ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

176, 465-471

(1976)

Stimulation of Adenylate Cyclase in Washed Pigeon Erythrocyte Membrane with Cholera Toxin and Its Subunits’ A. WODNAR-FILIPOWICZ Roche

Institute

of Molecular Received

AND

Biology,

Nutley,

March

29, 1976

C. Y. LAP New

Jersey

07110

Cholera toxin was found to stimulate adenylate cyclase activity in washed membrane of pigeon erythrocytes in the presence of dithiothreitol and NAD. When tested with isolated cholera toxin components, the stimulatory activity was found with subunit A or polypeptide Al derived from this subunit, but not with A2 or subunit B. On a molar basis, polypeptide Al was approximately four times more active than cholera toxin. Dithiothreitol was not required in the action of polypeptide Al, suggesting that the reagent was needed only to release Al from subunit A or the holotoxin for their action on adenylate cyclase. The single SH group in polypeptide Al was not involved in the activity of the peptide, since chemical modification of the thiol group did not alter the stimulatory activity of the peptide. The presence of NAD was, however, essential for the activation of adenylate cyclase with cholera toxin, subunit A, or polypeptide Al. Elevation of the adenylate cyclase activity was also observed when the intact pigeon erythrocytes were incubated with polypeptide Al, although a 30-fold molar excess of Al over that of holotoxin was required for the same level of activation.

Cholera toxin, the diarrhea-causing protein isolated from the culture filtrate of Vibrio cholerue (11, stimulates the adenylate cyclase activity ubiquitously in a variety of mammalian cells (for a review, see 2). The process of adenylate cyclase activation appears to take place in three stages: the rapid binding of cholera toxin to ganglioside c;MI on the cell surface, a characteristic lag period, and then the rise in the cyclase activity (3-6). It has been shown that cholera toxin consist of a subunit (A) and six smaller subunits of molecular weight 9000 (B) (7, 8). Subunit B, which also may be obtained in the purification procedure and is called choleragenoid (l), is responsible for the binding of the toxin to the cell. Subunit A, composed of two polypeptides Al (22,000 MJ and A2 (7500

M,) linked to each other through a single disulfide bond (8, 91, is considered responsible for the activity of cholera toxin. Gill and King have recently demonstrated that the effect of cholera toxin on the adenylate cyclase can be observed with lysates of pigeon erythrocytes (10). With the broken cells, activation of adenylate cyclase occurred immediately upon addition of the toxin, and the binding of the toxin to the cell surface was no longer necessary for the toxin action. They have also obtained evidence indicating that some cytoplasmic factors, one of which is NAD+ (ll), are required for the action of cholera toxin and that the active component of the toxin is polypeptide Al. In the present study, we show that cholera toxin stimulates the adenylate cyclase activity in washed membranes of pigeon erythrocyte in the absence of cytoplasm, provided that NAD+ and dithiothreitol (DTTj3 are

1 Taken in part from thesis to be submitted by A. Wodnar-Filipowicz to the Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland in partial fulfillment of the requirements for the Doctor of Philosophy degree. This work was carried out in Nutley under the auspices of Dr. C. Y. Lai. 2 To whom inquiries should be addressed.

3 Abbreviations used: NAD, nicotinamide adenine dinucleotide; Tris, tris(hydroxymethyl)amino methane; Hepes, N-2-hydroxyethylpiperazine N’-2ethanesulfonic acid; DlT, dithiothreitol; NEM, Nethylmaleimide. 465

Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

466

WODNAR-FILIPOWICZ

included in the assay system. With the isolated polypeptide Al @), only NAD is required for the activation of the membrane adenylate cyclase. The data obtained with the purified cholera toxin components indicate that direct interaction of polypeptide Al with the membrane is an obligatory step in the elevation of the adenylate cyclase activity by cholera toxin. MATERIALS

