained by subtracting the non-specific binding from the total binding. Ajinity

identijication of ABP in the enriched cardiac sarcolemmal fraction

Enriched cardiac sarcolemmal fraction (520 ,ug protein) were iodinated with 250 &i [‘251]Na in the presence of 50,~~g iodogen plated on a test tube, for 15 min at room temperature. Two different procedures were then used to solubilize membrane proteins: the non-ionic detergent octyl beta-D-glucopyranoside (1% OG), and a high strength ionic solution (2 M NaCl). The solubilization of proteins was completed during 30 min sonication (on ice), and shaking for 1 h at 4°C. The preparation was sedimented for 15 min at 10 000 g, and supernatant dialyzed against PBS. The solubilized proteins were interacted in a test tube containing 2pg/pl bovine IgG with 50,ul of albuminagarose (- 13.6 mg BSA/ml gel) for 17 h at 4°C on a shaking platform. After extensive washing in PBS with IgG (2 pg/pl), the gel was eluted in electrophoresis sample buffer and proteins analyzed by SDS-PAGE and autoradiography. Analytical procedures Protein method

concentration was assayed of Sheffield et al. [28].

by

the

Albumin

Binding

Proteins

993

of Cardiomyocytes kDc

Results

Characteristics of fieshty isolated and cultured neonatal cardiomyocytes The quality of the cardiomyocytes investigating their interaction with was assessed by several criteria.

used for albumin

Yield In nine different experiments (lo-20 pups per experiment), we obtained an average of 9.75 x IO6 freshly isolated cardiomyocytes per neonatal heart. These have been1 seeded at high density ( - 1.8 X 1O5 card&yocytes/ cm”) in order to diminish the influence of fibroblasts which during culturing usually invade the myocytes [27). Characteristic rod-shapes Microscopic examination of the cardiomyocyte preparation indicated that the rodshaped cells accounted for 92-95% of the total isolated cells and that the preparations obtained appeared to be largely homogeneous. However, the cross-striations of the neonatal cardiomyocytes were less visible at the resolution of light microscope as compared with similar preparations from adult rat hearts. Spontaneous contractions Freshly isolated myocytes contracted several times per minute. In culture, myocytes adhered to the substrate in few hours, and afirr 24 h approximately 75% of the cells showed rhythmic contractile beats. The rate of’ myocyte contraction counted visually with phase contrast optics ranged between 40 and 70 beatings/min. C’iability By the Trypan blue exclusion test, 85-95% of the cells appeared to be viable. In other experiments we radioiodinated the surface proteins of 3-day cultured cells and freshly isolated cardiomyocytes, in suspension. The isotope aimed to label (at 4°C) the surfaceexposed radioiodinatable proteins of viable cells was assumed to penetrate injured or dead c& (if present) and also label cytosolic proteins. Figure 1 shows that the autoradiographic pattern of myocytes in culture (lane CJ or suspension (lane d) differs considerably

0

b

0

d

FIGURE 1. Electrophoretic separation and autoradiography of cardiomyocyte proteins. Lane a, Molecular mass standards; lane h, Coomassie blur staining 01 cardiomyocyte proteins; lane c, autoradiograph) of cardiomyocyte surface radiolabeled proteins of 3 day cultured cells; and lane d, of freshly isolated cell suspension.

from the electrophoretic pattern of the whole cell homogenate stained with Coomassie blur ((lane b). These results suggest that the label was associated with surface proteins rather than with all cellular proteins, an indirect indication that cells were viable. Protein content When studying the interaction of all)umin with cardiomyocytes it was important to ascribe the results to a single cellular species (myocytes) especially when these cells were cultured. To this intent, myocytes were kept in culture only up to 3 days, after which nonmuscle cells often invaded the culture. ;\I 3 days in culture, myocytes contained 2-1-O350 pg protein/dish depending on the growth

994

D. Popov

characteristics experiments.

