Interaction of phallotoxins with actin

INTERACTION OF PHALLOTOXINS WITH ACTIN TH. WIELAND Max-Planck-Institut fiir Medizinische Forschung, Abteilung Naturstoff-Chemie, D-6900 Heidelberg, Germany

INTRODUCTION PhaUoidin is the main representative o f the phallotoxins, one family of the toxins o f the mushroom Amanita phalloides ( 1 , 2 ) . Chemical manipulations lead either to similar toxic derivatives, e.g. tritiated desmethylphalloin, or can annihilate the toxicity o f the molecule. In the formula of phalloidin depicted below the points essential for toxicity are marked by d o t t e d circles. T

-C-CH 3 I

OH s H H H2COH ..~-, I I I cH3~--C- CO-NH-CH--CO-NH-C -CH2-C-CH3 "J

I

I

HN

oc

I

~ .-.I

-

OH

I !1

"~ X~,.N,-9,,~' ~.N"

I

I

,.~ I

H2C'~."qHJ

- -N - - C O - C H ~

H_--~ ~H0 ' ~ I

I

H2C~

I

.I

3

l

^ ,

U. 3

HN-C0--CH-NH-C0 -^ I llU--~rl CH3

Points essential for toxicity in dotted circles: 1. Peptide bond, fissile by mild acidic treatment must be intact. The monocycfic seeophalloidin is nontoxic. 2. Sulfur bridge must be present. Molecule without sulfur, dethiophallotoxin, is nontoxic. Sulfoxide, S-O, is toxic, but only the (R)-diastereomer, not the (S)-diastereomer. 3. Indole-hydrogen may be substituted only by CH~ or C2Hs not by n-propyl, C3H,, or longer (3). 4. OH must be present and must be cis-standing (4). 5. H instead of CH~ leads to nontoxic Glys-phallotoxin (5). The symptoms o f phalloidin poisoning (2) point to an impairment of the cytoplasma membrane of hepatocytes: after administration o f 3 mg o f the toxin per kg the animals (white mice) will die within several hr with livers swollen to as much as double their weights. The swelling is due to an excessive accumulation o f blood in the liver caused by the formation o f numerous non285

286

TH. WIELAND

fatty vacuoles which have their origin in an endocytosis, which begins 2.5 min after addition of the poison to a perfused isolated rat liver preparation (6). An analogous uptake of water can also be produced without phalloidin by increasing the posthepatic pressure (7). So it seemed that phalloidin weakens the membrane in such a way as to take up by endocytosis outceUular liquid already under the pressure normally present in the liver. Since also an efflux of K+-ions had been observed as a consequence of phalloidin action (8) and a binding of the toxin to plasma membranes of liver cells had been reported (9), an electron microscopic study was undertaken on membrane fragments obtained by gradient eentrifugation from intoxicated rats (10). Numerous filamentous structures about 60 A in width and up to 1/lm in length were regularly observed, which were only occasionally present in similar preparations from control animals. Analogous structures could also be produced in vitro by incubation with phalloidin of cytoplasmic membrane preparations from the livers of unpoisoned rats. Using 3 H-labeled desmethylphalloin we proved that the toxin was bound mainly to the filaments (11). Later on it could be demonstrated by "decoration" with heavy meromyosin (HMM) that the phallotoxin-induced liver filaments consist of actin(12), which however proved resistant to degradation by 0.6 M KI, a reagent which is known to depolymerize normal F-actin. This prompted us to start an investigation on the interaction of actin from rabbit muscle with phallotoxins. MATERIALS

AND M E T H O D S

Materials

Actin was prepared from rabbit skeletal muscle. The actin used for the measurements of viscosity and ATP hydrolysis was prepared according to (13) and was free of the regulatory proteins tropomyosin and troponin. For the determination of the polymerization rates the water extract of the acetone actin powder (extracted for 20 min at 0°C) was used immediately after extraction in order to ensure that the experiments started with true G-actin. ATP was added to the extract to a final concentration of 0.1 mM. Some of these actin solutions contained minor traces of regulatory proteins as seen by sodium dodecyl sulfate gel electrophoresis. Phalloidin, 3H-desmethylphalloin, phalloidinsulfoxide A (S-form), phalloidinsulfoxide B (R-form), dethiophalloidin and seco(15)-phalloidin were samples from our laboratory. Cytochalasin B (CB)was purchased from Aldrich Chemical Company. DNase I (pancreatic deoxyribonuclease I, EC 3.1.4.5) and DNS were from Boehringer Mannheim. Subtilisin (EC 3.4.21.14) was from C. Roth, Karlsruhe. Methods