AND

METHODS

Cholera toxin was kindly provided by the cholera program of the National Institute of Allergy and Infectious Disease, through Dr. Carl E. Miller, or prepared from the lyophilized culture filtrate of V. ch0Zera.e 569B, obtained from the same source, by a modified procedure of Rappaport et al. (12). The preparations were checked for homogeneity by disc gel electrophoresis prior to use. Subunits A and B were separated by gel filtration in 5% formic acid as described previously (8). The separated subunits were dissolved in 8 M urea and dialyzed at 0°C against a linear gradient of urea from 4 to 0 M, in 0.05 M Tris-HCl buffer, pH 7.5 containing 0.1 M NaCl and 1 mM EDTA. This procedure “renatured” the proteins so that they remained in solution in neutral pH and showed reactivity toward antiserum (8). For the preparation of polypeptides Al and A2, subunit A (10 mg) was treated with 5 mM D’IT in 0.3 M Tris-HCl-0.015 M EDTA buffer, pH 8.5 containing 8 M urea (1 ml) for 2 h at room temperature, acidified by addition of 0.1 ml of 70% HCOOH and applied to a column of Sephadex G75 (1.5 x 200 cm) in 5% HCOOH containing 2 mM DTT. The recovery of Al and A2 was essentially quantitative. Pigeon erythrocytes were prepared as described by Gill and King (lo), and the packed cells were suspended with an equal volume of Buffer I (20 mM Hepes buffer, pH 7.3 containing 0.13 M NaCl, 5 mM KCl, and 2 mM MgCl,). To prepare the membrane fraction, 1 vol of the erythrocyte suspension was quickly frozen in a dryice ethanol mixture and then allowed to thaw; the procedure was repeated twice. ARer centrifugation at 10,OOOg for 10 min, the precipitate was washed with 2 vol of Buffer I three times and finally suspended in 0.5 vol of the same buffer. This crude membrane preparation is referred to simply as washed membrane or “membrane” in this report. The protein concentration ranged from 19-23 mg/ml. The following biochemicals were obtained from commercial sources: creatine phosphokinase, CAMP, NAD+, Hepes, DlT (Calbiochem, La Jolla, Calif.); phosphocreatine (Sigma Chemical, St. Louis, MO.); a-V*P]ATP (ICN, Waltham, Mass.); [“HIcAMP, [2-

AND

LA1

“Cliodoacetic acid ton, Mass.). Other purity commercially ther purification.

(New England Nuclear Co., Boschemicals were of the highest available and used without fur-

Methods Adenylate cyclase assay. This was carried out by the method of Salomon et al. (13) with modifications as described by Blume and Foster (14). The assay mixture (0.1 ml) contained 50 mM Tris-maleate buffer, pH 8, 0.5 mM ATP (containing 1 &i of a[=P]ATP), 0.5 mM CAMP, 6 mM MgCl,, 20 mM phosphocreatine, 0.3 mM R01724,“ 0.1 mg/ml of creatine phosphokinase, and about 2 mg/ml of washed erythrocyte membrane. The reaction was carried out at 37°C with gentle shaking for 30 min and terminated by addition of 0.2 ml of 1% HClO, containing L3H1cAMP (-10 nCi). It was then passed sequentially through small columns of Dowex 50 x 8 and alumina to separate CAMP for the radioactivity measurement. With this method, the production of CAMP was found to be linear with time up to 45 min, and with the added membrane enzyme up to 6.5 mg/ ml. Protein determination. The method of Lowry et al. (16) was used with bovine serum albumin as the standard. For cholera toxin and its constituent peptides, the values were calibrated against those from amino acid analysis.” Chemical modification of SH-groups. S-Carboxymethylation of the SH-group in polypeptide Al was carried out in 8 M urea and 5 mM DTI’, at pH 8.5 with [2-14Cliodoacetic acid (17). The amount of radioactivity bound indicated a complete reaction with the single cysteine residue in the protein (9). Reaction with N-ethylmaleimide (NEM) was carried out in a similar fashion except that iodoacetate was substituted with 10 mM NEM. After the modification reaction, the protein was dialyzed against linearly decreasing concentrations of urea for “renaturation” as described previously. RESULTS

Stimulation of adenylate cyclase in washed membranes of pigeon erythrocyte by cholera toxin. When washed mem-

branes of pigeon erythrocytes were treated with cholera toxin for 30 min at 37”C, only a small elevation of the adenylate cyclase activity was observed (Table I). A significant increase in the adenylate cyclase activity was obtained by the addition of cyto’ A phosphodiesterase inhibitor (15) obtained from Hoffmann-La Roche, Inc. a C. Y. Lai and D. Chang, manuscript in preparation.