of the cells isolated

in different

Interaction of cardiomyocytes with albumin To investigate the interaction between cardiomyocytes and albumin we have used ligand blotting and kinetic assays. Ligand blotting identz$cation of albumin binding proteins Experiments performed on proteins solubilized from neonatal rat hearts and from isolated cardiomyocytes have shown that two pairs of polypeptides of apparent M, 18 and 31 kDa reacted on blots with [‘251]Alb. Same pairs of polypeptides appeared on autoradiographs when blotted proteins were reacted with albumin of different species (i.e. bovine or mouse), as well as with chemically modified albumin (formaldehyde-treated or anionized BSA) (Fig. 2). In immunodiffusion experiments (not shown) bovine, mouse and rat albumin cross-reacted with an anti-BSA IgG. The binding of formaldehyde-modified albumin (fBSA) may indicate that the albumin binding site was not affected by the aldehyde treatment. However, with the data on hand, we cannot conclude that binding of fBSA to heart myocytes is specific. The specificity of the binding on myocytes remains to be tested by further experiments. Moreover, the polypeptides reacting with fBSA had an apparent Mr of N 18 and 30 kDa (Fig. 2, Panel A), the molecular mass of the latter corresponds to a fBSA-binding 30 kDa pepude otLecteo by Horiuchi et al. [15] in rat sinusoidal liver cells membranes. Figure 3 shows that in cultured myocytes, the 18 kDa ABP is relatively less expressed than in freshly isolated cells. An isolated membrane preparation obtained from neonatal cells also reacted on electroblots with [‘251]Alb, and 18 and 31 kDa ABP were revealed on autoradiographs (Fig. 4). The fact that ABP were extracted from the cells in the presence of detergents and were found in extracts of membrane fractions (and not in the cytosol), suggested that ABP are membrane-associated components. The ABP presence in the sarcolemma was further investigated on enriched sarcolemmal fraction (see below).

et al.

Kinetic assays on cardiomyocytes-albumin interactions As determined by counting the radioactivity associated with the cell layer (Fig. 5), cardiomyocytes incubated for 30 min at 37°C with increasing concentrations of [‘251]Alb (8 nM2.5 PM) bound the ligand in a saturable manner; the binding was specifically competed by unlabeled albumin. The amount of radioactivity confined to the cell layer may cumulate both the surface-bound and the internalized albumin. However, Stremmel [38] pointed out that albumin is not taken up by cardiomyocytes. To make an accurate quantification of the surface-bound albumin and to circumvent the possible damage of the myocytes at 4°C (viability of the cell preparations could be affected after incubation with ligand for 30 min at 4”C), additional kinetic experiments on enriched sarcolemmal fractions were performed.

Characteristics of the enriched sarcolemmal fraction The quality of the isolated sarcolemmal tion used for investigating the interaction albumin was assayed by several criteria.

fracwith

Floating density The isolated sarcolemmal fraction floated at a sucrose concentration of 1.1 g/ml during 30 min centrifugation at 73 400 g. Morphologic examination The sarcolemmal fraction consisted of roundshaped empty vesicles of various diameters (120-500 nm), almost half of the population having diameters in the range of 300-400 nm (Fig. 6). Rarely on electronmicrographs were structures originating probably from transverse tubules observed. The preparation did not seem to contain fibrillar materials or recognizable mitochondria. Protein yield in the isolated sarcolemma fraction was 6.03 f 0.5 mg/lOO g wet tissue. Enzymatic markers The assays showed a similar enrichment (up to lo-fold) in activity of both 5’-nucleotidase and K’-p-nitrophenyl-phosphatase with respect to the tissue homogenate. Since in the crude homogenates the (Na+ , K+ ) -ATPase

Albumin

Binding

Proteins

of Cardiomyocytes

Heart

Isolated

A

995

A

myocytes

\f

\

kDa

116-

67-

45

-

30 -

16.4-

(,,,. MSA

o6SA

BSA

I

fBSA

II

a

A FIGURE 2. Blotted proteins of neonatal [MSA), anionized bovine serum albumin blotted proteins obtained from preparative Ponceau staining of blotted proteins: (a) cardiomyocytes.