Viscosity (2 ml samples) has been measured at 20-22°C in a spiral capillary viscosimeter. The flow time for water was 40 s. *7spec. has been defined as usual

INTERACTION OF PHALLOTOXINSWITH ACTIN

287

as (t/to)-I with t = flow time of the sample and to = flow time of the corresponding protein free medium. Sonic vibration was performed at 20°C with a Branson sonifier B 12 using a microtip for 4.5 ml samples at 20 kc/s and 50 W. The inorganic phosphate produced during sonic vibration has been measured according to reference 14. Actomyosin ATPase was measured according to reference 13. The rate of polymerization of G-actin to F-actin has been observed by measuring (at 25°C) the increase of light scattering intensity of the actin solution at 4 0 0 n m in a Hitachi-Perkin-Elmer Fluorescence Spectrophotometer MPF 2 A. The scattered light has been measured perpendicular to the incident light. The values of light scattering intensity indicated at the ordinates of the figures are deflections of the registration device and are hence arbitrary values depending on apparatus parameters like gain, slit width etc. Besides the constituents indicated in the figures the actin solutions used for the measurements of the polymerization rate always contained 0.1 mM ATP and 5 mM Tris.HC1 buffer (pH8.0). Polymerization was always started by the addition of MgCI2. In the absence of MgCI2 no polymerization occurred during the time or measurement. Chromatography of all-labeled F-actin-phaUotoxin was performed with a column (1.5 X 40 cm) packed with Sephadex G-200 (Pharmacia) in a buffer of pH 8.5 consisting of 0.025 M Tris-HCl, 0.1 M NaC1, 1 mM EDTA and 5.0 mM /3-mercaptoethanol. Hydrolysis of DNA by DNase I was measured spectrophotometrically at 260 nm according to references 15 and 16 using an Aminco DW-2UV-VIS spectrophotometer. Difference spectra were taken in two tandem cuvettes with a total length of 0.875 cm.

RESULTS

AND DISCUSSION

Polymerization of G-Actin A polymerization of G-actin in presence of Cam or Mg÷÷ or K ÷ and ADP or ATP (which is split into ADP and inorganic phosphate during the attachment of the G-units), which takes 10-20 min at room temperature is finished already in a few minutes when phalloidin is added (17). The rate of polymerization of G-actin induced by different amounts of phalloidin and started by the addition of MgC12 (to 1 mM) has been measured by observing the increase of light scattering at 400 nm. Figure 1 shows the accelerating effect of the toxin on the rate of polymerization. As little as 5 nmoles (molar ratio toxin/actin 0.09) of the toxin distinctly increased the velocity as compared with the control and further gradual increase gave rise to a gradual increase of the rate. The maximal rate was reached not before phalloidin and actin were present in equimolar amounts.

288

TH. WIELAND

~a 0

~6

~2 t (mln) FIG. 1. Rate of polymerization of G-actin (58 m o l e s ) accelerated by increasing amounts of phalloidin, - o - o - without PHD, - * - * - with 100 nmoles PHD.

Stabilization of F-Actin Stabilization against depolymerization by potassium iodide. Whereas F - a c t i n is i n s t a n t a n e o u s l y d e p o l y m e r i z e d in 0 . 6 M KI - t h e viscosity, ~Tspez d r o p s f r o m a d e f i n i t e value t o zero - t h e a c t i n o b t a i n e d in p r e s e n c e o f p h a l l o i d i n p r o v e d r e s i s t a n t u n d e r these c i r c u m s t a n c e s ( P h - a c t i n ) . P o l y m e r i z a t i o n in p r e s e n c e o f t o x i c a n d n o n t o x i c derivatives o f p h a U o i d i n gave h i g h viscous s o l u t i o n s w h o s e viscosity o n a d d i t i o n o f KI r e m a i n e d n e a r l y c o n s t a n t o n l y w h e n t o x i c derivatives h a d b e e n a d d e d ( 1 8 ) ( T a b l e 1).

