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The cytotoxins of cobra venoms
Biochimica et Biophysica Acta, 393 (1975) 320-334 © Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands BBA 37050 T H E C Y T O T O X I N S OF COBRA VENOMS ISOLATION AND PARTIAL CHARACTERIZATION
SAMUEL J. DIMARI, KENNETH J. LEMBACH and VIRGINIA B. CHATMAN Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, Tenn. 37232 (U.S.A.) (Received October 14th, 1974)
SUMMARY Eight basic proteins which lyse virus-transformed mouse fibroblasts in culture have been isolated from the venoms of six Asian Naja naja subspecies. These cytotoxins appear to represent an homologous series of proteins, all within the molecular weight range of 7000-8000. They have been divided into three arbitrary types on the basis of amino acid composition, electrophoretic mobitities and elution order upon ion-exchange chromatography. The rate at which the toxins effect cell lysis: (1) appears to be a function of the basicity of each toxin; (2) is dependent upon toxin concentration; (3) is temperature dependent; and (4) is inhibited by heparin sulfate. In view of the physical changes, which the cell undergoes during lysis and of the various factors which affect the action of these proteins, it is proposed that interaction of membrane receptors with the toxin, leading to alteration of cell membrane structure, is the principal event which ultimately leads to the disruption of the cell.
INTRODUCTION Various workers have isolated basic protein fractions from the venom of the Indian cobra Naja naja naja and have named them according to the physiological action which they elicit in different test systems. These fractions have been called direct lytic factor (acts synergistically with phospholipase A in the lysis of erythrocytes [i, 2]), cobramines A and B (affect the permeability of thyroid slices to ions and Abbreviations: NNI-A and NNI-B (Type A and B cytotoxins from Naja naja naja (India) (NNI)); NNP-A and NNP-B (Type A and B cytotoxins from Naja naja naja (Pakistan) (NNP)); NNC-A and NNC-B (Type A and B cytotoxins from Naja naja ceylonicus (NNC)); NNS-B (major cytoxin from Naja naja siamensis (NNS)) ; NNO-C (major cytotoxin from Naja naja oxiana (NNO)); NNSe-C (major cytotoxin from Naja naja samarens& (NNSe)). Type A, B, and C have been used for the cytotoxins rather than Type I, II, and III in order to differentiate them from Type I and II venom neurotoxins. The different types of cytotoxin contain approximately the same number of amino acid residues and disulfides whereas the two types of neurotoxin differ from one another both in the number of amino acid residues and in the number of disulfides in each [27].
321 metabolites [3-5]), cytotoxin P6 (shows selective lysis of tumor cells over other cell types [6] and was reported to be a phospholipase C [7-9]), cardiotoxins (depolarize smooth muscle cells by inducing loss of K + [10]), and cytotoxins I and II (selectively lyse Yoshida sarcoma and ascites hepatoma cells [11, 12]). All of these various activities are assumed [11] to be different manifestations of the same two proteins present in the venom, with cytotoxin P6 a mixture of these plus perhaps a contaminating phospholipase. These proteins apparently induce alterations in membrane permeability or in membrane structural organization. They not only affect different membrane systems to different extents, but their action also appears to be sensitive to slight changes in membrane component content or to surface group configuration within different strains of the same cell type [2, 7]. In this respect, they resemble the plant lectins (e.g. concanavalin A), molecular probes used to detect membrane surface changes in erythrocytes and tumor cells [13]. These toxins would, therefore, appear to be not only potential antitumor agents, but are also potentially useful molecular probes for elucidating membrane ultrastructure and its role in biological functions of the cell. Three cytotoxins, the term used here for these proteins, have been isolated and sequenced: cytotoxins I and II (referred to in this paper as NNI-A and NNI-B, respectively) from the venom of the Indian cobra [12, 14, 15] and the cytotoxic [16] cardiotoxin from the venom of the Formosan cobra Naja naja atra [17]. The present study was undertaken to determine whether such membrane-active proteins are common components of Asian cobra venoms and if so, whether they vary in their abilities to lyse virus-transformed mouse fibroblasts in culture. METHODS
Isolation of Cytotoxins Cytotoxins were isolated from lyophilized whole venoms by repeated chromatography on the cation exchange resin sulphopropyl-Sephadex (C-25) followed by gel filtration on Sephadex G-50 (fine) as previously described [18]. Samples of cytotoxins used in experiments were either individual or combined fractions from the final sulphopropyl-Sephadex chromatography or from gel filtration. Homogeneity of isolated toxins was determined by both disc gel electrophoresis and amino acid analysis. Protein concentrations were determined by the method of Lowry et al. [19] with bovine serum albumin as standard and by amino acid analyses.