ADENYLATE

CYCLASE

STIMULATION

sol, but the effect of the toxin was even greater when NAD was also added to the system, confirming the results with whole erythrocyte lysate (10). However, DTT was found to substitute cytosol in the activation of adenylate cyclase by cholera toxin (Table I).

IN

PIGEON

ERYTHROCYTE TABLE

EFFECT

OF CHOLERA

ADENYLATE

TOXIN

CYCLASE ERYTHROCYTE

IN

I ON THE THE WASHED MEMBRANE”

Requirement of DTT and NAD for the stimulation of membrane adenylate cyclase by the cholera toxin components. The

results shown in Fig. 1 suggest that the requirement of DTT in the activation of adenylate cyclase by cholera toxin (Table I) is related to the dissociation of polypeptide Al from the rest of molecules (8). This appeared to be the case as both cholera toxin and subunit A required the presence of DTT for maximal stimulation of the membrane adenylate cyclase, whereas polypeptide Al did not. In fact, 1 mM DTT was somewhat inhibitory to the action of polypeptide Al (Table II). On the other hand, NAD appeared to be the absolute requirement in the stimulator-y action of all these proteins (Table II). No significant further increase in the adenylate cyclase activity was observed with concentration of added NAD or DTT higher than 1 mM (results not shown).

ACTIVITY

OF

PIGEON

Adenylate cyclase activity (pm01 of CAMP/ minimg of membrane protein)

Activities of subunits in the stimulation of the membrane adenylate cyclase. Chol-

era toxin subunit A as well as its polypeptide component Al was found to stimulate the adenylate cyclase activity in washed erythrocyte membranes in the presence of 1 mM DTT and 1 mM NAD, whereas subunit B and polypeptide A2 were completely inactive (Fig. 1). The amount of cholera toxin required for the half-maximal stimulation was approximately 90 pg (1.1 nmol)/ ml, and those for subunit A and polypeptide Al were 16 pg (0.53 nmol)/ml and 5 pg (0.23 nmol)/ml, respectively. On equimolar basis, polypeptide Al appeared to be more efficient than subunit A or whole toxin in the stimulator-y action. An experiment on the time course of stimulation (Fig. 2) indicated that cholera toxin or polypeptide Al acted on the adenylate cyclase in the “membrane” without an appreciable time lag. Similar observations were made by Gill and King (10) with the whole erythrocyte lysate.

467

MEMBRANE

No additions + cholera toxin + cholera toxin + cholera toxin + cholera toxin

1.47 1.79 2.54 15.42 13.22

+ cytosol + cytosol + NAD + DTT + NAD

” Pigeon erythrocyte “membrane” (8.3 mgiml) was incubated with cholera toxin (145 ~gimll, cytosol (29.1 mg/ml), NAD (1 mM), and DTT (1 mM1 as indicated, for 30 min at 37°C. Aliquots were then assayed for the adenylate cyclase activity as described in the Methods.

PROTEIN

CONCENTRATION

IpR/mll

FIG. 1. Activation of adenylate cyclase in the washed pigeon erythrocyte membrane by cholera toxin, subunit A, and polypeptide Al. The crude “membranes” (9.88 mg of protein/ml) were incubated with the indicated concentrations of cholera toxin (01, subunit A (A), subunit B (01, polypeptide Al (O), and polypeptide A2 (Al in the presence of 1 mM NAD and 1 mM DTT for 30 min at 37°C. Aliquots were taken for the duplicate assays of the adenylate cyclase activity as described in Materials and Methods. The basal adenylate cyclase activity of the washed pigeon erythrocyte membrane was 1.4 pmol of cAMP/min/mg of protein, which had been subtracted from all the values obtained with the activator proteins.

