Proc. Natl. Acad. Sci. USAVol. 85, pp. 2969-2973, May 1988Biochemistry

Purification and affinity labeling of dihydropyridine receptor fromrabbit skeletal muscle membranes

(Ca2l channel/transverse-tubule membrane/photoaffinity labeling/Ca2" antagonist)

UWE KANNGIESSER, PETER NALIK, AND OLAF PONGSLehrstuhl fur Biochemie, Ruhr-Universitat Bochum, 4630 Bochum-Querenburg, Federal Republic of Germany

Communicated by Helmut Beinert, October 5, 1987 (received for review January 21, 1987)

ABSTRACT Undegraded dihydropyridine (DHP)-re-ceptor (putatively a voltage-gated Ca2" channel) has beenpurified as a 340-kDa protein complex to -'80% homogeneity(2.4 nmol of DHP-receptor per mg of protein) from rabbitskeletal muscle by a rapid purification protocol. Transverse-tubule membranes were prepared in high yield by Ribi-presstreatment. The DHP-receptor complex was solubilized in 1%digitonin followed by a two-step chromatographic purificationprocedure. The equilibrium dissociation constant of [3H]( + )-PN200-110 binding (Kd; 0.9 nM) was not significantly changedby solubilization or purification. The purified DHP-receptor iscomposed of two subunits with apparent molecular masses of148 kDa and 195 kDa migrating in polyacrylamide gels undernonreducing conditions as a single moiety of -z300 kDa. The195-kDa subunit was affinity-labeled with [3Hlazidopine inboth transverse-tubule membranes and purified DHP-receptorpreparations. The subunit can be degraded by high-energyirradiation to a 26-kDa peptide and by proteolysis to a 32-kDapeptide. Thus, it is probably due to proteolytic cleavage and/orphotolysis that neither purification nor affinity-labeling studieshave previously identified a DHP-receptor subunit of compa-rable molecular mass (195 kDa).

The passive movement of Ca2" ions across membranes of avariety ofexcitable tissues is mediated by voltage-gated Ca2 +channels (1, 2), some of which are modulated by dihydropy-ridine (DHP)-receptor agonists and antagonists (3-5). DHPantagonists are used clinically in the treatment of cardiovas-cular disorders (6, 7). In addition to heart muscle, DHP-re-ceptors have been found in a variety of other tissues, asshown by saturable, reversible, and high-affinity binding of[3H]DHPs (8-11). Transverse-tubule (T-tubule) membranesof skeletal muscle are comparatively rich in DHP-bindingsites (12). Recent reports suggest that a voltage-gated Ca2"channel is an integral part of the DHP-receptor in skeletalmuscle (13, 14).

Reports on the isolation and purification of the DHP-re-ceptor are controversial because a variety of differentNaDodSO4 gel patterns are found for the purified DHP-re-ceptor protein and the photoaffinity-labeled membranes(15-18). The present report demonstrates the purification ofa DHP-receptor of -340 kDa, consisting of two subunits of195 and 148 kDa. The larger subunit was photoaffinity-labeled with [3H]azidopine in both T-tubule membranes andpurified DHP-receptor preparations. The 195-kDa subunit issensitive to proteolytic degradation and photolysis. Ourresults suggest that degradation might yield protein bands oflower molecular mass (32-170 kDa) that have been describedfrequently as DHP-receptor subunits (16, 17, 19-21).

MATERIAL AND METHODSMaterials. [3H]Nimodipine (160 Ci/mmol; 1 Ci = 37 GBq),

[3H]nitrendipine (71.6 Ci/mmol), [3H]azidopine (53 Ci/mmol), [3H]( + )PN200-110 (70 Ci/mmol), and unlabeledDHPs were kindly provided by Bayer (Wuppertal, F.R.G.).Their purity was routinely checked by thin-layer radiochro-matography. Wheat germ lectin-Sepharose 6MB and pepsta-tin A were from Sigma, DEAE-Sephadex A 25 was fromPharmacia, and Staphylococcus aureus V8 protease (staphy-lococcal serine proteinase; endoproteinase Glu-C, EC3.4.21.19) was from Paesel (Frankfurt, F.R.G.). All otherchemicals were from Roth (Karlsruhe, F.R.G.), Merck,Sigma, or Serva (Heidelberg) and all were analytical grade.