TABLE 1. SPECIFIC VISCOSITIES OF SOLUTIONS OF G-ACTIN (1.2 mg/ml 1 mM Tris-HCl, pH 7.4 + 0.7 mM Mg~ ) 30 min AFTER INCUBATION WITH DIFFERENT TOXIC AND NON-TOXIC CYCLIC PEPTIDES (80 #g) BEFORE AND AFTER ADDITION OF KI (0.6 M) Compound added

~ spec. Before KI After KI

None

0

KCI (0.1 M) Phalloidin Phallaeidin Desmethylphalloin (R)-Phalloidin-sulfoxide B (ff)-Phalloidin-sulfoxide A Dethiophalloidin Secophalloidin

1.04 1.04 1.0 1.2 1.1 1.05 1.0 1.0

-

0 0.93 0.70 0.94 0.94 0 0 0

Toxicity 0 0 + + + + -

INTERACTION OF PHALLOTOXINSWITH ACTIN

289

In order to find out a stoichiometric relation a viscosimetric assay was made of the protective effect of different amounts of the cyclic peptide on F-actin (17). From Figure 2 it can be seen that F-actin was completely protected only when phalloidin and the actin subunits were present in equimolar amounts, although a high degree of protection was already reached when the molar ratio of phaUoidin to actin was very small. Here it may be added that Ph-actin is also stable against 1 mM ATP in ion free medium (18). Phalloidin even brings about polymerization of G-actin in presence of KI. Whereas no polymerization occurred without the drug already in presence of 0.3 M KI, the toxin (2 moles per mol G-actin) induced Ph-actin formation even in 0.5 M KI. There exists something like an antagonism, for with decreasing concentration of iodide an increasing velocity of polymerization can be observed. L--

-

•

•

1.O. 0.8

~.0.6

f

0.4, 0.2

~b Amount of odded pholloidin (mo~/mol actin)

FIG. 2. Drop of viscosity by 0.6 M KI of F-actin stabilized with different amounts of phalloidin. Stabilization of F-actin by phalloidin was also observed in presence of 4 M urea (I. L6w, unpublished). Stabilization against depolymerization by DNAase L Deoxyribonuclease I has been shown to bind very specifically to actin, which accordingly is the long known naturally occurring inhibitor (19). The inhibition is due to the formation of a 1 : 1 complex of the enzyme with G-actin which is formed also from F-actin by the depolymerizing action of DNAase (20). This depolymerization can be prevented by phalloidin (21). The inhibitory effect of G-actin on DNAase was totally blocked when already 0.25 moles of phalloidin per mole of actin were incubated for 2 min before adding the enzyme. The ratio of 0.25 agrees very well with our former observation of 0.3 moles of the toxin being enough to produce 1 mol of actin filaments which withstand the depolymerizing treatment with 0.6 M potassium iodide (see Fig. 2).

290

TH. WlELAND

It is still not clear whether the interaction o f actin with DNAase has any significance as a regulatory principle. Although DNAase I is an extracellular enzyme produced by the pancreas, other DNA hydrolyzing enzymes are also known to occur inside of cells, which may be likewise inhibited b y G-actin. An interference of phalloidin on this level could plausibly be part o f its molecular action.

Stabilization against denaturation by heat (and alkali). Heat denaturation of actin (in absence of ATP) was followed by difference spectroscopy (22). On heating o f one of the two actin containing cuvettes (at 70 ° for 3 min) a strong absorption increasing with shorter wavelengths is produced due to a turbidity not remarkable in the visible light region. The difference of absorbance at 350 nm was used as a measure of the extent o f denaturation. Figure 3 shows the protective effect o f added phalloidin. Without the toxin a turbidity taken arbitrarily as 100% was obtained which fell down to about only 15% at a ratio of 2 moles toxin to one mole o f actin. At 60°C total protection o f the protein occurred under the same conditions.

t ~

50-

==

1

2 tool Phal/oidin tool F - A k t i n

FIG. 3. Protection from heat denaturation of F-actin (1 X 10-s M in 0.1 M KCI, 1 mM Tris, pH 7.4) by phalloidin (0.0-4.0 X 10-s M) at various temperatures as followed by difference spectroscopy at 350 nm.

