A cytolytic protein from the edible mushroom, Pleurotus ostreatus

451

Biochimica et Biophysica Acta, 585 (1979) 451--461 © Elsevier/North-Holland Biomedical Press

BBA 28919

A CYTOLYTIC PROTEIN FROM THE EDIBLE MUSHROOM, PLEUROTUS OSTREATUS

ALAN W. BERNHEIMER and LOIS S. AVIGAD

Department of Microbiology, New York University School of Medicine, New York, N Y 10016 (U.S.A.) (Received October 23rd, 1978)

Key words: Cytolysin; Hemolysin; Sphingomyelin; (Pleurotus ostreatus)

Summary Aqueous extracts of the edible mushroom, Pleurotus ostreatus, contain a substance that is lyric in vitro for mammalian erythrocytes. The hemolytic agent, pleurotolysin, was purified to homogeneity and found to be a protein lacking seven of the amino acids commonly found in proteins. In the presence of sodium dodecyl sulfate it exists as monomers of molecular weight 12 050 whereas under non-dissociating conditions it appears to exist as dimers. It is isoelectric at about pH 6.4. The sensitivity of erythrocytes from different animals correlates with sphingomyelin content of the erythrocyte membranes. Sheep erythrocyte membranes inhibit pleurotolysin-induced hemolysis and the inhibition is time and temperature dependent. Ability of membranes to inhibit hemolysis is abolished by prior treatment of membranes with specific phospholipases. Pleurotolysin-induced hemolysis is inhibited by liposomes prepared from cholesterol, dicetyl phosphate and sphingomyelin derived from sheep erythrocytes whereas a variety of other lipid preparations fail to inhibit. It is concluded that sphingomyelin plays a key role in the hemolytic reaction.

Introduction Aqueous extracts of edible mushrooms belonging to the genus Pleurotus are capable of lysing washed mammalian erythrocytes [1,2]. The potential commercial importance of Pleurotus and the development of methods for its cultivation [3--5] led us to investigate the nature and properties of the lytic agent here designated pleurotolysin. The results show that the substance responsible for cytotoxicity is a protein of unusual amino acid composition and that it differs in this and in other ways from cytolytic proteins that have been isolated from the basidiocarps of other kinds of gilled fungi.

452 Materials and Methods Pleurotus basidiocarps. Source A. Mycelium designated Pleurotus ostreatus and cultured in what appeared to be a mixture of hardwood sawdust and cereal grain [5] was purchased from Kinoko International {P.O. Box 6425, Oakland, CA 94621). Fruiting was induced by rehydrating and humidifying according to directions provided by the supplier. Source B. P. ostreatus strain Cloquet 3A, was aseptically cultivated at 20--23°C on p o t a t o dextrose agar (Difco Laboratories, Detroit, MI), 600 ml of m e d i u m / 2 8 0 0 ml Fernbach :]ask. Extracts from both sources contained approximately the same hemolytic activity, and the yield of purified p r o d u c t was approximately the same regardless of source. For the isolation o f pleurotolysin it was found necessary to use fresh material. Basidiocarps stored at --20°C for several months yielded no activity. Estimation o f hemolytic activity. Test solutions were diluted in 0.145 M NaC1/0.01 M Tris (pH 7.2) (buffer I). Volumes of toxin dilutions decreasing by a b o u t 25% were delivered into tubes (12 × 75 mm), and the volume in all tubes was brought to 1 ml by addition of the diluent. To each tube was added 1 ml washed sheep erythrocytes suspended in buffer 2. The density of the e r y t h r o c y t e suspension was adjusted to given an absorbance of 0.8 at 545 nm when complete lysis occurred. After mixing, the tubes were incubated at 37 ° C for 30 min and then briefly centrifuged. The percentage of hemolysis was estimated from the color of the hemoglobin in the supernatants as compared with that of standards. One hemolytic unit is that amount of test material needed to release the hemoglobin from 50% of the cells. Unless otherwise specified, sheep erythrocytes were used. Experiments were repeated at least once in order to test the reproducibility of the measurements. Inclusion in the system of either 5 mM CaC12 or 5 mM MgC12 did not affect the hemolytic activity. Amino acid analysis. Pleurotolysin (0.021 absorbance units) was hydrolyzed at l l 0 ° C for 22, 36 and 48 and 72 h in 6 N HC1 and approximately 0.05 mM phenol. Analysis was carried out in an automatic amino acid analyzer (Model D500, Durrum Instrument, Sunnyvale, CA), with the program set at 2.5 nm. Tryptophan was determined spectrophotometrically [6]. Erythrocyte membranes. Membranes from erythrocytes of sheep and rabbit were prepared osmotically according to method B of Ref. 7. They were stored at --20°C until used. Reagents. Staphylococcal sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12) was prepared as in Ref. 8. Phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) from Corynebacterium ovis [9] was purified as described [10]. Sphingomyelin from beef liver, phosphatidylethanolamine and gangliosides were purchased from Sigma Chemical Co. (St. Louis, MO), sphingomyelin from sheep erythrocytes from Supelco Inc. (Bellefonte, PA), phosphatidylcholine and diphosphatidylglycerol from Sylvana Co. (Milburn, NJ) and phosphatidylserine from Applied Science Laboratories (State College, PA). Gangliosides and sphingomyelin were used as dispersions in buffer I prepared with the aid of a teflon bead and a cyclo-mixer (Clay-Adams, Inc., Parsippany, NJ). Dispersions of phospholipids other than sphingomyelin were prepared by drying in vacuo ethanol or benzene solutions as thin films, and suspending the films at a con-