Effect of S-alkylation on the adenylate cyclase stimulating activity of polypeptide Al. Modification of the sulfhydryl group of

the single cysteine residue in polypeptide Al (9) by S-carboxymethylation or reaction with N-ethylmaleimide did not alter the activity of polypeptide Al toward the

468

WODNAR-FILIPOWICZ

AND

membrane adenylate cyclase (Table III). The results indicate that the SH-group in polypeptide Al is not essential for its activity to stimulate the adenylate cyclase activity in the erythrocyte membrane.

LA1

quent response of adenylate cyclase to catecholamines and NaF (15-17). The effect of diminished catecholamines was increased, while that of NaF was diminished by pretreatment of the cells with cholera toxin (18-20). With the washed membrane of pigeon erythrocyte, the effects of cholera toxin, subunit A, or polypeptide Al were similar -but more dramatic (Table IV). Over a threefold increase in the extent of stimulation was observed with epinephrine when the crude membrane preparation was pretreated with cholera toxin or its components, whereas the effect of NaF was diminished by 50%.

Response of the membrane adenylate cyclase to epinephrine and NaF after pretreatment with cholera toxin or its component proteins. It has been reported by sev-

eral authors that incubation of the intact cell with cholera toxin changes the subse-

Stimulation of the adenylate cyclase activity in the intact pigeon erythrocyte with TABLE STIMULATION ERYTHROCYTE MODIFIED

2 0

r

>I

20

:

II 40

f 60

>

i so

9

100

ETHYLMALEIMIDE”

-

Activator

FIG. 2. Time course of the adenylate cyclase activation in the washed erythrocyte membrane by cholera toxin and polypeptide Al. “Membranes” (8.87 mglml) were incubated with cholera toxin (130 pg/ml -O-), polypeptide Al (16.4 pg/ml, -0-1, or by itself (-O-j, in the presence of 1 mM DTI and 1 mM NAD at 37°C. At the indicated time, samples were withdrawn and diluted four times into the adenylate cyclase assay mixture for the direct measurement of the adenylate cyclase activity. It was assumed that the action of cholera toxin or Al on “membrane” diminished substantially upon dilution for the ade-, nylate cyclase assay, since the concentrations of D’IT and NAD were both also decreased to 0.25 mM.

OF DTT

AND

NAD

ON THE MEMBRANE

None Al unmodified Al-carboxymethylated Al treated with

-

+ DTI + NAD + D’PI’

+ NAD

0.88 8.09 9.91 8.46

NEM

II

OF ADENYLATE

BY CHOLERA

Cholera “Membrane”” “Membrane” “Membrane” “Membrane”

Adenylate cyclase activity (pmol of CAMP producediminimg)

” Pigeon erythrocyte “membranes” (8.3 mgiml) were incubated for 30 min at 37°C with a control preparation of Al (2.4 yglml), Al carboxymethylated (2.4 pg/ml), and Al treated with N-ethylmaleimide (2.0 pgiml). Adenylate cyclase activity was then determined in duplicate as described in Materials and Methods.

ACTIVATION

Adenylate

added

-

TABLE EFFECT

III

OF ADENYLATE CYCLASE IN PIGEON MEMBRANE BY Al POLYPEP~IDE WITH IODOACETIC ACID AND N-

TOXIN

cyclase toxin

1.5 -t 0.1 1.8 4.1 12.1 i 0.1

CYCLASE

IN

PIGEON

ERYTHROCYTE

COMPONENT”

activity

(pmol of cAMP/min/mg protein) Subunit

Polypeptide

A

1.4 f 0.1 1.6 10.2 13.7 * 0.3

” Washed erythrocyte membrane (6.4 mg/ml) was incubated with cholera pg/ml), or polypeptide Al (11 wg/mll for 30 min at 37”C, with or without indicated. Aliquots of the reaction mixture were taken for the adenylate Materials and Methods. h The crude preparation of pigeon erythrocyte membrane (see Materials). this preparation was approximately 1.4 pmol/min/mg of protein.

