Neuroscience Letters, 136 (1992) 123 126 ~ 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
123
NSL 08436
Immunologic identification of Na/Ca exchange protein in rat brain synaptic plasma membrane R i c k K. Yip", M o r d e c a i P. Blaustein a a n d K e n n e t h D. P h i l i p s o n b "Department of Physiology, University o[ Mao,land School of Medicine, Baltimore, MD 21201 ( USA ) and ~Departments ~/"Medicine and Physiology and the Cardiovascular Research Laboratoo,, UCLA School of' Medicine. Los Angeles, CA 90024 (USA)
(Received 13 September 1991; Revised version received 4 November 1991: Accepted 4 December 1991) Key words." Na/Ca exchanger; Cardiac; Sarcolemma;Synaptic; Antibody; lmmunoblot
Polyclonal antibodies raised against partially purified dog cardiac Na/Ca exchanger react with cardiac sarcolemmal proteins of 160, 120 and 70 kDa on SDS-PAGE. Using the same specific antiserum, we detected three prominent immunoreactive bands of about 150, 120 and 70 kDa on immunoblots with rat forebrain synaptic plasma membrane proteins. These data indicate that the Na/Ca exchange protein in rat brain synaptic plasma membrane is structurally and antigenically similar to the exchange protein in dog cardiac sarcolemma.
The Na/Ca exchanger is an integral plasma membrane protein that plays a key role in the regulation of intracellular Ca `,+ ions in m a n y cells [2]. Exchanger activity is prominent in cardiac muscle [12, 18] and in neurons [5, 8] including nerve endings [5]. Functional Na/Ca exchanger has been partially purified from decylmaltoside-solubilized dog heart sarcolemma by DEAE-Sepharose and wheat germ agglutinin affinity chromatography [16]. S D S - P A G E of the enriched protein shows two prominent bands: 120 k D a and 70 kDa; the latter appears to be a proteolytic fragment of the former. A single, prominent 160 k D a band is seen on non-reducing gels. Polyclonal antibodies raised against the partially purified exchanger recognize all three polypeptide bands [16], all of which are associated with the Na/Ca exchanger [15]. Molecular cloning of this exchanger indicates that the c D N A encodes for a 108 k D a protein [15] and can be used to express a functional Na/Ca exchanger [10, 15]. The difference between this deduced molecular weight and the apparent molecular weight observed on polyacrylamide gels, 120 kDa, may be due to glycosylation. In contrast, partially purified Na/Ca exchanger from rat or calf synaptic plasma membrane has a reported molecular weight of 70 k D a and exhibits a 33 k D a proteolytic fragment or subunit [3]. Purified N a / ( C a + K ) exchanger from cow retinal rod outer segment has a Correspondence: M.R Blaustein, Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, MD 21201, USA. Fax: (1) (410) 328-8341.
reported molecular weight of 230 kDa [7, 14], but this protein has a different stoichiometry and very little sequence homology with the cardiac Na/Ca exchanger protein [1]. The present report shows that polyclonal antibodies raised against partially purified dog cardiac sarcolemmal (SL) Na/Ca exchanger crossreact with rat brain synaptic plasmalemmal (SP) polypeptides of about 150, 120, and 70 kDa. Preparation o f sarcolemma. Highly purified SL was prepared from dog ventricles as described [17]. SL was resuspended in 140 m M NaCI, 10 m M MOPS/Tris (pH 7.4) and stored in liquid nitrogen. Preparation o f synaptic plasmalemma. SP was prepared from rat forebrains as described [19]. The SP was resuspended in 50 m M NaC1, 50 m M H E P E S (pH 7.4) and stored at -70°C. Protein concentrations were determined by the BCA protein assay (Pierce, Rockford, IL). Sample preparation and gel electrophoresis. SL and SP were solubilized with SDS buffer [2.3% (w/v) SDS, 62.5 m M Tris-HC1 (pH 6.8), and 10% (v/v) glycerol] containing either 15 m M dithiothreitol (DTT: 'reducing conditions') or 10 m M N-ethylmaleimide (NEM: 'non-reducing conditions'), and were analyzed by S D S - P A G E [10]. Prestained molecular weight standards (27 180 kDa, from BioRad, Richmond, CA; or 15-206 kDa, from BRE, Gaithersburg, MD) were included in each gel. In some experiments, SP was first solubilized by diluting the membrane suspension with an equal volume of