453

centration of 1 mg/ml buffer I with the aid of a cyclo-mixer. Liposomes were prepared as described [10]. Results

Isolation of pleurotolysin Fractionations were done at a b o u t 4 ° C. (A) 64 g of basidiocarps (source A) were cut into small pieces and homogenized 60 s with 100 ml 0.9% NaC1 in a blender (Cuisinarts Inc., P.O. Box 352, Greenwich, CT 06830). The mixture was centrifuged at 15 000 rev./min for 10 min, and the sediment was re-homogenized with a second 100 ml 0.9% NaC1. After centrifugation the t w o supernatant fluids were pooled. (B) 105 g of ammonium sulfate were dissolved in the crude extract to make it approximately 70% saturated. After 30 min the mixture was centrifuged at 14 500 X g for 10 min. The precipitate was extracted with t w o 50 ml portions of 43% saturated ammonium sulfate (Table I). (C) The combined extract was brought to 70% saturation b y dissolving in it 19.9 g ammonium sulfate. After 15 min the mixture was centrifuged at 12 000 × g for 10 min, and the supernate was discarded. The precipitate was dissolved in 10 ml 50% glycerol. (D) The glycerol solution was dialyzed against 20 volumes of 50% glycerol for 3 h followed by electrofocusing for 40 h. The latter was done with 1% (v/v) ampholine, pH 5--7, in a 440 ml column (LKB Produkter, Sweden), a 5--50% sucrose gradient and a final potential difference of 750 V with the cathode at the t o p of the column. Fractions (Fig. 1) of 16 ml each were collected. The four most active fractions (pH 6.2--6.5) were pooled. (E) The pool was dialyzed for 72 h against 600 ml saturated ammonium sulfate. The precipitate was collected b y centrifugation and suspended in 1 ml saturated ammonium sulfate for storage. Portions were freed of ammonium sulfate b y dialysis against distilled water as required. Amounts of pleurotolysin

TABLE I PURIFICATION SCHEME FOR PLEUROTOLYSIN Stage

Volume (ml)

A. C r u d e e x t r a c t B. 43% s a t u r a t e d ammonium sulfate extract C. 70% s a t u r a t e d ammonium sulfate e x t r a c t D. E l e c t r o f o c u s e d activity (fraction 19--22) E. A m m o n i u m sulfate precipitate

200 100

Total hemolytic units

Activity recovered (%)

A280/ ml

A 280 ] A260

250 225

50 0 0 0 22 5 0 0

100 45

17 6.1

0.49 0.66

14.7 36.9

11

2000

22 0 0 0

44

32.5

0.76

61.5

64

171

10 9 0 0

22

0.60

0.51

285

5800

6 670

13

4.94

1.50

1174

1.15

Hemolytic units/ml

Hemolytic units/ A 280

454 1i ~° DD

}8

c o (n

~,' II

I'I '~ /'D~

o E c 0

"

I,

oD

oJ 4

6

8

I0

12

14

FrCIctlon

16

18 2 0

22 2 4

26

28 50

number

Fig. 1. I s o e l e c t r i c f o c u s i n g o f p l e u r o t o l y s i n . l y t i c a c t i v i t y ( o ) a n d p H (a).

