The Cystine-Stabilized a-Helix: A Common Structural Motif of Ion-Channel Blocking Neurotoxic Peptides YUJl KOBAYASHI,’ * HlROYUKl TAKASHIMA,* H A R U H I K O TAMAOKI,’ YOSHIMASA KYOGOKU,’ PAUL LAMBERT,’ HISAYA KURODA,’ N A O Y O S H I CHINO,’ TAKUSHI X. WATANABE,’ TERUTOSHI KIMURA,3 SHUMPEI SAKAKIBARA,’ and LUIS M O R O D E R 4 ‘Institute tor Protein Research, Osaka University, Suita, Osaka 565, *Research Laboratories, Kuraray Co , L td , Kurashiki, Okayama 710, ’Peptide Institute, Inc , Protein Research Foundation, Ina, Minoh, Osaka 562, japan, and ‘Max Planck lnstitut fur Biochernie, 8033 Martinsried be1 Munchen, Federal Republic of Germany

SYNOPSIS

Neurotoxic peptides from venoms of scorpions and honey bees exhibit a consensus pattern in the two disulfide bridgings related to the sequence portions Cys-X-Cys and Cys-X-XX-Cys. A revised three-dimensional structure of charybdotoxin, as determined by twodimensional nmr spectroscopy, confirms that the consensus cystine dislocation generates in all these toxins a common structural element, i.e., the cystine-stabilized a-helical (CSH ) motif, which may be correlated with their common ion channel blocking activity.

INTRODUCTION From venoms of scorpions’ and honey bees,2 lowmass cystine-rich neurotoxic peptides have been isolated, which were recognized as potent inhibitors of ion channels of vertebrate tissues and are used as tools in the isolation of channel proteins and in studies concerning their physiological functions. These neurotoxins are classified principally into inhibitors of Na* and K + channels and their respective subclasses, as shown in Figure 1. A comparison of the amino acid composition of these neurotoxins a t first does not disclose any sequence homology, although their similar activity profile is expected to rely on some common structural features that would enable the toxins to bind to and thus to disturb the ion flows. Analysis of the three-dimensional structure of’ these peptides should possibly allow the answer to the question of whether common structural elements are involved in interfering the neurotransmission a t the ion channel level and of which structural features impart the selectivity for the various channels. Reduction and S-alkylation of the neuBiopolymers, \ < > I 31, 1213-1220 (1991) C, 1991 ,John Wiley & Sons, Inc.

CCC 0006-3.525/91/10lZ15-~R$04.00

* To whom correspondence should be addressed.

rotoxins is known to abolish their secondary structures and thus their channel-blocking a c t i ~ i t y . ~ . ~ Therefore the disulfide bridging conformationally restricts and stabilizes spatial structures that are essential for the biological activity. A comparative analysis of the cysteine pairings in the neurotoxins listed in Figure 1 reveals a n interesting consensus pattern consisting of one pair of half-cystines spaced by a tripeptide sequence, i.e., Cys-X-X-X-Cys, disulfide bonded to a second pair of half-cystines with only one amino acid residue in between, i.e., CysX-Cys. Thereby the way of cross-linking generates a parallel alignment of the peptide backbones of the two bridged sequence portions. A similar disulfide pattern was also observed for endothelin, a peptide factor of mammalian origin5 and for sarafotoxin of reptilian origin6;both exhibit biological activities unrelated t o the above-mentioned neurotoxins. For endothelin and sarafotoxin, however, the cysteine pairings reverse the direction of the peptide backbones in the two cross-linked sequence portions. T h e three-dimensional structure of endothelin in solution was determined in our laboratory by the combined use of nmr measurements and distance geometry calculations.’ It revealed the presence of a n a-helix spanning the Cys-X-X-XCys sequence and stabilized via disulfide bonding to 1213

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KOBAYASHI ET AL.