Isolation of T-Tubules from Rabbit Skeletal Muscle. Rabbitswere killed by cervical dislocation. Back muscles werewashed free of erythrocytes by infusing the muscles with 20ml of Ringer's solution (120 mM NaCl/5 mM KCl/2 mMCaCI2/20mM glucose/1 mM sodium phosphate, pH 7.4) (22).Muscles were then dissected and homogenized with 5 vol of250 mM sucrose/0.2 mM EDTA in a Waring Blendor at 4°C.All subsequent experiments were done at 4°C. The clearedhomogenate (8000 x g, 15 min) was filtered through Mira-cloth (Chicopee Mills, Milltown, NJ). Membranes weresedimented (1 hr, 140,000 x g), washed in 250mM sucrose/2mM L-histidine, pH 7.4 (buffer A), and finally resuspended inbuffer A (seven strokes in a loosely fitting Dounce homog-enizer). The membranes were centrifuged through a linear15-50%o (wt/wt) sucrose gradient in 2 mM L-histidine (72,000x g, 12 hr). The heavy microsomal fraction migrated as avisible band at 40% sucrose. It was collected with a pipetteby suction, sedimented, washed, and resuspended in bufferA. The membrane suspension (protein concentration, 10mg/ml) was passed through a Ribi-press (Ribi Cell Fractio-nator, Mod RF-1, DuPont) and the eluate was again centri-fuged through a second linear 15-50%o (wt/wt) sucrosegradient (141,000 x g, 5 hr). The membrane fraction thatmigrated at 25% sucrose was collected with a pipette asdescribed above, washed with buffer A, and then rapidlyfrozen in liquid nitrogen.

Isolation of DHP-Receptor Complex. The solubilized (1%digitonin) DHP-receptor complex was purified by chroma-tography on wheat germ agglutinin (WGA)-Sepharose andDEAE-Sephadex columns (18). DHP-receptor was used inexcess of the WGA-Sepharose binding capacity such thatbinding of DHP-receptor compared to other protein materialwas optimized. Phenylmethylsulfonyl fluoride (1 mM) andpepstatin A (1 ,uM) were present in all solutions. Experimentswith DHP-receptor complexes were carried out under so-dium light. Protein concentrations were determined accord-ing to Bradford (23).

Photoaffinity Labeling of DHP-Receptor. T-tubule mem-branes. Membranes (0.25 mg of protein per ml) were incu-

Abbreviations: DHP, 1,4-dihydropyridine; WGA, wheat germ ag-glutinin; T-tubule, transverse tubule.

2969

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

June

11,

202

0

2970 Biochemistry: Kanngiesser et al.

bated in buffer B (50 mM Tris-HCI, pH 7.4/1 mM phenyl-methylsulfonyl fluoride/1 ,uM pepstatin A) for 90 min with5-10 nM [3H]nimodipine or [PHiazidopine, respectively.Nonspecific binding was determined with a 100-fold excess ofunlabeled DHP (1 uM). [3H]DHP-labeled membranes werepelleted (65,000 x g, 20 min) and washed with buffer B toremove the unbound ligand. The [3H]nimodipine-labeledmembranes resuspended in buffer B were irradiated for 40sec with a 500W high-pressure mercury lamp at wavelengths>320 nm (24). [3HlAzidopine-labeled membranes were irra-diated for 2 min with a black-light lamp (25) followed bypelleting and resuspension in sample buffer [3% NaDodSO4/10% (vol/vol) glycerol/12 mM EDTA/30 mM Tris HCl, pH6.8].

Partially purified DHP-receptor. [3HjNiModipine and[3H]azidopine-receptor complex were solubilized from T-tubule membranes and chromatographically purified on aWGA-Sepharose column (18). [3H]DHP-receptor complexwas eluted with 0.1% digitonin/100 mM N-acetyl-D-glucosamine/iO% (vol/vol) glycerol/1 MM CaCl2, and im-mediately irradiated as described above.

