purification of functionally intact 1,4-dihydropyridine receptor from rabbit skeletal muscle by hplc

Purification of Functionally Intact 1,4-Dihydropyridine Receptor from Rabbit Skeletal Muscle by HPLC

H. Kalfisz 1 / Cs. Horvfith 2/P. L. Vaghy 1. 1Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0575, USA

2Department of Chemical Engineering, Yale University, P.O. Box 2159, New Haven, CT 06520, USA

Key Words Protein purification HPLC of receptor Calcium channel 1,4-Dihydropyridine receptor

exact location of the individual drug binding sites within the primary structure is still unknown. Current research in our laboratory is aimed at the deter- mination of the binding sites for calcium antagonists and requires a substantial amount of highly purified calcium channel. In order to meet this need we have examined the methods described in the literature [11- 13] and developed the present approach using HPLC.

Summary

Functionally intact 1,4-dihydropyridine receptors have been isolated from digitonin-solubilized skeletal muscle membranes with the combined use of Wheat- germ Lectin-Sepharose affinity chromatography and ion-exchange HPLC. Lectin affinity chromatography was used for the purification of glycoproteins containing N-acetyl-glucosamines residues. Separation of functionally intact 1,4-dihydrop~cridine receptor/ calcium channel complex from MgZ+-ATPase, one of the most abundant glycoproteins in skeletal muscle T- tubular membranes, was successfully performed by HPLC on a TSKgel DEAE-5PW column.

Introduction

The function of receptors is to induce a physiological response upon binding of neutrotransmitters, drugs, hormones or other biologically active substances. Most receptors are proteins and integral components of the cell membrane. Their purification without loss of function is often encumbered by lack of rapid and sufficiently gentle separation procedures.

Selectively acting calcium antagonist drugs have been successfully used for treatment of several different cardiovascular diseases such as hypertension, angina pectoris and supraventricular arrhythmias [1]. In a recent publication [2] they were also suggested for the treatment of AIDS. The primary structure of the calcium antagonist receptor, the ~1 subunit of the "L- type" voltage-dependent calcium channel [3-7], has been deduced from eDNA clones [8-10]. However, the

Experimental

Materials

Phenylmethanesulfonyl fluoride (PMSF) ultra-pure grade, Pepstatin A lyophylizate and Antipain-di- hydrochloride were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN, USA); iodoacetamide (IAA), N-2-hydroxyethyl-piperazine- N'-2-ethanesulfonic acid (HEPES), digitonin (approximately 50 % pure) and N-acetyl-D-glucosamine were purchased from Sigma (St. Louis, MO, USA). Wheat-germ Lectin-Sepharose 6MB (WGL) was obtained from Pharmacia LKB Biotechnology, Uppsala, Sweden.

(+)-[Methyl-BH]PN200-110 (isopropyl 4-(2,1,3-benzo- xadiazol-4-yl)-l,4-dihydro-5-methoxycarbonyl-2,6-di- methyl-3-pyridine carboxylate) was purchased from Amersham Corp. (Arlington Heights, IL, USA).

Solubilization Buffer: 10 mM HEPES-Tris buffer, pH 7.4 containing 185 mM KCI, 1.5 mM CaC12, 0.1 mM PMSF, 2 gM Pepstatin A, 1 mM IAA, 1 p_g/ml Anti- pain and 1% digitonin.

WGL-Elution Buffer A: 5 mM Tris-HC1, pH 7.4 buffer containing 1 mM CaC12, 0.1% digitonin 0.1 mM PMSF, 2 ~M Pepstatin A, 1 mM IAA, 1 ~tg/ml Anti- pain.

WGL-Elution Buffer B: WGL-Elution Buffer A containing 150 mM N-acetylglucosamine.

TSKgel DEAE-5PW Elution Buffer: Buffer A: 20 mM Tris-HC1 at pH 7.4, containing 0.1% digitonin. The Elution Buffer B is Elution Buffer A containing 1 M NaC1.

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0009-5893/90/11 0533-04 $ 03.00/0 �9 1990 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH

Methods

Membranes were prepared from rabbit skeletal muscle according to Glossmann and Ferry [14] with several modifications. Two male New Zealand white rabbits (each weighing about 1 kg) were sacrificed by cervical disloction; 300 g of back and leg muscles were quickly removed, and kept in ice-cold solution of 20 mM NaHCO3 and 0.5 mM PMSF. The work was done in a cold room at + 4 ~ and the individual steps of homogenization consisted of mincing the tissue with scissors (buffer: skeletal muscle ratio = 1:1), homogenization with Omnimixer (buffer: skeletal muscle ratio = 4:1, mincing time, 7 x 25 s, with 35 s intervals) and homogenization with Polytron (buffer: skeletal muscle ratio = 4:1; homogenization time, 7 x 25 s, with 35 s intervals between them). The homogenate was filtered through a single layer of cheesecloth and centrifuged at 1,500 g for 25 min. The supernatant was filtered through two layers of cheesecloth and centrifuged at 45,000 g for 25 minutes. The pellet was resuspended with ice-cold 50 mM Tris-HCl buffer, pH 7.4 containing 0.5 mM PMSF using a glass-Teflon homogenizer. The high-speed centrifugation and resuspension were repeated twice. The final pellet was resuspended in 50mM Tris-HCl buffer, pH7.4 containing 0.5 mM PMSF with hand-driven Thomas C size Teflon pestle in glass homogenizing vessel to yield a protein concentration of 10-15 mg/ml. The membrane suspension was frozen in a dry ice-ethanol mixture and stored at - 70 ~ until further use.