Channel*

Subclass**

Inhibitor H o n e v bee Apamin2 MCD2 T e r ti a p i n 2 6

QJCKA PETAL m

C QQH-NH2 I K m C KRHVI KPHIC RKICG KN-NH2 A L m C NRIII P H M m KKCGK K-NH2

K+ K+ K+

C a - a c t- 1 V-act V-act

K+ K+ K+

Ca - act -h Ca - act - h C a - a c t- 1

Na'

OL

Na+

P

Scomion NTx27 ChTX28 Leuirotoxin AaH 111 C s E v31

TIINV pEFTNV AFCNL VKDGY PYGNA KECYL CYAFA

KCTSP SCTTS RMCQL IVDDV CYDK VKKSD m m G

KQCSK KEmS SmSL NCTYF LPNHV GCKYG LPEST

PCKEL VCQRL GLLGK CGRNA RTKGP CLKLG PTYPL

YGSSA GAKCM NGKCK a X N

HNTSR GKCMN K K m C YS CIGDK C E p K H-NH2 YQJEE CTKLK GESGY CQWAS GRCH-NH2 ENEGC DTECK AKNQG GSYGY PNKSC

Human E n d o t h e l in5 ( E T - 1)

CSCSS LMDKE P Y F C H L D I I W

Snake Sarafotoxin6

CSCKE MTDKE CLYFC HQDVI W

(S6b)

Figure 1. Amino acid sequences of neurotoxic peptides from venoms of honey bees and scorpions and their classification according to their ion channel target. K + : K + channel; Na': Na' channel; Ca-act-1: low-conductance Ca'+ -activated K + channel; Ca-act-h: highconductance Ca2+-activated K' channel; V-dep: voltage-dependent K C channel; a: inactivation of Na' channel; @: interference in the activation phase. For comparison, other peptides with a similar disulfide bridging pattern are reported, i.e., endothelin, a vasoconstricting peptide from endothelial cells, and sarafotoxin, a toxic peptide from snake venom with vasoconstricting activity. The four half-cystine residues involved in the CSH motif are marked in boldface and are underlined. The toxins targeted against K' channels were reviewed in Ref. 29.

the Cys-X-Cys portion, itself folded in a n extended 0-strand conformation. This peculiar spatial arrangement was then also encountered in the solution structure of apamin' and in the crystalline CsE v3 scorpion toxin, although as mentioned above with a parallel alignment of the two disulfide-bridged sequence portions. This observation prompted us to propose it as a new structural i.e. as a cystine-stabilized a-helical ( C S H ) motif. The general character of this new structural motif was subsequently confirmed by the solution struc-

ture of the mast cell degranulating peptide ( MCD ) determined recently in our laboratory by nmr measurements" and by the tertiary structure of leiurotoxin I reported by Martins et al." It was therefore of great surprise that for charybdotoxin ( ChTX) a solution structure was determined by twodimensional nmr spectroscopy and distance geometry calculations, which despite the presence of the characteristic cystine pattern consists of three antiparallel P-strands without helical content 1 2 ; this proposed structure differs remarkably from those

Figure 2. A section of the NOESY spectrum of ChTX in water. Synthetic ChTX was dissolved in H 2 0 containing 5% D 2 0 and 10% deuterated acetic acid at a 5 m M concentration. The spectrum was obtained at 500 MHz on a Jeol GSX-500 spectrometer at 2OoC with a mixing time of 200 ms. In the lower part sequential assignments via intraresidual C,H-NH NOE connectivities and interresidual C,H-NH NOE connectivities within the adjacent residues are drawn by vertical and horizontal lines, respectively. The positions of intraresidual C,H-NH NOE cross peaks are indicated by the residue number. Intraresidual NOE connectivities of the Trp 14 residue were used as starting spin system for the sequential assignments of the spin systems detected in the DQF-COSY and HOHAHA spectra. In the upper part the NH-NH cross peaks of adjacent residues are labeled by the residue numbers and indicate the presence of the a-helical structure.

T H E CYSTINE-STABILIZEDa-HELIX 8.0

9.0

7.0

PPm

9.0

8.0

7.0

6.0

35

61

3

5 .O

4.0

3.0

1215

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KOBAYASHI ET AL.

already known for other neurotoxic peptides. From this spatial folding of ChTX, Massefsky e t a1.12have correlated shape, size, and charge distributions with a convincing mechanism of action of this neurotoxin a t the K+-channel level. In the context of our structural analyses of these series of neurotoxic peptides aimed a t disclosing common structural features possibly involved in a general mechanism of action a t membrane level, the three-dimensional structure of synthetic ChTX was determined adopting nmr procedures and distance geometry calculations similar to those used previously for the conformational analysis of endothelin and MCD." Since the structure resulting from this study again contained the CSH motif, but significantly differed from that meanwhile reported by Massefsky et al., l 2 a careful reexamination of all the spectroscopic data was carried out to allow for a definitive structure elucidation (for preliminary report, see Ref. 1 3 ) .