Fractions were examined

for 2 8 0 n m a b s o r b a n c e

(o), h e m o -

are expressed as either absorbance units (280 nm absorbance using 10 mm light path) or hemolytic units.

Analysis by gel electrophoresis Samples of pleurotolysin were subjected to electrophoresis in polyacrylamide gels. Under both non
Amino acid analysis The amino acid composition of pleurotolysin is shown in Table II. The absence of cystine, histidine, arginine, proline, tyrosine, phenylalanine and methionine is notable. Calculation of molecular weight by summation of the 114 residues of the 11 amino acids detected yields a molecular weight of 12 047. T A B L E II AMINO ACID ANALYSIS Residues/mol

OF PLEUROTOLYSIN

o f p r o t e i n are b a s e d o n m o l e c u l a r w e i g h t o f 13 6 0 0 ,

Amino acid

Residues/tool of protein (nearest integer) *

Asp Thr * Ser *

17 11 11 11 16 8 8 8 8 8 8

Glu Gly Ala

Val ne Leu Lys Trp

* Extrapolated to zero-time hydrolysis.

455

2OO

~" 150 y_

I00 .¢.~_ 5O E 35 5.0

6.0

7.0

8.0

9.0

pH Fig. 2. P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s . ( A ) P l e u r o t o l y s i n ( 0 . 0 2 a b s o r b a n c e u n i t ) w a s run in 7% gel a t p H 8 . 5 a n d 3 m A until t h e t r a c k i n g d y e had m o v e d 6 8 r a m . (B) Ple~trotolysin ( 0 . 0 3 7 a b s o r b a n c e u n i t ) , a f t e r 2 h a t 3 7 ° C in 1% s o d i u m d o d e c y l s u l f a t e and 1% 2 - m e r c a p t o e t h a n o l . w a s run in 7% gel a t p H 7 . 0 and S m A in the p r e s e n c e o f 0 . 1 % s o d i u m d o d e c y l s u l f a t e and 10% 2 - m e r c a p t o e t h a n o l (in the s a m p l e ) until the tracking d y e had m o v e d 6 8 m m . B o t h w e r e s t a i n e d w i t h C o o m a s s i e brilliant blue. Migration is f r o m top (cathode) to bottom. Fig. 3. H e m o l y t i c a c t i v i t y as a f u n c t i o n o f p H . o, 0 . 0 5 M c i t r a t e / p h o s p h a t e ; c e n t r a t i o n o f NaC1 was c o n s t a n t a t 0 . 1 2 M.

o, 0 . 0 5 M Tris-HC1. C o n -

Molecular weight Molecular weight was estimated (i) by Sephadex gel filtration [11] and (ii) by SDS-polyacrylamide gel electrophoresis [12]. (i) Pleurotolysin purified to stage C was fractionated on a 25 X 300 mm column of Sephadex G-100 equilibrated with 0.03 M sodium borate/0.1 M KC1/0.1 mg chymostatin/ml (pH 8.2). The column was calibrated with cytochrome c, soy bean trypsin inhibitor and ovalbumin. Pleurotolysin hemolytic activity yielded a single sharp peak corresponding to a molecular weight of 25 000. (ii) Three estimates done by the method of Weber and Osborn [12] on pure pleurotolysin, and using as standard proteins bovine serum albumin, ovalbumin, papain, pepsin, cytochrome c dimer, trypsinogen and cytochrome c momomer, gave values of 14 000, 14 500, and 12 300 (average = 13 600).