124 ice-cold buffer [140 mM NaC1, 10 mM MOPS/NaOH (pH 7.4) and 30 mM decylmaltoside] and incubating at 4°C for 15 min. Samples were centrifuged (12 rain, 10,000 × g) to remove non-solubilized material: the supernatants were then analyzed by SDS-PAGE. lmmunoblotting. Proteins were transferred to nitrocellulose [20], and incubated 2-5 h at 25°C in Tris-buffered saline with Tween (TBST) [10 mM Tris-HC1 (pH 8.0), 150 mM NaCI, and 0.05% (v/v) Tween 20] containing 2% bovine serum albumin (BSA) and 2% fetal calf serum. Nitrocellulose transfers were then incubated overnight at 4°C in TBST containing 1% BSA plus either preimmune serum or polyclonal antibodies (both at 1:300-1:500 final dilution) raised against the purified dog cardiac sarcolemmal Na/Ca exchanger [16]. Immunoblots were washed in TBST containing 0.1% BSA and incubated for 1 h at room temperature with peroxidase-conjugated secondary antibody (1:2500 final dilution; BioRad) with 3,Y-diaminobenzidine (DAB) as substrate, or for 2 h at 25°C with alkaline phosphatase-conjugated secondary antibodies (1:7500 final dilution: Organon Teknika, Durham, NC) with nitroblue tetrazolium and 5-bromo4-chloro-3-indoyl phosphate as substrate (Promega, Madison, WI). Fig. 1 shows an immunoblot obtained when SL and SP were incubated with rabbit polyclonal antiserum raised against purified dog cardiac sarcolemmal Na/Ca exchanger [16]. Three prominent immunoreactive bands were recognized by the antiserum in both SP (lane 3, at 150, 120, and 70 kDa) and SL (lane 4, at 160, 120, and 70 kDa) under reducing conditions. The antiserum also
1
2
M
3
4
Fig. 1. Immunoblot analysis of SL and SP proteins incubated with polyclonalanti-Na/Caexchangerantiserum.Immunoblotsfroma 7.5% polyacrylamidegel were probed with antiserum (1:500 final dilution), followedby peroxidase-conjugatedsecondaryantibodies. Lanes 1 (25 #g SP) and 2 (4 #g SL) were prepared with non-reducing SDS buffer (NEM present). Lanes 3 (SP) and 4 (SL) were prepared with reducing SDS buffer(DTT present). Lane M: molecularweightstandards.
crossreacted with an 87 kDa SL protein tha! may be a proteolytic fragment of 120 kDa protein. When SP and SL proteins were prepared with SDS non-reducing buffer, only one major immunoreactive band was observed: at about 150 kDa in the SP (lane 1) and 160 kDa in the SL (lane 2). Fig. 2A shows reactions of SP and SL proteins with preimmune serum obtained from the same rabbit as the antiserum. The preimmune serum crossreacted with two low molecular weight polypeptides (about 33 kDa and 40 kDa) in synaptic membranes solubilized with reducing SDS buffer (lane 1): crossreactivity was greatly diminished under non-reducing conditions (lane 3). To examine the reactions of the antiserum further, we increased the amount of SP protein in each gel lane fivefold, and overdeveloped the immunoblot (Fig. 2B). The antiserum crossreacted strongly and specifically with three polypeptide bands in both dog cardiac sarcolemmal membranes (at 160, 120, and 70 kDa) and rat synaptic membranes (at 150, 120, and 70 kDa) solubilized in SDS reducing buffer (Fig. 2B, lanes 1 -4). In addition, the antiserum crossreacted non-specitically with 40 kDa (SP and SL) and 33 kDa (SR only) protein bands. The effects of detergent solubilization on the membranes were also compared. The immunoreactivity patterns of only those SP and SL proteins solubilized by decylmaltoside (Fig. 2B, lanes 3 and 4, respectively) were similar to the SP and SL proteins solubilized directly with SDS (Fig. 2B, lanes l and 2, respectively). These data demonstrate that polyclonal antibodies raised against partially purified dog cardiac sarcolemmal Na/Ca exchanger proteins (70, 120 and 160 kDa) [15] crossreact with proteins of similar molecular weight from rat brain synaptic plasmalemma. The 120 kDa protein probably represents the fully mature, glycosylated protein [15], and the 70 kDa protein is likely to be a protelytic fragment [15]. The 150 and 160 kDa polypeptides of SP and SL, respectively, are most prominent under nonreducing conditons and may reflect a change in the conformational state of the exchanger during preparation for SDS-PAGE [9]. The 120 and 160 kDa SL polypeptides have identical amino-terminal amino acid sequences [9]. Brain has high endogenous protease activity [13, 21], and some of the minor immunoreactive bands may be due to proteolysis of the 120 kDa polypeptide during the lengthy purification process. Others, such as the 33 and 40 kDa protein bands, may be due to non-specific binding because they also cross-react with preimmune serum. The slightly different molecular weights of the large SL and SP immunoreactive proteins. 160 and 150 kDa, respectively, indicates that they are not identical. In addition, a monoclonal antibody recently raised against the