RESULTS AND DISCUSSION T h e synthetic ChTX used for the conformational studies was carefully analyzed in terms of the correct disulfide pairings as present in the natural compound, in order to avoid misinterpretation of the nmr data.I4 The sequence-specific resonance assignments were performed according to the procedure of Wuthrich l5 in two steps. Using the double-quantum filtered correlated spectroscopy ( DQF-COSY ) and homonuclear Hartmann-Hahn ( HOHAHA) spectra, the spin systems of each amino acid residue were identified by through-bond interactions. Then sequential nuclear Overhauser effect ( N O E ) connectivities between neighboring spin systems were identified from NOE spectroscopy ( NOESY ) spectra by through-space NOE interactions between C,H(i)-NH(i l ) , NH(i)-NH(i 1) and COH ( i) -NH ( i 1) . The results of the peak assignments are demonstrated in the NOESY spectrum of ChTX in water (Figure 2 ) , where the intraresidual C,H-NH coupling peaks are indicated by residue 1) numbers and the sequential C,H ( i ) -NH ( i NOE connectivities are traced with bold lines. T h e analysis of the NOESY spectra provides information about the interatomic distances in the molecule and these are summarized in Figure 3. The sequential and medium-range NOEs reported in Fig. 3 ( 1) allowed us to identify the secondary structure elements of the peptide backbone. Strong NOEs between N H ( i ) and N H ( i 1) were observed ho-

+ +

+

+

+

mogeneously in the sequence region Lysll-Glnl8, suggesting a n a-helical folding of this part of the molecule. Supportive for this conformation are the medium-range NOEs between C,H ( i ) and CBH( i 3 ) and between C,H ( i) and N H ( i 3 ) observed in this sequence region. Furthermore, the H --* D exchange experiments revealed low-exchange rates for amide protons involved in the hydrogen bonds in this segment of ChTX. Successive strong NOEs between C,H (i) and N H ( i 1) are indicative for p-sheet or p-turn structures in the Arg25-Ser37 region. Additionally, the relatively low chemical shifts of C,H protons observed in this sequence portion, particularly for the residues Gly26, Lys32, Cys33, and Cys35, support the existence of a p-sheet structure. In order to further characterize the chain folding of the whole molecule, the mutual proximities between residues, a s detected by NOEs on their protons, are reported in the triagonal distance map l6 shown in Figure 3 ( 2 ) . The small distances along the diagonal agree with the above-mentioned helical structure and the line of the small distances perpendicular to the diagonal reveals the antiparallel arrangement of the /I-strands. T h e pattern of the psheet drawn in Figure 3 ( 3 ) was constructed taking into account the strong interstrand NOEs between C,H ( i) -C,H ( j ) protons; the slow exchange rates observed for the backbone amide protons of Va15, Cys7, Lys27, Met29, Lys32, Cys33, and Arg34 support the existence of this p-sheet. The NOE information map of Figure 3 ( 2 ) , which contains the experimentally determined specific and short interproton distances, i.e., sequential, medium-, and long-range NOEs, was then used for a quantitative treatment of the nmr data by distance geometry calculations with the DADAS program17 following procedures described p r e v i o ~ s l y . ' ~For ~'~ side-chain proton NOEs, not assigned stereospecifically, interpretation by the pseudoatom method was performed as suggested by Wuthrich.20The shortrange NOEs observed between intraresidual protons or between C,H-NH, NH-NH, and COH-NHin adjacent residues were calculated with a rigid model, whereas the long-range NOEs between pairs of protons in the molecule were assumed t o be within 4 as the postulated threshold distance value for detectable connectivity." T h e tertiary structure was determined by minimizing the differences between interatomic distances in the calculated structure and the distance constraints derived from the nmr data. For this purpose 227 interresidual NOEs, i.e., 115 sequential and 112 long-range NOEs, as well as the three disulfide constraints, were used. For the minimization trials, 75 random structures served as