Solubilization of the calcium channel was performed as described previously [3, 6, 7]. Membranes (500 to 1000 mg) were thawed at room temperature, homo- genized using a glass-Teflon homogenizer, and centrifuged at 100,000 g for 30 min. The pellet was diluted to 5 mg of protein/ml with the solubilization buffer containing 1% digitonin. The mixture was suspensed in glass-Teflon homogenizer and kept on ice for 40 min; the insoluble material was removed by centrifugation at 100,000 g for 45 min.

Affinity Chromatography on WGL-Sepharose. The digitonin-solubilized membrane proteins were incubatdd for 2 hours with 30 ml of WGL-Sepharose at 4 ~ The WGL-Sepharose was packed into a column (50 cm x 1 cm i.d.) and washed with 100 ml of solubilization buffer containing 1% digitonin and then with 400 ml of solubilization buffer containing 0.1% digitonin. Glycoproteins were eluted from the column by a 0-150 mM linear N-acetylglucosamine gradient using WGL Buffers A and B.

Operational Mode of HPLC: Elution was performed by a 0-1 M NaC1 gradient. The elution was started with TSKgel DEAE-5PW Elution Buffer A (zero NaC1), holding it for 2 min, then a linear NaC1 gradient was applied within 28 rain. Buffer B (containing 1 M NaC1) was administered for 5 rain, then a linear gradient to zero NaC1 followed which lasted for 1 min.. Finally, the column was conditioned with 100 % TSKgel DEAE-5PW Elution Buffer A (zero NaC1) for 4 min. The flow rate was 1 ml/min; 2 ml fractions were collected and stored at the temperature of melting ice and functional assays were performed

534

immediately following fractionation-. Purified calcium channel proteins were frozen in a dry ice-ethanol mixture and stored at - 70 ~

Protein Concentration: Glycoproteins eluted from the Lectin-Sepharose column were concentrated by ultrafiltration using CentriCell (Polysciences Inc., War- rington, PA, USA.) which had a molecular weight cutoff at 30 kDa.

Measurement of Receptor Activity by Radioligand Binding: The 1,4-dihydropyridine binding activity of the purif ied protein was de te rmined using (+) [3H]PN200-110 as labelled ligand [7, 14, 15]. The nonspecific binding was measured in the presence of 2 gM unlabeled PN200-110.

Mga+-ATPase Activity: Measurements were carried out as described by Kirley [16].

Protein was determined by the method of Bradford using Bio-Rad Protein Assay Kit [17].

Instrumentation

The liquid chromatograph of LDC/Milton Roy (Riviery Beach, FL, USA) was used consisting of Model I and IIIG Constametric Pumps, Spectro- monitor D, Accessory Control Module, Chromato- graphy Control Module and Dual Drive Floppy Disc. Rheodyne injector (Rheodyne, Berkeley, CA, USA), equipped with a 1-ml sample loop, and Model 2110 Fraction Collector (Bio-Rad Laboratories Richmond, CA, USA) were also used.

Omnimixer 17105 (Omni Corporation Interantional, Waterbury, CT, USA) with 16,000 rpm. and Polytron (Brinkman Instruments, Westbury, CT) with rotation speed 7 of the total 10 were used.

Column: TSKgel DEAE-5PW ion-exchanger (Toyo Soda, Yamaguchi, Japan) was used.

Results For high-performance ion-exchange chromatography a partially purified 1,4-dihydropyridine receptor/calcium channel complex was used. This was prepared by using previously described procedures [6, 7]. Skeletal muscle membranes containing 5 to 10 pmol 1,4-dihydropyridine receptor per mg protein were solubilized with l % digitonin, a detergent which does not denature the receptor. The glycoproteins containing N-acetyl-glucosamine residues were purified by Wheatgerm Lectin-Sepharose affinity chromatography. Protein concentration and receptor binding activity was measured in each fraction. The affinity-purified fractions enriched in 1,4-dihydropyridine binding sites were pooled and concentrated to about 1.3 ml by ultrafiltration using CentriCell. The concentrated sample contained 0.5 to 1.0 mg protein per ml.