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exchange protein, Biochim. Biophys. Acta, 945 (1988) 298 306. 17 Philipson, K.D. and Ward. R., Modulation of Na'-Ca e~ exchange and Ca > permeability in cardiac sarcolemmat vesicles by deoxylstearic acids, Biochim. Biophys. Acta, 897 (1987) 152 158. 18 Reeves, J.P. and Philipson, K.D., Sodium-calcium exchange activity in plasma membrane vesicles. In T.J.A. Allen, D. Noble and H. Reuter (Eds.), Sodium-Calcium Exchange, Oxford University Press, Oxford, 1989, pp. 27 53. 19 Salvaterra, RM. and Matthews, D.A., Isolation of rat brain subcellular fraction enriched in putative neurotransmitters and synaptic junctions, Neurochem. Res., 5 (1980) 181-195. 20 Towbin, H. and Gordon, J., Immunoblotting and dot immunobinding - current status and outlook, J. lmmunoll Methods, 72 (1984) 313 340. 21 Zimmerman, U.-J.R and Schlaepfer, W., Calcium-activated neutral protease (CANP) in brain and other tissues, Prog. NeurobioL, 23 (1984) 63 78.
123
NSL 08436
Immunologic identification of Na/Ca exchange protein in rat brain synaptic plasma membrane R i c k K. Yip", M o r d e c a i P. Blaustein a a n d K e n n e t h D. P h i l i p s o n b "Department of Physiology, University o[ Mao,land School of Medicine, Baltimore, MD 21201 ( USA ) and ~Departments ~/"Medicine and Physiology and the Cardiovascular Research Laboratoo,, UCLA School of' Medicine. Los Angeles, CA 90024 (USA)
(Received 13 September 1991; Revised version received 4 November 1991: Accepted 4 December 1991) Key words." Na/Ca exchanger; Cardiac; Sarcolemma;Synaptic; Antibody; lmmunoblot
Polyclonal antibodies raised against partially purified dog cardiac Na/Ca exchanger react with cardiac sarcolemmal proteins of 160, 120 and 70 kDa on SDS-PAGE. Using the same specific antiserum, we detected three prominent immunoreactive bands of about 150, 120 and 70 kDa on immunoblots with rat forebrain synaptic plasma membrane proteins. These data indicate that the Na/Ca exchange protein in rat brain synaptic plasma membrane is structurally and antigenically similar to the exchange protein in dog cardiac sarcolemma.
The Na/Ca exchanger is an integral plasma membrane protein that plays a key role in the regulation of intracellular Ca `,+ ions in m a n y cells [2]. Exchanger activity is prominent in cardiac muscle [12, 18] and in neurons [5, 8] including nerve endings [5]. Functional Na/Ca exchanger has been partially purified from decylmaltoside-solubilized dog heart sarcolemma by DEAE-Sepharose and wheat germ agglutinin affinity chromatography [16]. S D S - P A G E of the enriched protein shows two prominent bands: 120 k D a and 70 kDa; the latter appears to be a proteolytic fragment of the former. A single, prominent 160 k D a band is seen on non-reducing gels. Polyclonal antibodies raised against the partially purified exchanger recognize all three polypeptide bands [16], all of which are associated with the Na/Ca exchanger [15]. Molecular cloning of this exchanger indicates that the c D N A encodes for a 108 k D a protein [15] and can be used to express a functional Na/Ca exchanger [10, 15]. The difference between this deduced molecular weight and the apparent molecular weight observed on polyacrylamide gels, 120 kDa, may be due to glycosylation. In contrast, partially purified Na/Ca exchanger from rat or calf synaptic plasma membrane has a reported molecular weight of 70 k D a and exhibits a 33 k D a proteolytic fragment or subunit [3]. Purified N a / ( C a + K ) exchanger from cow retinal rod outer segment has a Correspondence: M.R Blaustein, Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, MD 21201, USA. Fax: (1) (410) 328-8341.