+

+

+

w

T H E CYSTINE-STABILIZED a-HELIX 5

1

1

pE

1

10

15

20

25

30

1217 35

F T N V S C T T S K E C W S V C Q R L H W T S R G K C M W K K C M N K K C R C Y S @O@ 00 000 @ @ @ @ @

10

5

15

20

25

33

35

Residue

Figure 3. ( 1 ) Sequential and medium-range NOEs determined in the NOESY spectrum of ChTX. dNN: NOE between NH ( i ) and NH ( i 1 ) where ( i ) represents the residue number; daN: NOE between C,H(i) and N H ( i 1 ) ;dPN: NOE between C,H(i) and NH ( i 1); dab ( i, i 1) : NOE between C,H ( i) and CBH( i + 3 ) as index of a-helical structures. The thickness of the lines reflects the relative intensities of cross peaks in the NOESY spectrum. Residues with slowly exchanging backbone amide protons in H -+ D exchance experiments at 2OoC are marked with 0 for detectable NH resonance 1 h after dissolving the sample in D,O, and with @ for detectable NH resonance more than 5 h after dissolution of the sample in D20. ( 2 ) Distance map resulting from the NOE connectivities determined in the NOESY spectrum of ChTX. Solid squares at cross-positions between residues i and j indicate NOEs between backbone-backbone and/or backbone-side-chain protons of these residues. ( 3 ) /I-Sheet pattern of ChTX. This was constructed by the use of the sequential NOEs summarized in part 1 and of the detected interstrand long-range NOEs indicated with arrows. Slowly exchanging backbone amide protons involved in hydrogen bonding are marked with circles. The chiralities of the C,H’s are indicated with solid triangles, thus defining the orientation of the side chains of the corresponding residues. The conserved residues in the three scorpion toxins (see text) are marked with squares.

+

+

starting conformers. Among the resulting conformers, the five fitting best to the constraints were selected and compared in order to judge their convergence. The root mean square distance (rmsd) for

+ +

the peptide backbone atoms of the five structures was 1.46 A, a value that supports the correctness of the proposed structure. The resulting three-dimensional structure of

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KOBAYASHI ET AL

ChTX is reported in Figure 4* by a ribbon drawing of the peptide backbone of the best fitting conformer. Its folding pattern consists of the following suc cessive secondary structure elements: extended P-structure (Va15-Cys7), P-turn (Thrg-ThrS), a-helix ( L y s l l - G l n l 8 ) , P-turn (His21-Asn22), P-structure (Arg25-Met29),P-turn (Asn30-Lys31), P-structure (Lys32-Tyr36). The existence of the a-helix and @-sheet structures is fully consistent with the results of the sequential NOEs analysis discussed above. The presence of the three turns agrees with the strong NH-NH NOE connectivities observed in the corresponding peptide bonds as shown in Figure 2. T h e extended part of the peptide in the N-terminal region seems to constitute a Pstrand involved in a three-strand P-sheet; on one side it contacts the 0-strand Lys32-Tyr36 and on the other side it is disulfide-bonded via Cys7 /Cys28 to the @-strandArg25-Met29. The a-helical part is placed above the three-stranded @sheet and linked to it via the disulfide bridges Cys13/Cys33 and Cysl7 / cys35. This solution structure of ChTX differs significantly from that reported by Massefski et al., l 2 e.g., in the position of the P-sheet and in the presence of the a-helical stretch. The reason for this discrepancy is difficult to detect, since the one-dimensional spectra in H 2 0 and D 2 0 and the HOHAHA spectrum in D 2 0 are very similar to those of our measurements. Differences, however, are observed in the assignments. While in our sequential assignment no disconnection was encountered and the residues 14, 21, and 36 could be unambiguously assigned by clearly detected intraresidual NOEs between the ring protons and C,H, C,H, and NH, the sequential assignment of Massefsky et a1.12 is disconnected a t five residues and the above-mentioned residues 14, 21, and 36 are differently assigned. On the other side, the structure proposed in the present study fully agrees with those determined for the other members of the family of neurotoxins and exhibits the CSH motif as expected from the characteristic disulfide pattern. Furthermore, it is confirmed by a similar nmr conformational analysis performed independently from us by Bontemps et al.*l With the resolution of the tertiary structure of the last member of the known neurotoxic peptides capable of inhibiting ion channels, it is reasonable t o attribute to the CSH motif a crucial function for the juxtaposition of specific residues involved in binding of the toxins to the channel proteins. In this context the amino acid composition of the interven-

* See color art.