Proteolysis of[3H7azidopin'e-labeled T-tubule membranes.[3H]Azidopine-labeled membranes (140 ,ug) were treatedwith 0.5 ,ug of S. aureus V8 protease (1 hr at 40C), trans-ferred immediately into sample buffer, and submitted toPAGE.PAGE. Samples (pelleted membrane material or solubi-

lized DHP-receptor protein) were suspended and denaturedin sample buffer either under reducing conditions in thepresence of 20 mM dithiothreitol or under nonreducingconditions in the presence of 20 mM N-ethylmaleimide.PAGE was performed according to ref. 26. Gels for fluo-rography were stained with Coomassie blue, impregnatedwith enhancer (New England Nuclear) for 1 hr, size-reducedwith polyethylene glycol (27), dried, and exposed to pre-flashed Fuji x-ray film at -70°C. Gels were silver stainedaccording to the method of Merril et al. (28).

Binding Assays. DHP-binding activity of T-tubule mem-branes (1 mg/ml) and of purified DHP-receptor (100ng/assay) was measured as described (29). DHP-receptorconcentration (B.,,.) and equilibrium binding constants (Kd)were calculated by plotting bound/free DHP (B/F) vs. boundDHP (B) according to Scatchard (30).

A BA *~--A2W0

30-0.025-

120[

RESULTST-Tubule Membrane Preparation. T-tubule membranes

were prepared by means of Ribi-press homogenization.Breakup of triads was controlled by using sucrose gradientcentrifugation (Fig. 1). By applying a pressure of 9000 psi (1psi = 6.89 kPa), two membrane fractions with DHP-bindingcapacity were obtained (Fig. IA). The lighter fraction con-tained T-tubule membranes, and the heavier one containedapparently undisrupted triads. When a pressure of 15,000 psiwas applied, detachment ofT-tubules from terminal cisternaewas complete (Fig. 1C). This isolation technique yielded6-fold more T-tubule membranes (20 mg from 200 g of rabbitskeletal muscle) than the common Ca2' phosphate-loadingprocedure (3.5 mg from 200 g of rabbit skeletal muscle) (31).Compared to the latter procedure, T-tubule membranesobtained by Ribi-press treatment had a similar density ofDHP-binding sites (2-6 pmol per mg of protein) and a similarequilibrium dissociation constant (Kd) for binding of [3H]-azidopine'(0.32 nM) and [3H](+)PN200-110 (0.9 nM). There-fore T-tubule membranes prepared in this way are a suitablesource for further purification of DHP-receptor. The solu-bilized (1% digitonin) DHP-receptor sedimented in a 5-20%6linear gradient at 21 s2o,w (Fig. 2), which is consistent with svalues reported previously for solubilized DHP-receptor ofrabbit skeletal muscle (18), rat brain (29), and chicken heartmuscle (32).

Subunit Composition of DHP-Receptor. The solubilizedDHP-receptor complex was purified by WGA-Sepharoseand DEAE-Sephadex chromatography (18). The DHP-re-ceptor complex was eluted with a linear NaCl gradient fromthe DEAE-Sephadex column as a single peak at 75 ± 25 mMNaCl. Fig. 3A (lanes 4a, 4b, and 5) demonstrates that twopeptides of 195 and 148 kDa were copurified with the[3H]DHP-binding activity. In the native receptor, thesepeptides are probably linked by disulfide bridges. The DHP-receptor migrated as a single band with an apparent size ofapproximately 300 kDa under nonreducing conditions ofPAGE (Fig. 3A, lane 4c).

Specific binding of [3H](+)PN200-110 to the purifiedDHP-receptor preparation yielded a K of 0.9 nM-i.e., thesame value as obtained for binding of [ H](+)PN200-110 toT-tubule membranes. An apparent Bm value of 2.4 nmol ofreceptor per mg of protein was derived from Scatchardanalysis (Fig. 3B). Thus, if a molecular mass of 340 kDa for

-A280 C -.. A280

.40 W

02O0

- co)

FRACT ION

FIG. 1. Isolation of T-tubule membranes by Ribi-press treatment of microsomes. Aliquots of a crude microsomal fraction (5 mg of protein)were fragmented by Ribi-press treatment under different pressures (A, 9000 psi; B, 12,000 psi; C, 15,000 psi). Broken fragments were separatedin 2 mM L-histidine (pH 7.4) on a linear sucrose gradient [15-50%o (wt/wt); o]. Centrifugation was for 5 hr at 141,000 x g at 40C. Aliquots offractions of the sucrose gradient were assayed for DHP-binding activity (e) with 0.5 nM I3t](+)PN 200-110.