reported molecular weight of 230 kDa [7, 14], but this protein has a different stoichiometry and very little sequence homology with the cardiac Na/Ca exchanger protein [1]. The present report shows that polyclonal antibodies raised against partially purified dog cardiac sarcolemmal (SL) Na/Ca exchanger crossreact with rat brain synaptic plasmalemmal (SP) polypeptides of about 150, 120, and 70 kDa. Preparation o f sarcolemma. Highly purified SL was prepared from dog ventricles as described [17]. SL was resuspended in 140 m M NaCI, 10 m M MOPS/Tris (pH 7.4) and stored in liquid nitrogen. Preparation o f synaptic plasmalemma. SP was prepared from rat forebrains as described [19]. The SP was resuspended in 50 m M NaC1, 50 m M H E P E S (pH 7.4) and stored at -70°C. Protein concentrations were determined by the BCA protein assay (Pierce, Rockford, IL). Sample preparation and gel electrophoresis. SL and SP were solubilized with SDS buffer [2.3% (w/v) SDS, 62.5 m M Tris-HC1 (pH 6.8), and 10% (v/v) glycerol] containing either 15 m M dithiothreitol (DTT: 'reducing conditions') or 10 m M N-ethylmaleimide (NEM: 'non-reducing conditions'), and were analyzed by S D S - P A G E [10]. Prestained molecular weight standards (27 180 kDa, from BioRad, Richmond, CA; or 15-206 kDa, from BRE, Gaithersburg, MD) were included in each gel. In some experiments, SP was first solubilized by diluting the membrane suspension with an equal volume of
124 ice-cold buffer [140 mM NaC1, 10 mM MOPS/NaOH (pH 7.4) and 30 mM decylmaltoside] and incubating at 4°C for 15 min. Samples were centrifuged (12 rain, 10,000 × g) to remove non-solubilized material: the supernatants were then analyzed by SDS-PAGE. lmmunoblotting. Proteins were transferred to nitrocellulose [20], and incubated 2-5 h at 25°C in Tris-buffered saline with Tween (TBST) [10 mM Tris-HC1 (pH 8.0), 150 mM NaCI, and 0.05% (v/v) Tween 20] containing 2% bovine serum albumin (BSA) and 2% fetal calf serum. Nitrocellulose transfers were then incubated overnight at 4°C in TBST containing 1% BSA plus either preimmune serum or polyclonal antibodies (both at 1:300-1:500 final dilution) raised against the purified dog cardiac sarcolemmal Na/Ca exchanger [16]. Immunoblots were washed in TBST containing 0.1% BSA and incubated for 1 h at room temperature with peroxidase-conjugated secondary antibody (1:2500 final dilution; BioRad) with 3,Y-diaminobenzidine (DAB) as substrate, or for 2 h at 25°C with alkaline phosphatase-conjugated secondary antibodies (1:7500 final dilution: Organon Teknika, Durham, NC) with nitroblue tetrazolium and 5-bromo4-chloro-3-indoyl phosphate as substrate (Promega, Madison, WI). Fig. 1 shows an immunoblot obtained when SL and SP were incubated with rabbit polyclonal antiserum raised against purified dog cardiac sarcolemmal Na/Ca exchanger [16]. Three prominent immunoreactive bands were recognized by the antiserum in both SP (lane 3, at 150, 120, and 70 kDa) and SL (lane 4, at 160, 120, and 70 kDa) under reducing conditions. The antiserum also
1
2
M
3
4
Fig. 1. Immunoblot analysis of SL and SP proteins incubated with polyclonalanti-Na/Caexchangerantiserum.Immunoblotsfroma 7.5% polyacrylamidegel were probed with antiserum (1:500 final dilution), followedby peroxidase-conjugatedsecondaryantibodies. Lanes 1 (25 #g SP) and 2 (4 #g SL) were prepared with non-reducing SDS buffer (NEM present). Lanes 3 (SP) and 4 (SL) were prepared with reducing SDS buffer(DTT present). Lane M: molecularweightstandards.