ing sequences could possibly act merely as spacers as long as the characteristic consensus pattern of disulfide bridging is retained. This working hypothesis is also supported by superimposition of the ChTX primary and secondary structure with those portions of the CsE v3 scorpion toxin and leiurotoxin I, which contain the CSH motif as shown in Figure 5 . A surprising convergence of the secondary structure elements is observed, thus strongly suggesting that even in the higher molecular weight scorpion toxin only this portion may act as binding head. Positively charged amino a n d / or guanido groups are known to strongly contribute to the binding of the channel entryway via salt bridges with functionally important negative charges2*;therefore a particular spatial array of such basic residues may play a n essential role. In fact, a comparison of the tertiary structures leads to the interesting observation that in apamin and leurotoxin I two arginine residues are located on the same side of the respective a-helices"; in MCD a lysine and a n arginine residue are positioned on the same face of an amphiphilic helix. Similarly, in ChTX the side chains of LyslO located in the helix and of Argl9 positioned a t the end of this helical segment are also displaced on the same face as shown in Figure 4. Furthermore, it is noteworthy that the four positively charged residues of the P-sheet of ChTX, i.e., Arg25, Lys27, Lys32, and Arg34, are clustered on the same side of the sheet, but opposite to that where the helix is mounted on. On this side the contact to the helix relies on Gly26 and on the three half-cystine residues 28,33, and 35. These four contact residues are conserved in the three toxins from scorpion venom (see Figure 5 ) , which act on the same subclass of C a 2 + activated K channels. Particularly Gly26 is interesting in this context, since the absence of a side chain allows the close contact of the P-sheet to the helix whereas the contact surface of the helix results from the two half-cystine residues. T o conclude, a clustering of positive charges as dictated by the folding of the molecules is observed and this may provide the specific interactions with the channel proteins. From this study we also learned that the CSH motif represents a highly stable local structure, which may act as a protein subdomain and thus be implicated, whenever present, as a n initiation structure in protein folding processes. This hypothesis is further confirmed by the observation that air oxidation of the synthetic neurotoxic cysteine peptides leads almost quantitatively to the correct disulfide pairing as long as conditions are used that assure establishing of thermodynamic eq~i1ibria.l~ This ~ ' ~ fact may facilitate, together with other statistical method^,'^ the prediction of cysteine +

T H E CYSTINE-STABILIZEDa-HELIX

Figure 4. ( 1 ) Ribbon drawing of the peptide backbone structure of the ChTX conformer with the smallest difference values between calculated and experimentally obtained interatomic distances. The side chains are represented in ball-and-stick type. ( 2 ) The arginine and lysine side chains are drawn in purple and blue, respectively, whereas the other residues are represented in yellow to better illustrate the clustering of the positively charged side chains of Arg25, Lys27, Lys32, and Lys34 on one face of the P-sheet, and the second set with Lys20 and Argl9 protruding from the a-helical segment into the opposite direction. The disulfide bonds are represented in green.

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KOBAYASHI ET AL.

1

K

CSl.: v 3

li

G

Y

5 I.

10

V

K

K

S

Charybdotoxin I.ciurotoxin I

D pu

G I'

hclix

turn

_ _ . ~ ~ __-__ _ ~ ~ _ . . _

35

K-

N L -

Q II S

G N L

T G G

S S L

Y R

TG

L

G

iY K

lC C

MY I

~

turn

P

E

S

55 T P

shcct

T

Y

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60 L P

K i b R i Y :'

N A

F

A

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W

C

E

G

I.

G

D

K

C

K

C

V

K

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-

sheet

turn

N

K

S

65 C

Figure 5. A comparison of the three scorpion toxins CsE v3, leiurotoxin I, and ChTX in terms of secondary structure elements by aligning the sequences according to the four conservative half-cystine residues marked with squares. Insertions or gaps were necessary for this alignment and the common secondary structure elements are underlined by bars.

pairings in proteins, but also the de novo design of proteins with particular functions. Note added inproof. After the present study was completed the publication of the three-dimensional structure of CHTX by W. Massefski et a1.I' was retracted by the aut h o r ~ and ~ ~a structure analysis with results similar to ours was reported by F. Bontems et LM gratefully acknowledges the receipt of a visiting professorship in 1990 from the Osaka University.

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2. 3. 4. 5.

6. 7.