Proc. Natl. Acad. Sci. USA 85 (1988)

Dow

nloa

ded

by g

uest

on

June

11,

202

0

Proc. Natl. Acad. Sci. USA 85 (1988) 2971

5

4

0o

0 2

10 20FRACTION

30

I ZA

165x\155'118- _90- -

39- ON

M 1 2 M 3 4a 4b 4c

B

FIG. 2. Sucrose-density centrifugation of solubilized DHP-re-ceptor. T-tubule membranes (1 mg/ml) were incubated for 90 min at40C with 10 nM [3H]nimodipine in the absence (e) or presence (o) of1 j.M nimodipine. The labeled membranes were sedimented,washed, and solubilized with 1% digitonin in 185 mM KCI/1 mMphenylmethylsulfonyl fluoride/1 jLM pepstatin A/10 mM Hepes/Tris, pH 7.4, buffer. An aliquot of 0.25 ml of solubilized protein (1mg/ml) was layered on a 5-20% sucrose gradient. The gradient wascentrifuged for 80 min at 50,000 rpm (4°C) in a Sorvall TV 865 B rotor.Protein-bound radioactivity was determined in fractions by liquid-scintillation counting. Marker proteins for the s20,w value: thyroglob-ulin (19,2 s20,), immunoglobulin (7 s2o w).

the DHP-receptor is assumed, the receptor preparation hada purity of -80%. The purification yielded a 300-fold enrich-ment of DHP-receptor (Table 1). The data indicate thatcompared to previously reported purification protocols (17,18), a two step chromatographic procedure is sufficient topurify DHP-receptor. The protein pattern of purified DHP-receptor did not change after a second WGA-Sepharosechromatography (Fig. 3A, compare lanes 4 and 5). Anadditional peptide of -120 kDa was observed in somepreparations shown in Fig. 3A (lanes 4a and 4b). This peptideis not an integral part of the DHP-receptor, since it corre-sponds to an abundant protein in the starting material. Weconclude that the undegraded, purified DHP-receptor con-sists of two subunits of 148 and 195 kDa.

Affinity Labeling of DHP-Receptor. T-tubule membraneswere incubated with [3H]azidopine as described. [3H]Azido-pine was irreversibly and specifically incorporated into thelarge subunit of the DHP-receptor upon irradiation (Fig. 4A,lanes 1 and 2). A similar photoreaction was obtained withsolubilized [3H]azidopine-receptor complex irradiated afterWGA-Sepharose chromatography (Fig. 4B, lane 5). Labelingefficiencies of DHP-receptor were -2% in T-tubule mem-branes and -6% after solubilization and subsequent chro-matography.

Results presented here are in contrast to previous reportsas neither purification not affinity-labeling studies (15-19, 32)have previously identified a DHP-receptor subunit of com-parable molecular mass. It appears feasible that the reportedDHP-receptor peptides of smaller size have been generatedby proteolysis during purification. This may be concludedfrom the fact that the large [3H]azidopine-binding subunit israther susceptible to proteolysis in T-tubule membrane prep-

I

E

Ec

-s

M 5

3H-PN200-110 [nM]

FIG. 3. (A) PAGE of DHP-receptor preparations. The DHP-re-ceptor complex was isolated as described. Aliquots of the differentpurification steps were incubated with sample buffer under reducingconditions (except in lane 4c) and analyzed on an 8.75% polyacryl-amide gel (5 V/cm, 14 hr). Lanes 1-4b, stained with Coomassie blue;lanes 4c and 5, silver stained (28). Lanes: 1, T-tubule membraneproteins (20 j&g of protein); 2, digitonin extract (20 ,ug of protein); 3,eluate from the first WGA-Sepharose column (2 ,g of protein); 4a,eluate from the DEAE-Sephadex column (1 Zg of protein); 4b, eluatefrom the DEAE-Sephadex column, supplemented with thyroglobulin(indicated by an asterisk) as an internal marker protein (-330 kDa);4c, eluate from the DEAE-Sephadex column, PAGE was undernonreducing conditions (60 ng of protein); 5, eluate from the secondWGA-Sepharose column (100 ng of protein); M, marker proteins assize standards. Numbers at left correspond to kDa. (B) Binding[3H]( + )PN200-110 to purified DHP-receptor. Specific binding (B) of[3H](+)PN200-110 to purified DHP-receptor was measured as de-scribed. Bound [3H](+)PN200-110 (nmol per mg of protein) wasplotted against the respective [3H] radioligand concentration. (Inset)Plot of bound/free [3H]( + )PN200-110 (B/F) vs. bound [3H]( + )PN-200-110 according to Scatchard (30).