INTERACTION OF PHALLOTOXINSWITH ACTIN

291

The protecting effect was also studied for other toxic and nontoxic derivatives of phalloidin. The diagram of Figure 4 shows the relative turbidities in heating experiments with 5 different phaUopeptides and a control value (100%). One notices a strong protecting effect of the toxic compounds, phallacidin, phaUoidin and phaUoidin sulfoxide A (R-form), and the ineffectivity of the nontoxic secophalloidin. Intermediate protection was exhibited by the sulfoxide B (S-form), which shows no toxicity in the experimental animal. This contradictory phenomenon could be explained by a ten times lower affinity of sulfoxide B as compared with the toxic phallopeptides (see below).

I00

-

>,

I Phollacidin 2 Phalloidin 3 (Rl-Phalloidinsulfoxide (B) 4 (S)-Phalloidinsulfoxide (A) 5 Seco-phalloidin 6 Control

-io JD

50

N

,!! I

2

3

FIG. 4. Protective effect of phallopeptides (1.0 × 10-s M) on F-actin from denaturation by heating to 70°C for 3 rain in 0.1 M KC1, 1 mM Tris, pH 7.4, 1) phallacidin, 2) phalloidin, 3) phalloidin sulfoxide B, 4) phalloidinsulfoxide A, 5) secophalloidin, 6) actin alone. The curves in Figure 5 show that there is also exerted some protection by phalloidin to F-actin against denaturation by alkali. F-actin, as measured by its ability to split adenosintripiaosphate when sonically vibrated, is totally denatured on exposition to pH 12 within 1 min. After addition of 1 mol of phalloidin per actin total destruction is not reached before 30 min (I. LOw, unpublished results). Stabilization against hydrolysis by subtilisin. G-actin is degraded by subtilisin at a relatively high rate. Also F-actin is susceptible to enzymatic hydrolysis although in a slower reaction (Fig. 6). Phalloidin will retard the hydrolysis when present in a less than 1:1 molar ratio and stop it completely at equimolecular amounts (J. X. de Vries, unpublished results).

292

TH. WIELAND 100 -

\ "",,::.°

m u~

o

0

? 30

Min

FIG. 5. Denaturation of F-actin (1.0 X 10-s M) at 20 ° in 2 mM ATP, 9 mM NaOH (pH ~ 12) without ( - - - ) and with added phalloidin (1.0 X 10-5 M), equimolar ratio). Intactness measured by ultrasonic ATPase at pH 4 (31).

010

3b

~o

9~mi.

FIG. 6. Digestion of F-actin (1 × 10-s M in 0.1 mM Tris, pH 7.4; 0.1 M KC1) by subtilism (0.3 × 10-s M) at 30°C in presence of different amounts of phalloidin (as noted at the curves). Followed by dual wave length spectroscopy at 285 nm and reference 320 rim.

Influence o f phalloidin on F-actin as an ATPase. F-actin displays the p r o p e r t y to catalyze the hydrolysis o f ATP yielding ADP and inorganic phosphate (Pi) when exposed to local loosenings of its structure. This allows the exchange of b o u n d ADP with ATP o f the medium. The subsequent healing of the perturbation induces the hydrolysis o f ATP in a way reminiscent o f the ATP splitting during polymerization o f G-actin. The first author to describe this effect was Asakura (23) who observed as early as 1961 that ATP was hydrolyzed b y F-actin during ultrasonication. We found that phalloidin F-actin also stabilizes against breaking b y sonic vibration(17). Figure 7 shows that the ATPase activity o f F-actin generated by ultrasonication can be completely inhibited by the toxin. The ATP hydrolysis, like the susceptibility for depoly-

INTERACTION OF PHALLOTOXINSWITH ACTIN

293

.... tOO

.,~

ao

•,~ ,~o"a ~ 20.

g:

de

02

d5

ds

Amount of added pholloldin (rnol/mol octin)