of membrane Al

1.4 i 0.1 1.1 24.4 19.2 i 0.9

toxin (140 pg/ml), subunit A (31 1 mM DT’I and 1 mM NAD as cyclase assays as described in The

basal

enzyme

activity

in

ADENYLATE

CYCLASE TABLE

EFFECTS MEMBRANE CHOLERA

OF EPINEPHRINE

STIMULATION

IV AND

NaF

ON THE

ADENYLATE CYCLASE PRETREATED TOXIN. SUBUNIT A OR POLYPEP~IDE

Incubation

WITH

Al”

Adenylate cyclase activity of CAMP formedlminimg)

Assay No addition NaF L-Epinephrine

IN

Control

Cholera toxin

0.70 70.16 7.11

5.17 47.45 21.39

(pmol

A

Al

6.22 36.39 25.18

6.97 29.98 22.72

(i Washed “membranes” (9.7 mg/ml) were incubated for 30 min at 37°C with cholera toxin (140 pg/ ml), A subunit (31 pg/ml), or polypeptide Al (7 pg/ ml) in the presence of 1 mM NAD, and in the case of cholera toxin and A, 1 mM DTT. Aliquots were then added to the adenylate cyclase assay mixture containing, as indicated, either 10 mM NaF or 0.1 mM Lepinephrine for assay.

0

2

4 6 6 PROTEINCONCENTRATION l&l)

IO

ERYTHROCYTE

Al. According to current concepts, intoxication of intact cells with cholera toxin requires binding of the toxin to the surface receptors of the cell, the presence of subunit B being necessary for this binding (2,lO). Our results show that polypeptide Al, though requiring a high concentration, causes a significant increase in the adenylate cyclase activity in the intact pigeon erythrocyte (Fig. 3). The adenylate cyclase activity reached a plateau at a

MEMBRANE

469

cholera toxin concentration of 1 pglml (per milligram of membrane protein). For the same level of activation, 7 kg/ml of polypeptide Al was required. This is a 30-fold molar excess over the amount of holotoxin. The study of the time course of the adenylate cyclase activation in intact pigeon erythrocyte showed that a lag existed with both cholera toxin and polypeptide Al. With Al, the lag was about three times longer than with the whole molecule (Fig. 4). DISCUSSION

Our results show that cholera toxin stimulates adenylate cyclase in the crude membrane preparation of pigeon erythrocytes in the absence of cytosol and that DTT as well as NAD are required for the activation reaction. With this simplified system, studies on the mode of action of cholera toxin would be greatly facilitated. The availability of the purified subunits and component polypeptides of cholera toxin (8) has permitted us to confirm and extend the results of Gill and King (10) and to show quantitatively that polypeptide Al is the active moiety of cholera toxin in its stimulator-y action on the membrane adenylate cyclase. Other compo-

12

FIG. 3. Stimulation of adenylate cyclase in intact pigeon erythrocytes by cholera toxin and polypeptide Al. Washed pigeon erythrocytes were incubated for 60 min at 37°C with the increasing amounts of cholera toxin (0) and polypeptide Al (0). After the incubation, cells were washed twice with Buffer I and lysed and “membranes” were prepared as described in Materials and Methods. Adenylate cyclase activity of the “membrane” was assayed in duplicate as described in Materials and Methods.

polypeptide

PIGEON

“>

4c

./+-I

FIG. 4. Time course of adenylate cyclase activation by cholera toxin and polypeptide Al in intact pigeon erythrocytes. Pigeon erythrocytes were incubated at 37°C with 6.8 pg/ml of cholera toxin CO), 6.1 pg/ml of polypeptide Al (O), or without any additions (0) in the presence of 1 mM NAD. At times indicated, portions of the cells were withdrawn, washed with Buffer I, and lysed and “membranes” were prepared and assayed for adenylate cyclase activity as described in Materials. Essentially the same results were obtained when NAD was omitted from the incubation mixture (not shown).