b B

c

II

fBSA

BSA

aBSA

MSA I

C

heart and freshly isolated myocytes reacted with mouse serum albumin (aBSA), BSA and formaldehyde-treated BSA (fBSA). Panels A and C, gels reacted with the radiolabeled ligands and autoradiographed. Panel B, total neonatal heart; (b) molecular mass standards; (cl freshly isolated

activity could be affected by. a number of factors such as inhomogeneity of suspensions, interference of other ATP-splitting cellular components, and formation of vesicular structures [II], we did not assay the enzyme in the initial extracts. In the final sarcolemmal fraction (Na+, K+)-ATPase activity was 25.6 f 1.4 pmol/mg prot. h; this was increased up to 75.2*2.5,~mol/mg prot. h by SDS treatment of vesicles. The contamination of

the sarcolemmal fraction with sarcoplasmic reticulum and mitochondrial membranrs represented only 3-4%. In the assay conditions used, no angiotensin converting enzyme activity was detected in the sarcolemmal fraction. This indicated that the preparation was not contaminated with endothelial cell membranes.

996

D. Popov et al. kDa

116 97

67

67

-

3a

I-

18.4

a

Ia A

B

FIGURE 3. Comparison between the ABP expression in freshly isolated OS cultured cardiomyocytes. Panel A, Ponceau-stained electrotransferred proteins from extracts of isolated (lane b) and cultured cells (lane c). Panel B, Autoradiography of blotted proteins reacted with [1251]Alb: a more intense 18 kDa band is expressed by the freshly isolated cells (lane d) as compared with the cultured myocytes (lane e).

Interaction

of sarcolemma fraction

with albumin

Ligand blotting experiments Ligand blotting experiments have been conducted on proteins solubilized from the cardiac sarcolemma by 1% SDS or 1% OG. As shown in Figure 6, the sarcolemmal interaction with [‘251]Alb was stronger as compared

b

c

FIGURE 4. Ligand blotting identification of ABP in a membrane preparation isolated from neonatal cardiomyocytes. Lane a, molecular mass standards; lane b, blotted proteins stained with Ponceau; lane c, autoradiography of blotted proteins reacted with [‘2iI]Alb.

with the total cellular membrane represented in [Fig. 4, lane (c)].

preparation

Kinetic experiments In preliminary assays we established that the optimal experimental conditions to be used for kinetic investigation of [‘251]Alb binding to cardiac sarcolemma were 20~9 sarcolemma1 protein, [‘251]Alb with a specific activity higher than 2,uCi/,ug protein, the presence of protease inhibitors during incubations and of 2,~g/pl bovine IgG as quencher for nonspecific binding. To ensure that during the filtration procedure there was no loss of

Albumin

0

0.5

Binding

1.0 Free

FIGURE 5. Binding of [‘2SI]Alb non-specific binding. (b) Competition 50 PM of unlabeled protein.

[‘25~]-album~n

Proteins

1.5

2.0

of Cardiomyocytes

2.5

(PM)

to freshly isolated cardiomyocytcs of [‘251]Alb binding; approximately

Unlabeled ia)

albumm

(PM)

Saturation curve: sb, specific 90% of radio12 ibeled ligand is diplacrd

h)

LDa

0

FIGURE constituted sarcolemmal with [“‘I]Alb.

6. Cardiac sarcolemma enriched fraction. f.ej, by electron microscol of various size membranous vesicles. Bar=0.2pm. Rig&, ligand fraction. Lane a, Ponceau staining of blotted proteins; lane h, autorad

b

>y the fra rtion appears to ix mostly blotting identification of ABP in liography of hlottrd proteins rrartrd

998

D. Popov et al.

sarcolemmal material, we monitored filtration conditions with iodinated sarcolemma. The amount of [‘251]Alb bound to the filter in the presence of sarcolemma fractions was minimal and only when using [‘251]Alb with high specific activity (more than 2 ,Li/pg). By incubating 2Opg protein of enriched sarcolemmal fraction with increasing concentrations of [‘251]Alb (from 24.9 to 397.8 nM), a saturable binding was obtained. The specific binding was calculated by subtracting the non-specific binding (in the presence of 79.6pM cold albumin) from the total binding. Using the data of the saturation curve, we have constructed the Scatchard plot and obtained an apparent II, for the interaction of sarcolemma fraction with albumin of 3.66 x lo-‘M. This plot gave an estimate of n = 10.60 p.moles/mg protein (Fig. 7). This data represented the contribution of sarcolemma to the interaction with [‘251]Alb at 4°C. identzjication of ABP in cardiac sarcolemmal fraction

Ajinity

Kinetic assays provided evidence that cardiac sarcolemmal fraction binds saturably [‘251]Alb with an affinity in the range of lo’lo8 M-I. On blots, the proteins responsible for

KD= 3.66 n= 10.60

x IO-’ Y p.moles/mg

prot.