456 The data indicate that the basic subunit of pleurotolysin is a polypeptide chain of molecular weight 12 047 (13 600 by sodium dodecyl sulfate polyacrylamide gel electrophoresis). Under non-dissociating conditions it appears to exist in the form of dimers of 2 X 12 047 or 24 094 (25 000 found).

pH optimum The hemolytic activity of a solution of pleurotolysin was measured using sheep erythrocytes in 0.15 M NaC1 buffered at appropriate pH values. The results (Fig. 3) show that the optimum pH for lytic activity is in the region of 7.0.

Relative sensitivity of erythrocytes from different animals A preparation of pleurotolysin was titrated using various species of erythrocytes suspended in buffer I. The density of all suspensions was adjusted to the same hemoglobin content as that of the standard sheep erythrocyte suspension. The results are shown in Table III. It can be seen that the more sensitive kinds of erythrocytes are those having a relatively large content of sphingomyelin whereas the most resistant have the least.

Inhibition of hemolysis by erythrocyte membranes On the assumption that inhibition of lysis by a membrane constituent might provide a clue to the nature of the cellular receptor for pleurotolysin, decreasing quantities of osmotically dispersed sheep erythrocyte membranes were mixed with 3 hemolytic units of pleurotolysin. After 10 min at 20°C the mixtures were tested for lytic activity by adding washed sheep erythrocytes followed by incubation for 30 min at 37°C. It was found that 2.7--4pl of membrane preparation inhibited 2/3 the test amount of pleurotolysin. Rabbit erythrocyte membranes also inhibited pleurotolysin but comparable inhibition required

TABLE III SENSITIVITY OF ERYTHROCYTES SOLUTION OF PLEUROTOLYSIN

OF DIFFERENT

ANIMAL SPECIES TO LYTIC ACTIVITY OF A

H e m o l y t i c u n i t s / m l are d i l u t i o n s o f a s o l u t i o n of p l e u r o t o l y s i n t h a t will l i b e r a t e t h e h e m o g l o b i n cont a i n e d in a n a p p r o x i m a t e l y 0 . 3 5 % ( v / v ) s u s p e n s i o n o f e r y t h r o c y t c s u n d e r s t a n d a r d c o n d i t i o n s . Species

Number of individual animals

Hemolytic units/ml

S p h i n g o m y e l i n as p e r c e n t o f total phospholipid *

Sheep Ox Goat Man Cat Rabbit Swine Mouse Rat Horse Dog Guinea-Pig

4 2 3 3 2 2 i i 1 2 2 2

6000--10 000 2600-- 3 300 380-- 2 800 1500-- 2 600 730-- I 050 750-- I 0b0 750 500 400 <100-350 <100-430 <100-330

51.0 46.2 45.9 26.9 26.1 19.0 26.5 ? 12.8 13.5 10.8 11.1

* F r o m R o u s e r et al. [ 1 3 ] .

457

approximately 100 times as much as for sheep membranes. Inhibition by sheep erythrocyte membranes was time dependent, the volumes of membranes needed for inhibition at 37°C being 22, 40, 2.5, 2.0 and 1 pl when the times of interaction between membranes and pleurotolysin were 0, 5, 10, 15 and 45 min, respectively. The reaction was dependent to some extent on temperature as well, the volumes of membranes needed for inhibition at 0, 20 and 37°C, for 10 min, being 7.0, 4.0 and 2.5 pl, respectively. As erythrocyte membranes are known to possess proteolytic activity [14] it seemed possible that the foregoing results could be explained by proteolytic inactivation of pleurotolysin. However, inclusion in the system of each of a series of protease inhibitors (antipain, chymostatin, elastinol, leupeptin, pepstatin and phosphoramidon) at 0.5 mg/ml, did not affect the capacity of membranes to inhibit.