For control HPLC experiments 0 .9ml Lectin- Sepharose elution buffer was used and the materials adsorbed to the TSKgel DEAE-5PW column were eluted with 0 to 1.0 M linear NaC1 gradient at a flow rate of 1 ml/min. Figure 1 (broken line) shows the elution profile of the buffer not containing glyco-

Chromatographia Vol. 30, No. 9/10, November 1 9 9 0 Originals

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Figure 1 Elution profile of glycoproteins from the TSKgoI DEAE-5PW column. Glycoproteins purified by Wheat-germ lectin affinity chromatography (1.2 mg protein) were injected and the proteins adsorbed to the column were eluted with 0 to 1 M linear NaCI gradient (dashed-dotted line). The light-absorbance at 280 nm was recorded. The absorbance measured after injection of proteins (solid line) is shown together with the absorbance change measured after injection of the same volume of the buffer not containing glycoproteins (broken line).

eight and sixteenth minutes of elution, corresponding to 0.15 to 0.5 M NaCI, a sharp increase in the UV absorbance was observed (Figure 1). Direct measurement of protein in the corresponding fractions in- dicated that this UV absorbance represents proteins eluted from the TSKgel DEAE-5PW column. The peak absorbance corresponds to 180 lag protein/ml. Further increase in the salt concentration did not remove more proteins from the column.

Based on the protein profile alone, one may conclude that separation of .glycoproteins did not occur. However, measurements of 1,4-dihydropyridine binding and Mg2+-ATPase activities in the corresponding fractions clearly show that a fair-to-good overall separation of these two structurally unrelated proteins occurred. In fact, fraction 5 which contained the peak Mg2+-ATPase activity, did not contain 1,4-dihydropyridine binding activity suggesting complete separation. Also, fraction 8 which contained the peak 1,4-dihydropyridine binding activity, had little or no Mg2+-ATPase activity (Figure 2). These data suggest that measurement of UV absorbance at 280 nm alone is not always sufficient to determine the degree of separation and functional assays may also be required.

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Figure 2 Dihydropyridine binding and Mg2+-ATPase activities in fractions collected during HPLC. Dihydropyridine binding (closed circles) represents specific binding of 5 nM (+) [3H]PN200-ll0 to 50-pJ

2 + aliquot taken from the corresponding fractions. Relative Mg - ATPase activities in the corresponding fractions is shown by the open squares.

proteins. The UV absorbance recorded under these conditions is unrelated to 1,4-dihydropyridine receptors and can be attributed to digitonin and protease inhibitors present in the control buffer. When the same volume of buffer containing glycoproteins was injected and the salt elution was performed in the same way as under the control conditions, multiple peaks were obtained (Figure 1, solid line). The small peaks obtained during the first seven minutes of elution, corresponding to salt concentration from 0 to approximately 0.15 M, were not related to either Mg 2+- ATPase activity or 1,4-dihydropyridine binding. This was proven by direct measurement of these activities in the individual fractions (Figure 2). In between the

Discussion Many different low-pressure chromatographic methods have been used for the purification of the functional calcium channels from skeletal muscle transverse tubular membranes [3-7, 14, 18-27]: ion- exchange chromatography on DEAE-Sephadex and DEAE-Trisacryl; affinity chromatography on WGL- Sepharose, WGA-Affi-Gel 10; size-exclusion chromatography on Sephacryl S-400 and Sucrose Density Gradient Centrifugation have been employed. In this paper we describe the application of ion-exchange HPLC for the separation of functionally intact calcium channel proteins from Mga§ We have found that the elution of digitonin and protease inhibitors present in WGL sample buffer is completed during the first five minutes and, therefore, precedes the elution of glycoproteins from the TSKgel DEAE-5PW column. Glycoproteins appear to be eluted together forming a single large protein peak. Based on the protein profile alone, separation is not obvious. How- ever, with direct measurement of 1,4-dihydropyridine binding and Mg2+-ATPase activities in all fraction collected during the elution, we have been able to prove that partial separation of these two glycoproteins has occurred. Ion-exchange HPLC resulted in fast and gentle separation within a short period of time. The results of HPLC have been comparable to or better than these obtained by conventional separation methods [6, 18].

Conclusions HPLC methods have been generally used for the separation of denatured proteins. Our results show the successful employment of TSKgel DEAE-5PW for the

Chromatographia Vol. 30, No. 9/10, November 1990 Originals 535

puri f icat ion of funct ional ly intact 1 ,4-dihydropyridine/ calcium channe l complex f rom Mg2+-ATPase . H o w - ever, separa t ion f rom Mg2+-ATPase and o the r glyco- p ro t e in s is incomple te . T h e r e f o r e , a l t e rna t ive separa t ion p r o c e d u r e s such as s ize-exclus ion c h r o m a t o - g raphy and sucrose dens i ty -g rad ien t cen t r i fuga t ion remain impor tan t purif icat ion steps.

Acknowledgements We thank Ms. Ka th leen E. R o w a d e r and Drs. T e r e n c e L. Ki r l ey and Maur i c io Cace re s for the i r va luab le ass is tance and advice dur ing the cou r se of these exper imen t s . This work was s u p p o r t e d by Na t iona l Inst i tute of Hea l th Gran t R01 HL41088 ( to P.L.V.).

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Received: July 6, 1990 Accepted: July 16, 1990 A

536 Chromatographia Vol. 30, No. 9/10, November 1990 Originals

purification of functionally intact 1,4-dihydropyridine receptor from rabbit skeletal muscle by hplc

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