470

WODNAR-FILIPO

nents of the toxin, polypeptide A2, and subunit B were both completely inactive for the stimulation of the membrane enzyme. The levels of adenylate cyclase activity per milligram of membrane protein reached with cholera toxin or the active components were approximately the same and were about 12-16 times the basal activity of the erythrocyte membrane. In terms of the number of folds of activation, the value is comparable to that obtained by Gill and King with the concentrated erythrocyte lysates (10). The relative amounts of polypeptide Al, subunit A, and cholera toxin required for the half-maximal activation were 0.23, 0.48, and 1 nmol, respectively. Since 1 mol of cholera toxin contains 1 mol of subunit A which, in turn, contains 1 mol of polypeptide Al (8), the results indicate that polypeptide Al is more active, on an equimolar basis, than subunit A or the holotoxin in the stimulation of adenylate cyclase. Recently, Sahyoun and Cuatrecasas (22) reported a small but significant stimulation of adenylate cyclase by cholera toxin in a membrane preparation of fat cells. With 0.5 pg/ ml of cholera toxin, an approximately 50% increase in the adenylate cyclase activity was observed in 100 min, after a 20-min lag (22). In our experiments with washed pigeon erythrocyte membrane, no appreciable lag phase was observed with cholera toxin or peptide Al (Fig. 2). The role of DTT in the activation of membrane adenylate cyclase by cholera toxin appeared to be merely that of releasing polypeptide Al from the toxin. DTT was not required, and in fact somewhat inhibitory, to the activation of adenylate cyclase by isolated polypeptide Al. The thiol group in this peptide was not essential for the activity, as chemical modification of the functional group did not alter the effect of Al on the adenylate cyclase. In a separate experiment not shown in this report, cholera toxin was pretreated with 1 mM DTT for 30 min at 37°C and then added to 20 vol of the “membrane” suspension containing 1 mM NAD. Stimulation of the adenylate cyclase activity comparable to that reported here (“membrane” + DTT + NAD + cholera toxin) was observed. This

WICZ

AND

LA1

result further supports the above conclusion. Bitensky et al. (21) reported the formation of a “macromolecular cyclase activating factor, MCAF” from cholera toxin when the latter was incubated with DTT, NADH, or NAD, then with the membranes. The MCAF was found to activate adenylate cyclase in membranes from mouse ascites cancer cells, but not membranes from normal cells (21). We suggest that “MCAF” is none other than polypeptide Al. The effect of cytosol on the extent of stimulation by Al was not studied in detail, but a preliminary test indicated that a further enhancement of the adenylate cyclase activity was possible. It is currently a subject of further investigation. It is interesting that polypeptide Al alone can activate the adenylate cyclase activity in intact pigeon erythrocytes. The stimulation observed was probably not due to the action of Al on some cells lysed during incubation, since the extent of stimulation of adenylate cyclase was dependent upon the amount of Al added (Fig. 3) and followed a nonlinear time course (Fig. 4). The possibility that polypeptide Al also causes hemolysis still remains to be carefully tested, however. In any event, our experiments indicated that an approximately 30-fold molar excess of Al over cholera toxin was required to attain the same level of the adenylate cyclase activity. Moreover, the lag before the elevation of the cyclase activity was found to be three times longer with polypeptide Al than with the holotoxin. The data suggest that polypeptide Al enters the cell, or reaches the site of interaction with adenylate cyclase, via a passive transport mechanism such as diffusion. The time lag observed in the action of Al on the intact erythrocytes (Fig. 4) would argue against the possibility that Al bound nonspecifically to the cells and remained bound through washing and disruption of the cells. The effect of the holotoxin appears to be mediated by binding with the surface receptors of the cell, the amount of which determines the level of activation of adenylate cyclase. Sahyoun and Cuatrecasas (22) reported that subunit A directly stim-