‘l ‘\ I I

I 2

I 3 Bound

I 4

I 5 ( p. moles

I 6

I 7 /mg

I 6

I 9

\

\

\ I\ IO

prot. 1

FIGURE 7. Scatchard plot for [‘251]Alb binding to isolated sarcolemmal fraction. Abscissa: bound [‘251]Alb; co-ordinate: the ratio of bound to free ligand x 10 *.

albumin binding had apparent M, of 18 and 31 kDa. In order to circumvent the possible cross-reactivity occurring on blots reacted with [1251]Alb, we performed an affinity chromatography of sarcolemmal proteins on albumin-agarose. To enhance the sensitivity of detection of specifically reacting proteins, we used radioiodinated sarcolemma. The proteins were solubilized either in 1% OG or in 2 M NaCl. Proteins which were retained by the albumin-agarose matrix appeared on autoradiographs at the position of the apparent molecular mass of 18 and 31 kDa ABP (Fig. 8). The most prominent was a 16 kDa polypeptide (especially after extraction with 2 M which is part of the 18 kDa pair NaCl), revealed on electroblots (Fig. 6). The 31 kDa polypeptide reacting with albumin-agarose was less pronounced. Discussion The data reported in this work demonstrate that at 37°C radioiodinated albumin interaction with cardiomyocytes is saturable and can be competed with unlabeled albumin (Fig. 5). The ligand blotting assays (Figs 2 and 3) revealed that the polypeptides involved in this interaction have the same apparent A4, of 18 and 3 1 kDa as the ABP identified in endothelial cells [II, 331. The binding specificity was assessed by first, the kinetic assays in which cold albumin competed with the [1251]Alb binding to isolated and cultured cardiomyocytes as well as to the sarcolemmal fraction; and second, isolation of ABP by affinity chromatography on an albumin-agarose matrix. However, it is important to note that by ligand blotting, the cardiomyocyte ABP interact with albumins of different species (bovine and mouse) and also with formaldehydetreated or anionized albumins in which the free amino groups of the molecules have been blocked. The latter type of interaction may nat bt specific. The likelihood that cardiomyocyte ABP will interact with various affinity with other members of the multigene family to which albumin belongs such as Vitamin D-binding protein and alpha-fetoprotein [4, 25, 401 is very high. The enriched sarcolemmal fraction binds at 4°C [‘251]Alb with an apparent Kd= 3.66 x lo-’ M, showing that interaction

Albumin

Binding

Proteins

kDo

66-

lo A

FIGURE 8. Affinity identification of ABP on albumin-agarose beads. Before the interaction with the matrix the surface radiolabeled sarcolemmal proteins have been solubilized in I % OG (Panel A) or in 2 M NaCl (Panel B). Lanes a, autoradiography of sarcolemmal proteins prior to incubation with albumin-agarose; lanes b. proteins specifically retained hy the affinity matrix. l‘he faint bands present at 66 k& may represent traces of endogenous albumin.