crossreacted with an 87 kDa SL protein tha! may be a proteolytic fragment of 120 kDa protein. When SP and SL proteins were prepared with SDS non-reducing buffer, only one major immunoreactive band was observed: at about 150 kDa in the SP (lane 1) and 160 kDa in the SL (lane 2). Fig. 2A shows reactions of SP and SL proteins with preimmune serum obtained from the same rabbit as the antiserum. The preimmune serum crossreacted with two low molecular weight polypeptides (about 33 kDa and 40 kDa) in synaptic membranes solubilized with reducing SDS buffer (lane 1): crossreactivity was greatly diminished under non-reducing conditions (lane 3). To examine the reactions of the antiserum further, we increased the amount of SP protein in each gel lane fivefold, and overdeveloped the immunoblot (Fig. 2B). The antiserum crossreacted strongly and specifically with three polypeptide bands in both dog cardiac sarcolemmal membranes (at 160, 120, and 70 kDa) and rat synaptic membranes (at 150, 120, and 70 kDa) solubilized in SDS reducing buffer (Fig. 2B, lanes 1 -4). In addition, the antiserum crossreacted non-specitically with 40 kDa (SP and SL) and 33 kDa (SR only) protein bands. The effects of detergent solubilization on the membranes were also compared. The immunoreactivity patterns of only those SP and SL proteins solubilized by decylmaltoside (Fig. 2B, lanes 3 and 4, respectively) were similar to the SP and SL proteins solubilized directly with SDS (Fig. 2B, lanes l and 2, respectively). These data demonstrate that polyclonal antibodies raised against partially purified dog cardiac sarcolemmal Na/Ca exchanger proteins (70, 120 and 160 kDa) [15] crossreact with proteins of similar molecular weight from rat brain synaptic plasmalemma. The 120 kDa protein probably represents the fully mature, glycosylated protein [15], and the 70 kDa protein is likely to be a protelytic fragment [15]. The 150 and 160 kDa polypeptides of SP and SL, respectively, are most prominent under nonreducing conditons and may reflect a change in the conformational state of the exchanger during preparation for SDS-PAGE [9]. The 120 and 160 kDa SL polypeptides have identical amino-terminal amino acid sequences [9]. Brain has high endogenous protease activity [13, 21], and some of the minor immunoreactive bands may be due to proteolysis of the 120 kDa polypeptide during the lengthy purification process. Others, such as the 33 and 40 kDa protein bands, may be due to non-specific binding because they also cross-react with preimmune serum. The slightly different molecular weights of the large SL and SP immunoreactive proteins. 160 and 150 kDa, respectively, indicates that they are not identical. In addition, a monoclonal antibody recently raised against the
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exchange protein, Biochim. Biophys. Acta, 945 (1988) 298 306. 17 Philipson, K.D. and Ward. R., Modulation of Na'-Ca e~ exchange and Ca > permeability in cardiac sarcolemmat vesicles by deoxylstearic acids, Biochim. Biophys. Acta, 897 (1987) 152 158. 18 Reeves, J.P. and Philipson, K.D., Sodium-calcium exchange activity in plasma membrane vesicles. In T.J.A. Allen, D. Noble and H. Reuter (Eds.), Sodium-Calcium Exchange, Oxford University Press, Oxford, 1989, pp. 27 53. 19 Salvaterra, RM. and Matthews, D.A., Isolation of rat brain subcellular fraction enriched in putative neurotransmitters and synaptic junctions, Neurochem. Res., 5 (1980) 181-195. 20 Towbin, H. and Gordon, J., Immunoblotting and dot immunobinding - current status and outlook, J. lmmunoll Methods, 72 (1984) 313 340. 21 Zimmerman, U.-J.R and Schlaepfer, W., Calcium-activated neutral protease (CANP) in brain and other tissues, Prog. NeurobioL, 23 (1984) 63 78.