arations. To prove proteolysis [3H]azidopine-labeled T-tu-bule membranes were exposed to a limited amount of S.aureus V8 protease, and the proteolytic degradation of theaffinity-labeled DHP-receptor subunit was monitored byPAGE (Fig. 4C). Two specific degradation products (32 and81 kDa) were generated. Suspiciously, the molecular massesof both peptides are similar to values reported previously forDHP-receptor fragments (15, 18, 33).

Photoaffinity labeling of DHP-receptor using high-energyirradiation has been described (19). Such conditions, how-

19,2S

---A280

Biochemistry: Kanngiesser et al.

I

"I

II01

cr

olol

0.0.0.0-0.0-0-0-alO-e

Dow

nloa

ded

by g

uest

on

June

11,

202

0

2972 Biochemistry: Kanngiesser et al.

Table 1. Purification of DHP-receptorDHP-receptor Protein, Specific activity, Scatchard analysis,

Purification step pmol % mg pmol per mg of protein B,,n, pmol per mg of proteinT-tubule 120 100 40 3.0 8.1Digitonin extract 50 42 18 2.8 NDWGA-Sepharose 18 16 0.16 110 NDDEAE-Sephadex 4 3 0.01 400 2400DHP-receptor was labeled with 8-10 nM [3Hlnitrendipine prior to solubilization. Values are the mean of five different

experiments and are not corrected for dissociation of [3Hlnitrendipine-receptor complex during the purification. Bm a valueswere determined by binding of [3H](+)PN200-110 as shown for purified receptor in Fig. 3B. ND, not done.

ever, may damage the DHP-receptor. In particular, specificincorporation of [3H]nimodipine into a single T-tubule mem-brane protein was not observed under these conditions (Fig.5, lanes 1 and 2). Most of the major peptides reacted with[3H]nimodipine upon irradiation. Subsequently, [3H]nimodi-pine-receptor complex was partially purified by the sameprocedure as the [3H]azidopine-receptor complex describedabove. Irradiation of the partially purified [3H]nimodipine-receptor complex resulted in labeling an %26-kDa peptide(Fig. 5, lane 3). The results suggest that high-energy irradi-ation causes photolysis of the [3H]nimodipine binding pro-tein, while low-energy irradiation yields specific incorpora-tion of [3H]azidopine into the 195-kDa subunit of the DHP-receptor.

DISCUSSIONPrevious data on the isolation and purification of the DHP-receptor suggested that the DHP-receptor consists of onelarge (130-142 kDa) and one or two smaller (32-50 kDa)peptides (15, 18). In contrast, we have purified a DHP-re-ceptor consisting of only two large polypeptides of highmolecular mass: 148 kDa and 195 kDa, respectively (Fig. 3).The 148-kDa polypeptide corresponds to the 130- to 142-kDa

A B

165155Z

118- i90-

39-~~~.