with this ligand takes place at the level of plasma membrane of cardiomyocytes (Fig. 7). Ligand blotting experiments on sarcolemmal proteins had revealed the presence of two ABP peptides of 18 and 31 kDa (Fig. 6). These peptides which react on electroblots with

of Cardiomyocytes

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[‘251]Alb originate from sarcolemmal proteins which were previously subjected to denaturating solubilization conditions of SDS-PAGE. One can speculate that in the kinetic assays used for Kd estimation, [‘251]AIb interacts probably with a certain domain of native RBP molecule(s) exposed on the sarcolemma surface, giving a single Kd value. It can be presumed that 18 and 31 kDa ABP revealed on electroblots may represent components or subunits of the native ABP molecule(s), which are not accessible to albumin interaction in the non-denaturating conditions of the kinetic assays (cardiomyocytes or sarcolemmal fraction). In an attempt to confirm 18 and 31 kDa polypeptides as specific sarcolemmal albumin binding proteins, we have interacted radioiodinated sarcolemmal proteins with albumin-agarose; the proteins bound specificalIy to the affinity matrix were in the range of 18 and 31 kDa ABP pairs. The most prominent was a 16 kDa polypeptide that is part of the 18 kDa ABP pair. The 3 1 kDa peptide was lrss expressed, and possibly susceptible to proteolytic degradation. In vivo, the interaction of albumin with cardiomyocytes involves a complex route. Plasma albumin binds non-covalently free FA. Upon interaction with the luminal endothelial surface, some Alb-FR complexes are dissociated, allowing FA to diffuse passively through endothelial cells, while albumin rt‘turns to plasma. A fraction of Al b-FA molecules are transcytosed by endotheiial plasmalemma1 vesicles i by a receptor-mediated process) [29, 30] to the interstitial fluid, thus becoming available for interaction with the cardiomyocytes (for reviews see [9] and [31]j. After binding to sarcolemmal ABP, the dissociated FR diffuse across the latter membrane, to he finally carried by the cytosolic FABP to mitochondria where it is used as main fuel for oxidative phosphorylation [9]. Oxidation of fatty acids provides up to 70% of the metabolic energy of the myocardium [7]. After thr FA dissociation, albumin not being internalized by the cardiomyocytrs [38] returns to the interstitial fluid. It is interesting to point out that the fatty acids have similar affinities ( lo8 M- ’ ) fbr both albumin molecule and FABP of cardiomyocyte sarcolemma [31i, 38f. As shown hy out

1000

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data, the interaction of albumin with sarcoiodinated sarcolemmal proteins interacted lemma1 ABP occurs with an affinity of 3 x IO6 ,with albumin-agarose; (iii) at 37°C [‘251]Alb M-’ (x,=3.66x 10e7 M). Cardiomyocyte sarbinding to cardiomyocytes was saturable and colemma binds albumin with slightly higher, competed by excess unlabeled albumin; (iv) at affinity, as compared with other cell types 14°C the sarcolemmal fraction bound [‘251]Alb I with K, of 3.66 x 10e7 M. These results suggest such as hepatocytes and erythrocytes which bind albumin with a Kd in the order of 10e6 M that albumin interacts with relatively high [21, 4.21. The role of the sarcolemmal ABP affinity with cardiomyocytes sarcolemma; might be to serve as a docking molecule for these albumin binding proteins could serve as the albumin-FA complexes; these complexes docking molecules for albumin-FA comwill dissociate so as to allow the FA to enter plexes. the cardiomyocytes either by a simple diffusion process [23, 241 or by the use of a transmembrane FABP carrier [34, 381. AssoAcknowledgements ciation of albumin with transverse tubule vesicles isolated from skeletal muscle was The authors gratefully acknowledge the conreported [19]. tributions of Dr A. Hillebrand and Dr G. On the physiological significance of the Costache throughout the experiments, and the presence on sarcolemma of both ABP and excellent technical assistance of V. Craciun, FABP we can hypothesize that one recognizes M. Toader, C. Dobre (biochemistry), I. the albumin (ABP) and the other facilitates Manolescu and E. I. Georgescu (cell culture), F. Georgescu, D. Taflan (radioisotopic techtranslocation of fatty acids into the cell (FABP). niques), E. Stefan (photography), M. Schean The data reported here showed that: (i) in (graphics) and L. Barbulescu (word processelectroblots of cardiomyocytes or sarcolemma ing). This project was supported by the Minisextracts, two pairs of polypeptides of M, of 18 try of Education and Science and the Roma31 kDa react with [‘251]Alb; (ii) these polynian Academy, and by the National Institutes peptides were affinity isolated from radioof Health, USA, Grant HL-26343.

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Albumin

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1001

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