/.0

FIG. 7. ATPase induced at F-actin by ultrasonic vibration is suppressed by increasing amounts of phaUoidin. merization by KI (page 289), was already diminished considerably when only a small proportion of the actin units could have combined with the toxin. The line drawn in the Figure represents the probability that groups of three actin units remain free from phaUoidin if the total added phalloidin were bound in a purely statistical manner. This implies that the binding of one phalloidin to one actin unit inhibits the contribution of three actins to the ATPase activity of the filaments. Cytochalasin B (CB), a metabolite of the fungus H e l m i n t h o s p o r i u m d e m a t i o i d e u m , obviously also weakens the F-actin structure. This can be concluded from the fact that CB inhibits cellular functions linked to actin-like microfilaments (for reviews see (24~ 25)), and that it decreases the viscosity of F-actin solutions(26). Following our observation that CB prevented the phalloidin-induced formation of microfilaments in cell membrane preparations of rat liver (27), we inspected more closely the system F-actin-CB-phaUoidin. We learned that phalloidin is able to antagonize the weakening effect which CB exerts on the F-actin structure (28). This was demonstrated by viscosimetry and again by measuring the ATPase activity. F-actin in the presence of CB exhibits an ATPase activity of a similar rate to that induced by sonic vibration (28). The activity is observable only when KC1 is absent. The ATPase activity observable in the absence of K ÷, like that produced by sonic vibration, is not very high: the rate of liberation of inorganic phosphate is around 0.2 mol Pi per mole actin per minute. A curve similar to that of Figure 7 shows that the ATPase decreases with increasing amounts of phalloidin. As in the case of sonic vibration (Fig. 7) one can conclude that ATPase activity in the presence of CB is only possible when all actin subunits are free from phalloidin but that in order to inhibit the contribution of about 3 subunits to the ATPase activity only 1 of them needs to be combined with phalloidin.

294

TH. WlELAND

F-actin also becomes a (weak) ATPase when the pH is adapted to around its isoelectrical point of pH 4.7 (29, 30). In this case ATPase activity is associated with the precipitation of actin in a paracrystal-like form. This spontaneous "acidic" ATPase effect of F-actin decreases on going to lower pH values as also does the above mentioned ultrasonic ATPase. Whereas phaUoidin at a pH less acidic than the isoelectric point inhibits both ATPase activities, it enhances them at pH values between 3 and 4. Figure 8 shows that in the presence of phalloidin both ATPase activities increased with increasing acidity of the medium (31).

lO. .5 ~ 'g6.

i

PHD

2'

HD 3.0

,~.0 pH

5.0

FIG. 8. Ultrasonic (US) and steady state (no US) ATPase activity of polymeric actin (1.2 mg/ml, 26 I~M) in 1 mM ATP between pH 3 and pH 5 in the presence (40 t~M) or absence of phalloidin (PHD). Inorganic phosphate measured after 30-rain sonication. Conditions noted at the curves. For experimental details see (31). We can interpret these apparently paradoxical findings as being due to a stabilizing effect of phalloidin on the structure of polymeric actin if we make the following assumptions. (1) Polymeric actin can go through a relatively large range of structural transitions from a rather "closed" structure to a rather "opened" one. (2) A particular state of actin is not solely determined by phaUoidin but results from the interaction of all factors which can influence the structure of the actin polymer. Factors acting in the opposite destabilizing direction (as compared to phalloidin) are evidently hydrogen ions and sonication. (3) ATPase activity is possible only in a limited range of actin filament structure, which must be neither too "closed" nor too "opened". Figure 9 presents diagrammatically in which way the different combinations of presence or absence of phalloidin and presence or absence of sonication may influence actin structure at different values of pH. According to this scheme there is inhibition of ultrasonic ATPase activity by phalloidin at neutral pH,

INTERACTION OF PHALLOTOXINSWITH ACTIN

295

"closed" R

I Rangeof AYPaseactiyity C

"K "opened" pH 7

I Lh

pHi.5

pH~'.O-3.5

FIG. 9. Schematic presentation of the influences which are presumably exerted (in the presence of ATP) by pH, sonieation and phalloidin (PHD) on the polymer structure of actin. US means ultrasonieation.

because actin structure remains too "closed" for ATPase activity but there is activation by phalloidin (both of the spontaneous as well as of the ultrasonic ATPase) at lower pH values because without phaUoidin actin structure would be too "opened". In the absence of both ATP and phaUoidin acidic pH "opens" actin structure to such an extent, that irreversible denaturation occurs. Since phalloidin even evokes ATPase activity at pH 3 4 this "acidic" ATPase can be used as a probe for intactness of F-actin after exposure to different conditions in presence of the toxin. In this way the retarding effect of phalloidin on denaturation by high pH values (Fig. 5) has been tested.