Architecture of Proteins and Enzymes, Bradshaw, R. A. & Tang, J., Eds., Academic Press, Orlando, FL, p. 65. Gauldie, J., Hanson, J. M., Shipolini, R. A. & Vernon, C. A. (1978) Eur. J. Biochem. 83,405. Miroshnikov, A. I., Elyakova, E. G., Kudelin, A. B. & Senyavina, L. B. (1978) Bioorg. Khim. 4, 1022. Walde, P., Jackle, H., Luisi, P. L., Dempsey, C. J. & Banks, B. E. C. (1981) Biopolymers 20, 373. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K. & Masaki, T. (1988) Nature 3 3 2 , 411. Takasaki, C., Tamiya, N., Bdolha, A., Wollberg, Z. & Kochva, E. (1988) Toxikon 26,543. Tamaoki, H., Kobayashi, Y., Nishimura, S., Ohkubo, T., Kyogoku, Y., Nakajima, K., Kumagaye, S., Kimura, T. & Sakakibara, S. ( 1991) Protein Engng, 4,

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9. Almassy, R. J., Fontecilla-Camps, J. C., Suddath, F. L. & Bugg, C. E. ( 1983) J . Mol. Biol. 170, 497. 10. Kobayashi, Y., Sato,A., Takashima, H., Tamaoki, H., Nishimura, S., Kyogoku, Y., Ikenaka, K., Kondo, T., Mikoshiba, K., Hojo, H., Aimoto, S. & Moroder, L. ( 1991) Neurochem. Int. 1 8 , 525. 11. Martins, J. C., Zhag, W., Tartar, A., Lazunski, M. & Borremans, F. A. M. (1990) FEBS Lett. 260, 249. 12. Massefski, W., Redfield, A. G., Hare, D. R. & Miller, C. (1990) Science 249, 521. 13. Takashima, H., Kobayashi, Y., Tamaoki, H., Kyogoku, Y., Lambert, P. F., Kuroda, H., Chino, N., Watanabe, T. X., Kimura, T. & Sakakibara, S . ( 1991) in Peptides 1990, Giralt, E. & Andreu, D., Eds., ESCOM, Leiden, p. 557. 14. Lambert, P., Kuroda, H., Chino, N., Watanabe, T. X., Kimura, T. & Sakakibara, S. (1990) Biochim. Biophys. Res. Commun. 170,684. 15. Wuthrich, K. (1986) in N M R of Proteins and Nucleic Acids, John Wiley & Sons, New York. 16. Levitt, M. & Greer, J . (1977) J. Mol. Biol. 114, 181. 17. Braun, W. & Go, N. (1985) J . Mol. Biol. 25,611. 18. Ohkubo, T., Kobayashi, Y., Shimonishi, Y. & Kyogoku, Y. (1986) Biopolymers 25, s123. 19. Kobayashi, Y., Ohkubo, T., Kyogoku, Y., Nishiuchi, Y., Sakakibara, S., Braun, W. & Go, N. (1989) Biochemistry 28, 4853. 20. Wuthrich, K., Billeter, M. & Braun, W. (1983) J. Mol. Biol. 169, 949. 21. Bontemps, F., Roumestand, C., Gilquin, B.,Boyot, P., Menez, A. & Toma, F. ( 1990) in 14th International Conference of Magnetic Resonance in Biological Systems, Abstract P7. 22. Smith, C., Phillips, M. & Miller, C. (1986) J . Biol. Chem. 2 6 1 , 14607. 23. MacKinnon, R. & Miller, C. ( 1989) Biochemistry 28, 8087. 24. Nakajima, K., Kumagaye, S., Nishio, H., Kuroda, H., Watanabe, T. X., Kobayashi, Y., Tamaoki, H., Kimura, T. & Sakakibara, S. (1989) J . Cardiouasc. Pharmacol. 13, s8. 25. Muskal, S. M., Holbrook, S. R. & Kim, S.-H. (1990) Protein Engng. 3, 667. 26. Hider, R. C. & Ragnarsson, V. ( 1981) Biochim. Biophys. Acta, 667, 197. 27. Possani, L. D., Nartin, B. M. & Svendensen, I. ( 1982) Carlsberg Res. Commun. 47, 285. 28. Gimenez-Gallego, G., Navia, M. A., Reuben, J. P., Katz, G. M., Kaczyorowski, G. J. & Garcia, M. L. (1988) Proc. Natl. Acad. Sci. USA, 85, 3329. 29. Moczydlowski, E., Lucchesi, K. & Ravindran, A. (1988) J . Membrane Biol. 105,95. 30. Massefski, W., Redfield, A. G., Hare, D. R. & Miller, C. (1991) Science, 252, 631. 31. Bontems, F., Roumestand, C., Boyot, P., Gilquin, B., Doljansky, Y., Menez, A. & Toma, F. (1991) Eur. J . Biochem. 1 9 6 , 19. Received April 10, 1991 Accepted May 8, 1991