ADENYLATE

CYCLASE

STIMULATION

ulates the adenylate cyclase in the intact fat cells. The present studies suggest that binding of cholera toxin to cell surfaces is still the first step in the process of intoxication of the cell. When bound, the release of polypeptide Al from the toxin as well as its entry into the membrane apparently is facilitated at the receptor sites. Polypeptide Al then stimulates the adenylate cyclase in the presence of NAD. Calculations based on the stimulatory activity of polypeptide Al indicate that a complete dissociation of the cholera toxin molecule bound to the cell surface has occurred. The mechanism of the activation reaction is currently under investigation. The effects of pretreatment of the crude membrane preparation with cholera toxin, subunit A, or Al on its response to epinephrine and NaF (Table IV) were found to be similar to those reported for pretreatment of the intact cell with cholera toxin (15-17). With the washed membrane of pigeon erythrocyte, the effect was more dramatic. Epinephrine stimulated the adenylate cyclase activity of the pretreated “membrane” further, fourfold over the prestimulated level. However, in terms of number of fold of stimulation, pretreatment with cholera toxin appeared to reduce the effect of epinephrine (from tenfold stimulation in the untreated “membrane” to fourfold in the prestimulated adenylate cyclase). Adenylate cyclase in “membrane” pretreated with cholera toxin, subunit A, or Al was stimulated by NaF to a level only 50% of that reached with the untreated “membrane.” ACKNOWLEDGMENTS We are grateful to Drs. A. Blume and S. Levinson of this department for their help in the adenylate cyclase assay, and to Dr. Felix G. Garcia of Hoffmann-La Roche for supplying us with pigeon blood.

IN

PIGEON

ERYTHROCYTE

MEMBRANE

471

REFERENCES

5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 15. 16.

17. 18. 19. 20. 21.

22.

FINKELSTEIN, R. A., AND LOSPALLUTO, J. J. (1969) J. Exp. Med. 130, 185-220. FINKELSTEIN, R. A. (1973) Crit. Rev. Microbial. 2, 553-623. VAUGHAN, M., PIERCE, N., AND GREENOUGH, W. B., III (1970) Nature (London) 226,658-659. KIMBERG, D. V., FIELD, M., JOHNSON, J., AND HENDERSON, E. (1971) J. Clin. Invest. 50, 1218-1230. CUATRECASAS, P. (1973) Biochemistry 12, 35583566. VAN HEYNINGEN, S. (1974)Science H&656-657. LONROTH, I., AND HOLMGREN, J. (1973) J. Gen. Microbial. 76, 417-427. LAI, C. Y., MENDEZ, E., AND CHANG, D. (1976) J. Infect. Dis. 133, S23-S30. MENDEZ, E., LAI, C. Y., AND WODNAR-FILIPOWICZ, A. (1975) B&hem. Biophys. Res. Commun. 67, 14351443. GILL, D. M., AND KING, C. A. (1975) J. Biol. Chem. 250, 6424-6432. GILL, D. M. (1975)Proc. Nut. Acad. Sci. USA 72, 2064-2068. RAPPAPORT, R. S., RUBIN, B. A., AND TINT, H. (1974) Infection and Immunity 9, 294-303. SALOMON, Y., LONDOS, C., AND RODBELL, M. (1974) Anal. B&hem. 58, 541-548. BLUME, A. J. AND FOSTER, C. J. (1975) J. Biol. Chem. 250, 5003-5008. SHEPPERD, H., AND WIGGAN, G. (1971) B&hem. Phurmucol. 20, 2128-2130. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. CRESTFIELD, A. M., MOORE, S., AND STEIN, W. H. (1965) J. Biol. Chem. 238, 622-627. FIELD, M. (1974) Proc. Nut. Acud. Sci. USA 71, 3299-3303. BENNETT, V., AND CUATRECASAS, P. (1975) J. Membrane Biol. 22, l-28. GANGULY, U., AND GREENOUGH, W. B., III (1975) Proc. Nat. Acud. Sci. USA 72, 3561-3564. BITENSKY, M. W., WHELLER, M. A., MEHTA, H., AND MIKI, N. (1975) Proc. Nat. Acud. Sci. USA 72, 2572-2576. SAHYOUN, N., AND CUATRECASAS, P. (1975)Proc. Nut. Acud. Sci. USA 72, 3438-3442.