Inhibition o f hemolysis by sphingomyelin A series of dispersions of lipids were tested for inhibition of pleurotolysin under conditions similar to those of Ref. 15. Liposomes prepared from sphingomyelin obtained from beef brain as well as liposomes prepared from sphingomyelin obtained from sheep erythrocytes were also tested. The results (Table IV) show that the only preparation that was inhibitory was that consisting of liposomes made from sheep erythrocyte sphingomyelin. Failure of beef brain sphingomyelin liposomes to inhibit suggests that the nature of the variable fatty acid moiety is a determinant of inhibition, and some support is provided for this view by the fact that there is a rather profound difference in fatty acid composition of sheep brain and bovine brain sphingomyelin [16]. Effect o f pretreatment of membranes with phospholipase on capacity o f membranes to inhibit hemolysis 0.1 ml amounts of sheep erythrocyte membranes were mixed with 0.1 ml each of a series of 10-fold dilutions of staphylococcal sphingomyelinase C and of corynebacterial phospholipase D in the presence of 0.145 M NaC1, 0.01 M Tris buffer (pH 7.2), 0.01 M MgC12 and 0,2% gelatin. After 60 min at 37°C the mixtures were diluted in 4.8 ml buffer I and centrifuged at 12 000 × g for 10 min. The sedimented membranes were resuspended in 5 ml buffer I and TABLE IV EFFECT OF LIPID DISPERSIONS ON PLEUROTOLYSIN Test preparation

Concentration needed for inhibition of lysis (~g/ml)

1 2 3 4 5 6 7 8 9

>250 >250 :>250 :>250 :>250 :>250 :>250 :>250 25

Phosphatidylcholine Ph osphatidyle thanolamine Phosphatidylserine Dipho sphatidylgly cerol Gangliosides from bovine brain Sphingomyelin from bovine brain Liposomes prepared from 6 plus cholesterol and dicetylphosphate Sphingomyelin from sheep erythrocytes Liposomes prepared from 8 plus cholesterol and dicetylphosphate

458 TABLE V A B O L I T I O N O F C A P A C I T Y O F M E M B R A N E S T O I N H I B I T P L E U R O T O L Y S I N BY P R I O R T R E A T MENT OF MEMBRANES WITH PHOSPHOLIPASES Enzyme with which membrane were treated

Enzyme concentration (pg/ml)

Volume of m e m b r a n e preparation needed f o r i n h i b i t i o n of lysis (pl)

Phospholipase D

100 10 1 0.1 0

>10 >10 3.5 3.0 3.0

Sphingomyelinase C

0.2 0.02 0.002 0.0002 0

>10 >10 3.2 2.7 2.7

tested for capacity to inhibit pleurotolysin. The results (Table V) show that between I and 10 pg phospholipase D significantly reduced the inhibitory capacity of the membranes and that as little as 2--28 ng sphingomyelinase C p r o d u c e d a similar effect. Other biological effects Many agents that lyse erythrocytes also cause in vitro lysis of platelets [17]. Dilutions o f pleurotolysin were added to samples of human platelets that had been separated from plasma and suspended in buffer I containing 0.1% bovine serum albumin. At a concentration of 46 hemolytic units/ml, pleurotolysin did not produce gross lysis o f platelets in 30 min at 22°C. However, phase contrast examination revealed the m o r p h o l o g y of the platelets to be drastically altered to largely e m p t y spherical structures each containing an eccentric aggregate of phase
459

Fig. 4. P h a s e - c o n t r a s t m i c r o g r a p h s o f h u m a n b l o o d p l a t e l e t s a f t e r 3 0 m i n a t 2 2 ° C ( A ) in b u f f e r I cont a i n i n g 0.1% b o v i n e s e r u m a l b u m i n , a n d (B) in t h e s a m e d i l u e n t c o n t a i n i n g 4 6 h e m o l y t i c u n i t s o f p l e u x o t o l y s i n / m l . M a g n i f i c a t i o n : 2 7 0 0 X . See t e x t .