123 5 yA

C

: 165.155-118- 90

polypeptide reported earlier (15, 18). The 195-kDa polypep-tide has not been documented previously. Assuming a stoi-chiometry ad3 the DHP-receptor would have a molecularmass of =340 kDa, which is in agreement with results oftarget size analysis (20). The purification protocol (Table 1)differs from previous ones in two major aspects: (D) T-tubulemembranes were prepared by a Ribi-press treatment and notby the Ca2" phosphate loading procedure (31); (it) DHP-re-ceptor protein was used in excess of the WGA-Sepharose-binding capacity to optimize binding of DHP-receptor ascompared to other protein material.The 195-kDa DHP-receptor subunit was affinity-labeled

with [3H]azidopine in T-tubule membranes and in purifiedDHP-receptor preparations. The experiments in Fig. 4Cshow that this DHP-receptor subunit is susceptible to pro-teolysis in T-tubule membranes. A 32-kDa peptide wasgenerated by S. aureus V8 protease. It is similar in size to asmall DHP-receptor fragment reported earlier (15, 18).We have identified high-energy irradiation as a further

source of degradation of the DHP-receptor in affinity-labeling experiments (Fig. 5). Photolysis might thereforeexplain the results of previous affinity-labeling experimentsthat have suggested various DHP-binding peptides in therange of 32 to 170 kDa (16, 17, 19-21). As a furtherconsequence, discrepancies in the peptide compositions ofpurified DHP-receptor may also derive from proteolyticcleavage that readily occurs during isolation of membraneproteins (34). Therefore, the DHP-receptor protein of 340kDa described here has been eluted from DEAE-Sephadex at2-fold higher salt concentrations than reported earlier (18). In

BA

155'l..

11 8-- 39

..'4w,

': -165155

-118

- 90

39-6 7 8

FIG. 4. Photoaffinity labeling of DHP-receptor with [3H]azido-pine. (A) T-tubule membranes were incubated with 5 nM [3H]azido-pine in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 1AtM nitrendipine. After irradiation, [3H]azidopine-labeled peptideswere separated by PAGE in an 8.75% polyacrylamide gel underreducing conditions. The gel was stained with Coomassie blue (lanes3 and 4) and was subsequently fluorographed (lanes 1 and 2).Exposure time was 7 days. Molecular size standards are the same asin Fig. 3. (B) Partially purified [3H]azidopine-receptor complex wasirradiated. Fluorography (lane 5) and PAGE as well as staining (lane6) of [3Hlazidopine-labeled peptides were the same as inA. Exposuretime was 40 days. (C) [3H]Azidopine-labeled and irradiated T-tubulemembranes were treated with S. aureus V8 protease for 1 hr at 40C.Proteolytic fragments were separated by PAGE for fluorography asdescribed above. Peptides were labeled with [3H]azidopine in theabsence (lane 7) and in the presence (lane 8) of 1 IAM (+ )-PN200-110.Exposure time was 40 days.

- 39

1 2 3 4

FIG. 5. Photoaffinity labeling of DHP-receptor with [3Hlnimo-dipine. (A) T-tubule membranes were incubated with 10 nM[3H]nimodipine in the absence (lane 1) or presence (lane 2) of 1 /AMnimodipine. After irradiation, [3H]nimodipine-labeled peptides wereseparated by PAGE, stained, and fluorographed as described in Fig.4. Exposure time was 40 days. Molecular size standards are the sameas in Fig. 3. (B) Partially purified [3H]nimodipine-receptor complexwas irradiated. Fluorography (lane 3) and PAGE (lane 4) of[PHinimodipine-labeled peptide (lane 3) were the same as in A.

Proc. Natl. Acad Sci. USA 85 (1988)

Dow

nloa

ded

by g

uest

on

June

11,

202

0

Proc. Natl. Acad. Sci. USA 85 (1988) 2973

conclusion, undegraded DHP-receptor does not containsmall peptides as subunits. The DHP-receptor complex iscomposed of only two subunits (a, f3) with molecular massesof 195 and 148 kDa.

The authors are indebted to Dr. N. R. Brandt for advice in theRibi-press treatment of membrane fractions. Special thanks are dueto Dr. Bellemann of Bayer, Wuppertal, F.R.G., for his extensivetechnical support and continuously stimulating advice and discus-sion. Many thanks to Prof. Dr. A. Maelicke, Max-Planck-Institut,Dortmund, F.R.G., for fruitful discussions. This work was supportedby a grant (SFB 168) of the Deutsche Forschungsgemeinschaft.