Binding of Phallotoxins to F-Actin The effects described above are due to a binding of the phallotoxins to F-actin. This has been shown using a H-desmethylphaUoin, a toxic label, in ultrafiltration experiments (32). Firmly bound toxin, which was not dissociated from the protein by aqueous buffer systems, showed exchange of label with cold phalloidin. This experiment and the fact that the bound toxin is easily set free on treatment with water soluble organic solvents like methanol proves a noncovalent combination. Recently we obtained direct evidence for a proteinactin conjugation by difference spectroscopy(33). Figure 10 shows the difference spectra of phalloidin, its (S)-sulfoxide (A) and (R)-sulfoxide (B) combined with F-actin as measured against the separated components. Clearly at

296

TH. WIELAND 004-

3

AA 003-

002-

I

001-

ZXA

O-

-00t-

-002-

FIG. 10. Difference spectra of mixtures of F-actin (1.15 × 10-5 M), with 1) phalloidin (1.48 × 10-s M), 2) nontoxic phalloidinsulfoxide A (1.41 X 10-s M) and 3) phaUoidinsuffoxide B (1.55 X 10-s M).

295 nm and 305 nm for phalloidin and at 300 nm for the sulfoxides the u.v. spectra are perturbed by the interaction of the chromophoric systems of the respective molecules with the protein, which evidently participate in the interaction. As one sees also the nontoxic sulfoxide A binds to F-actin, so seemingly running counter to the theory of only the toxins stabilizing the structure of F-actin. In the heating experiments referred to in Figure 4, however, the sulfoxide (A-form) had only a limited protecting effect. It is the magnitude of the dissociation constant (KD) which makes a phallotoxin a more or less protecting (nontoxic) agent. A striking difference of K D between the toxic sulfoxide B and the non-toxic sulfoxide A could be demonstrated by qualitative displacement experiments (22). Here both tandem cuvettes contained equimolar mixtures of F-actin plus the respective sulfoxide in one and an equimotar amount of phalloidin in the second compartment. After adjustment of the zero line in both experiments the contents of the compartments of one cuvette were mixed. In the case of sulfoxide A immediately the difference spectrum of bound phalloidin emerged, whereas in the analogous experiment with sulfoxide B the zero line remained unaltered, an indication that here no displacement had taken place. The order of magnitude of the difference of both KD was estimated to about ten fold. G-Actin does not combine with phalloidin as can be concluded from the absence of a difference spectrum in a system analogous to that with F-actin, but with conditions under which no polymerization occurs (low ionic strength, absence of Mg+÷ or Ca*+).

INTERACTION OF PHALLOTOXINSWITH ACTIN

297

The combination of phallotoxins with F-actin is not antagonized by antamanide (34), a cyclic decapeptide which counteracts the lethal toxicity of phaUoidin in vivo when given prior to the administration to experimental animals.