460

trypsin inhibitor. The existence of proteolytic activity in cultures of Pleurotus has been recorded earlier by others [ 20,21], and among the gilled fungi generally the protease of Armillaria mellea has been the subject of detailed study [22]. Discussion

Hemolytic toxins in higher fungi were detected long ago by Kobert [23] and Ford [24]. Relatively recently their occurrence was comprehensively surveyed by Seeger and Wiedmann [2]. But few of them have been studied biochemically. Phallolysin, from Amanita phalloides, has been intensively investigated by Seeger and coworkers [25--27] and shown to be a basic protein of molecular weight 33 000, whose mechanism of lysis is thought to involve binding to N~cetylglucosamine [28]. Flammutoxin from Flamrnulina volutipes is a strongly hemolytic protein of molecular weight 22 000 having the amino acids usually present in proteins except for those containing sulfur [29,30], and volvatoxin from Volvariella volvacea has been isolated as a lytic protein of molecular weight 24 000 (volvatoxin A2) having all of the usual amino acids [31]. Pleurotolysin differs most strikingly from the foregoing toxins in its amino acid composition, specifically in the absence of histidine, arginine, proline, tyrosine, phenylalanine, cystine and methionine. The basic subunit of pleurotolysin is a polypeptide of 12 050 daltons which apparently gives rise to dimers. In amino acid composition pleurotolysin is somewhat similar to melittin, the cytolytic polypeptide of bee venom, which lacks six of the usual amino acids [32], to staphylococcal delta toxin which lacks five [33--35] and to streptolysin S which lacks six [36]. Melittin has a minimum molecular weight of 2840 and a micellar weight of approximately 12 000 [32] while delta toxin has a minimal molecular weight of 5100 [35] and appears to exist as aggregates of various sizes up to 195 000 [34,35]. Streptolysin S is a polypeptide of 32 amino acids complexed with an oligonucleotide, and exists as a dimer of the polypeptide-oligonucleotide complex [36]. The order of decreasing lytic sensitivity of erythrocytes of different animals approximates the order of animals according to decreasing membrane sphingomyelin (Table III). Comparison of our data with those of Seeger and Burkhardt [27] shows that there is a roughly inverse correlation between sensitivity of erythrocytes to pleurotolysin and to phallolysin. Of the species tested, erythrocytes from sheep and ox are most sensitive to pleurotolysin and most resistant to phallolysin whereas the most phallolysin-sensitive erythrocytes, those of mouse, rabbit and guinea-pig, are among those least sensitive to pleurotolysin. The foregoing information suggests that pleurotolysin probably functions as a detergent rather than as an enzyme, and that sphingomyelin is the target of its cytolytic action. The importance of sphingomyelin is further supported by the observation that capacity of sheep erythrocyte to inhibit pleurotolysininduced hemolysis is abolished by pretreatment of the membranes with sphingomyelin-specific phospholipases. It also appears very significant that sphingomyelin is the only lipid tested that inhibits pleurotolysin, and that in order to do so the sphingomyelin must be derived from sheep (? erythrocytes) and that it must be in the form of liposomes. We interpret these results as indi-

461

cating that the nature of the variable fatty acid of the sphingomyelin moiety may be an important determinant of specific inhibitory capacity and that an equally decisive determinant is its molecular orientation. Acknowledgements We are grateful to Dr. J. San Antonio and Mr. M.W. Miller for strain Cloquet 3A of P. o s t r e a t u s and for information regarding its aseptic cultivation. We are grateful to Dr. I. Schenkein for the amino acid analyses, to Dr. K.S. Kim for the phase-contrast micrographs, and to the United States-Japan Cancer Program for samples of protease inhibitors. This study was supported in part by Public Health Career Program Award 5K06-AI-14-198 (to A.W.B.), from the National Institute of Allergy and Infectious Diseases. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