1. Tsien, R. W. (1983) Annu. Rev. Physiol. 45, 341-358.2. Reuter, H. (1983) Nature (London) 301, 569-574.3. Janis, R. A. & Triggle, D. J. (1983) J. Med. Chem. 26, 775-785.4. Schramm, M. & Towart, R. (1985) Life Sci. 37, 1843-1860.5. Bellemann, P. (1986) in Innovative Approaches in Drug Re-

search, ed. Harms, A. F. (Elsevier Science, Amsterdam), pp.23-46.

6. Stone, P. A., Antman, E. M., Muller, J. E. & Braunwald, E.(1980) Ann. Int. Med. 93, 886-904.

7. Fleckenstein, A. (1977) Annu. Rev. Pharmacol. Toxicol. 17,149-166.

8. Bellemann, P., Ferry, D. R., Lubbecke, F. & Glossmann, H.(1981) Arzneim.-Forsch. 31, 2064-2067.

9. Gould, R. J., Murphy, K. M. M. & Snyder, S. H. (1982) Proc.Nati. Acad. Sci. USA 79, 3656-3660.

10. Bellemann, P., Schade, A. & Towart, R. (1983) Proc. Nati.Acad. Sci. USA 80, 2356-2360.

11. Ferry, D. R. & Glossmann, H. (1982) FEBS Lett. 148, 331-337.12. Fosset, M., Jaimovich, E., Delpont, E. & Lazdunski, M. (1983)

J. Biol. Chem. 258, 6086-6092.13. Curtis, B. M. & Catterall, W. A. (1986) Biochemistry 25,

3077-3083.14. Flockerzi, V., Oeken, H. J., Hofmann, F., Pelzer, D., Cavalid,

A. & Trautwein, W. (1986) Nature (London) 323, 66-68.

15. Borsotto, M., Barhanin, J., Norman, R. I. & Lazdunski, M.(1984) Biochem. Biophys. Res. Commun. 122, 1357-1366.

16. Ferry, D. R., Kdmpf, K., Goll, A. & Glossmann, H. (1985)EMBO J. 4, 1933-1940.

17. Galizzi, J.-P., Borsotto, M., Barhanin, J., Fosset, M. &Lazdunski, M. (1986) J. Biol. Chem. 261, 1393-1397.

18. Curtis, B. M. & Catterall, W. A. (1984) Biochemistry 23,2113-2118.

19. Campbell, K. P., Lipshutz, G. M. & Denney, G. H. (1984) J.Biol. Chem. 259, 5384-8387.

20. Venter, J. C., Fraser, C. M., Schaber, J. S., Jung, C. Y.,Bolger, G. &Triggle, D. J. (1983) J. Biol. Chem. 258,9344-9348.

21. Kirley, T. L. & Schwartz, A. (1984) Biochem. Biophys. Res.Commun. 123, 41-49.

22. Lau, Y. H., Caswell, A. H. & Brunschwig, J.-P. (1977) J. Biol.Chem. 252, 5565-5574.

23. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254.24. Gronemeyer, H. & Pongs, 0. (1980) Proc. Natl. Acad. Sci.

USA 77, 2108-2112.25. Ferry, D. R., Rombusch, M., Goil, A. & Glossmann, H. (1984)

FEBS Lett. 169, 112-118.26. Laemmli, U. K. (1970) Nature (London) 227, 680-685.27. Palumbo, G. & Tecce, M. F. (1983) Anal. Biochem. 134,

254-258.28. Meml, C. R., Goldmann, D., Sedman, S. A. & Ebert, M. H.

(1981) Science 211, 1437-1438.&

29. Curtis, B. M. & Catterall, W. A. (1983) J. Biol. Chem. 258,7280-7283.

30. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51, 660-672.31. Rosemblatt, M., Hidalgo, C., Vergara, C. & Ikemoto, N. (1981)

J. Biol. Chem. 256, 8140-8148.32. Rengasamy, A., Ptasienski, J. & Hosey, M. M. (1985) Bio-

chem. Biophys. Res. Commun. 126, 1-7.33. Goll, A., Ferry, D. R. & Glossmann, H. (1983) FEBS Lett. 157,

63-69.34. Casadei, J. M., Gordon, R. D. & Barchi, R. L. (1986) J. Biol.

Chem. 261, 4318-4323.

Biochemistry: Kanngiesser et al.

Dow

nloa

ded

by g

uest

on

June

11,

202

0