DISCUSSION The features essential for strong binding of a phallotoxin as mentioned in the formula on page 1 and the maxima in the u.v. difference spectrum (Fig. 10) point to a lipophilic region including the thioether moiety and a methyl group, and to an allo-hydroxy group at the proline residue. In a molecular model constructed according to detailed n.m.r, data and lowest energy calculations (35) these groupings could form a binding entity (36). From synthetic analogs, on the other hand, it can be seen that the side chains on the "right side" of the formula are less crucial, if at all, for toxic activity. The remaining side chain of D-threonine (which may be altered to D-a-aminobutyric acid(5) or D-erythro-/3-hydroxyaspartic acid in phallacidin (1) without loss of toxicity of the molecule, but causes drop of toxicity when provided with bulkier groups (37) is in some way a candidate for the function as a second attaching arm. The molecular mechanism of the stabilizing effect of the phallotoxins on F-actin is by far not elucidated until today. G-Actin is one of the most sided proteins. In the double helical chain of F-actin every G-actin unit has 3 neighbour molecules, i.e. 3 different binding sites. In addition there exists a binding site for an adenine nucleotide and at least one for a metal ion (Ca *+, Mg÷÷, K÷). Moreover for the reaction with myosin and the regulatory proteins several further binding sites are necessary. None of all seem to be identical with the binding site(s) for the phallotoxin, since, e.g., the binding to myosin and most probably to all regulatory proteins is not influenced by the toxin. Besides more complicated suggestions one can consider two alternative modes of action of the phallotoxins on actin, (1) the toxin binds to one actin unit each; (2) the toxin binds to two actin units so functioning as a cross linking molecule. As to 1): A binding of phalloidin to G-actin could not be observed by difference spectroscopy (as for F-actin, page 296) until now. Since, however, phalloidin binds firmly to F-actin, it is conceivable that it also binds to each G-actin dimer as it is continuously formed in a solution of G-actin. By this attachment one of the G-units of the dimer would be (allosterically) altered in such a way as to fix irreversibly an adjacent molecule. Since in this way the number of nuclei would increase, the acceleration of polymerization, as observed, would be obvious as well as the stability of Ph-actin.

298

TH. WlELAND

As to 2)" The assumption of a bifunctionality of the phallotoxin would obviate an allosteric phenomenon and would likewise account for the acceleration of the polymerization and for the strengthening of the micro filaments. A decision between both the possibilities can be tried by attempts to bind covalently the toxin molecule to its receptor(s). Such experiments are currently being performed in our laboratory. SUMMARY Phalloidin (PHD) like its toxic relatives from the mushroom Amanita phaUoides and like toxic derivatives obtained by chemical modifications combines with F-actin, thus stabilizing its polymer structure. Since there is no dissociation of F-actin to G-actin in presence of PHD F-actin filaments have been found in poisoned liver cells. The stabilization of F-actin from rabbit muscle by PHD has been established experimentally by its behavior towards several chemical and mechanical stresses. PHD-treated actin (Ph-actin) is not depolymerized by 0.6 M KI, it withstands the depolymerizing action of DNAase I, denaturation by heating to 70°C for 3 min, and retards destruction by alkali (pH 12) about 30 fold. Ph-actin is also resistant to the hydrolyzing action of subtilisin. Phallotoxins prevent the ATPase activity of F-actin which normally is induced by ultrasonic vibration or by cytochalasin B around neutral pH values. Whereas on going to acidic pH without PHD the ultrasonic ATPase decreases and completely disappears at pH 3.5, PHD even under the same conditions preserves the structure to such an extent as to still retain ATPase activity. At all effects observed a ratio of 1 mol PHD per 1 mol of G-actin seems sufficient for full protection. Phallotoxins are firmly bound to F-actin as observed by difference spectroscopy in the u.v. light. As to their stabilizing action either an allosteric effect enforcing the coherence between the actin units or crosslinking by bifunctionality of the phallotoxin molecules has been postulated. REFERENCES 1. T. WIELAND, Poisonous principles of mushrooms of the genus Amanita, Science 159, 946-952 (1968). 2. T. WIELAND, Phallotoxins and microfflaments, pp. 203-214 in 26. Colloquium Mosbach 19 75 Molecular Basis o f Motility, (L. HEILMEYER, I. C. ROEGG and T. WIELAND, eds.), Springer Verlag, Berlin, Heidelberg, New York (1976). 3. H. FAULSTICH and T. WIELAND, Relation of toxicity and conformation of phallotoxins as revealed by optical rotatory dispersion studies, Europ. J. Biochem. 22, 79-86 (1971). 4. H. FAULSTICH, E. NEBELIN and T. WIELAND, Peptid-synthesen LIV. Synthesan einiger Analoga des Norphalloins, Liebigs Ann. Chem. 50-58, (1973). 5. H. HEBER, H. FAULSTICH and T. WIELAND, Syntheses of further analogues of norphalloin. Gly I -, L-VaP and D-Abu2-norphalloin and (~-Trideutero)-Ala s -norphalloin. Intern. J. Peptide Protein Res. 6, 381-389 (1974).

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