Bernheimer, A.W. and Farkas, M.E. (1953) J. I m m u n o l . 7 0 , 1 9 7 - - 1 9 8 Seeger, R. and Wiedmann, R. (1972) Arch. Toxikol. 2 9 , 1 8 9 - - 2 1 7 Eugenio, C.P. and Anderson, N.A. (1968) Mycologia 60, 6 2 7 - - 6 3 4 Anderson, N.A., Wang, S.S. and Schwandt, J.W. (1973) Mycologia 65, 28--35 Jong, S.C. and Peng, J.T. (1975) Mycologia 67, 1 2 3 5 - - 1 2 3 8 Edelhoch, H. (1967) Biochemistry 6, 1 9 4 8 - - 1 9 5 4 Bernheimer, A . W , Avigad, L b . and Avigad, G. (1975) Infect. I m m u n . 11, 1312--1319 Bernheimer, A.W., Avigad, L.S. and Kim, K.S. (1974) Ann. N.Y. Acad. Sci. 236, 292--305 Souckov~i, A. and Souc~k, A. {1972) Toxicon 10, 501--509 Linder, R. and Bernheimer, A.W. (1978) Biochim. Biophys. Acta 5 3 0 , 2 3 6 - - 2 4 6 Andrews, P. (1964) Biochem. J. 9 1 , 2 2 2 - - 2 3 3 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4 4 0 6 - - 4 4 1 2 Rouser, G , Nelson, G.J., Fleischer, S. and Simon, G. (1968) Biological Membranes Physical Fact and F u n c t i o n (Chapman, D., ed.), pp. 5--69, Academic Press, New Y ork Schrier, S.L. (1977) Blood 5 0 , 2 2 7 - - 2 3 7 Bernheimer, A.W. and Avigad, L b . (1978) Biochim. Biophys. Acta 541, 96--106 Barenholz, Y., Su ur kuusk, J , Mountcastle, D., Thomp s on, D.E. and Biltonen, R.L. (1976) Biochemistry 15, 2441--2447 Bernheimer, A.W. and Schwartz, L.L. (1965) J. Pathol. Bacterlol. 8 9 , 2 0 9 - - 2 2 3 Bernheimer, A.W. and Avigad, L.S. (1976) Proc. Natl. Acad. Sci. U.S. 73, 467--471 Tomarelli, RAM., Chaxney, J. and Harding, M.L. (1949) J. Lab. Clin. Med. 34, 428--433 Bukhalo, A.S., Bilai, T.I. and Besarab, B.N. (1971) Mikrobiol. Zh. (Kyyiv) 33, 663--666 Fedorova0 L.N. (1973) Mikol. F i t o p a t o l . 7 , 5 4 2 - - 5 4 4 Lewis, W.G., Basford, J.M. and Walton, P.L. (1978) Biochim. Biophys. Acta 5 2 2 , 5 5 1 - - 5 6 0 Kobert, R. (1891) St. Petersburger Med. Wochenschr. 1 6 , 4 6 3 - - 4 7 1 Ford, W.W. (1911) J. Phaxmacol. Exp. Ther. 2 , 2 8 5 - - 3 1 8 Seeger, R. (1975) Naunyn-Schmiedeberg's Arch. Phaxmacol. 2 8 8 , 1 5 5 - - 1 6 2 Seeger, R. (1975) Naunyn-Schmiedeberg's Arch. Phaxmacol. 2 8 7 , 2 7 7 - - 2 8 7 Seeger, R. and Bttrkhardt, M. (1976) Naunyn-Schmiedeberg's Arch. Pharmacol. 293, 163--170 Faulstich, H. an d Weckauf, M. (1975) Hoppe-Seyler's Z. Physiol. Chem. 356, 1187--1189 Lin, J.Y., Lin, Y~I., Chert, C.C., Wu, H.L., Shi, G.Y. and Jeng, T.W. (1974) Nature 2 5 2 , 2 3 5 - - 2 3 7 Lin, J.Y., Wu, H.L. and Shi, G.Y. (1975) T o x i c o n 1 3 , 3 2 3 - - 3 3 1 Lin, J.Y., Jeng, T.W., Chert, C.C., Shi, G.Y. and Tung, T.C. (1973) Nature 2 4 6 , 5 2 4 - - 5 2 5 Hab ermann, E. (1972) Science 1 7 7 , 3 1 4 - - 3 2 2 Heatley, N.G. (1971) J. Gen. Microbiol. 6 9 , 2 6 9 - - 2 7 8 Kreger, A ~ . , Kim, K.S., Z a b o r e t s k y , F. and B e m h e i m e r , A.W. (1971) Infect. I m m u n . 3 , 4 4 9 - - 4 6 5 Kantor, H,$., Temples, B. and Shaw, W.V. (1972) Arch. Biochem. Biophys. 1 5 1 , 1 4 2 - - 1 5 6 Lai, C.Y., Wang, M.-T., deFaria, J.B. and Ako, T. (1978) Arch. Biochem. Biophys. 191, 804--812