Characterization of Cytotoxins Molecular Weight Determination. Molecular weights of the cytotoxins were estimated by gel filtration on a calibrated 2.5 x 70 cm column of Sephadex G-50 (fine) eluting with 0.05 M sodium phosphate buffer, pH 6.0. Flow rate varied from 19 to 22 ml/h. Disc gel Electrophoresis. The apparatus, gel percentages, conditions for electrophoresis, gel staining and destaining procedures were those previously described [18]. Amino Acid Analysis. Aliquots of combined fractions obtained from gel filtration and to which norleucine had been added as a standard were lyophilized
322 to dryness in combustion tubes and were subjected to 24 h methanesulfonic acid hydrolysis [20] in vacuo at 115 °C. Amino acid contents were determined using a Beckman amino acid analyzer, Model 120C updated to a Model 121 and equipped with an automatic sample applicator. Assay for Cytotoxicity: Growth of SVIO1 Cells. A simian virus 40 (SV40)transformed derivative of mouse 3T3 cells, designated SV101 [21], was used to assay cytotoxicity. This cell line was kindly provided by Dr Howard Green, Department of Biology, Massachusetts Institute of Technology. Cells were maintained in Dulbecco's medium [22] supplemented with 5 ~ calf serum. Experimental cultures (25 cm 2) were initiated from trypsinized cell suspensions and were incubated at 37 °C for 48 h before use. Approx. 2 • 106-4 106 cells in 5 ml of medium were utilized for each experiment unless otherwise stated. •
Lactate Dehydrogenase Assays General Procedure. The release of the cytoplasmic marker enzyme lactate dehydrogenase was used to monitor lysis of SV101 cells. Units of enzyme released were determined spectrophotometrically by measuring the rate of N A D H oxidation (pyruvate substrate) using the reagents and experimental procedures described by Kornberg [23]. Reaction was initiated by the addition of the enzyme and was followed for 3 min using a Model 240 Gilford spectrophotometer (0.1 absorbance unit full scale) fitted with automatic sample changer and a Honeywell Model 6040 recorder. Standard curves correlating the rate of N A D H oxidation with units of lactate dehydrogenase were prepared using serial dilutions of commercial rabbit muscle enzyme. The rate of reaction was linear within the range of enzyme concentration present in the cultured cells. One unit of lactate dehydrogenase activity is defined as that quantity of enzyme necessary for the oxidation of 1 nmol of N A D H per rain at non-limiting substrate and N A D H concentrations. Determination of Lactate Dehydrogenase Content of SVlO1 Cells. SV101 cells were subcultured at different densities using the above procedure. At the end of the growth period, medium was removed, the cells adhering to the culture flasks were washed twice with 37 °C phosphate-buffered saline, pH 7.2, and were removed from the flasks by treatment with 5 ml of 0.25 ~ trypsin in phosphate-buffered saline for 5 min at 37 °C. Cells were collected by centrifugation at 1500 rev./min using a Sorvall Model RC-3 refrigerated centrifuge. The packed cells were washed twice by centrifugation with 5 ml of phosphate-buffered saline and were then suspended in 1-3 ml of phosphate-buffered saline to give approximately the same number of cells per ml in the different systems. The total number of cells in each suspension was determined using a Coulter Counter. Each cell suspension was sonicated for 30 s at 4 °C with a Sonifier Cell Disrupter, Model WI85D (Heat Systems-Ultrasonics, Inc., Plainview, L.I., N.Y.) fitted with a microprobe. The supernatant solutions obtained after centrifugation (5000 rev./min for 30 min) were assayed for lactate dehydrogenase activity by the above method. Protein content of each supernatant was determined by the method of Lowry et al. [19].
Determination of Lactate Dehydrogenase Released .from SVIO1 Cells by Cytotoxins. A 0.2-ml aliquot of the serum-containing medium in which the cells had been cultured was withdrawn from each system to serve as a "zero time" sample. Flasks were inverted to remove medium from direct contact with cells, and the
323 cytotoxin was added to the medium. The cytotoxin-cell systems were equilibrated at the indicated temperatures for 5 to 10 min and were then inverted to bring the cytotoxin-containing medium into contact with the cells ("zero time point"). All of the experiments presented in each figure were conducted on the same day with cells grown and treated in the same manner. At various time intervals, 0.2-ml aliquots of supernatant media were withdrawn from each system, and samples (50-1130/A) were assayed for lactate dehydrogenase. The total units of enzyme shown in the figures have been corrected for volume changes during the experiment, for units of enzyme removed with each aliquot and for the units of enzyme present in the system at time "zero". MATERIALS Lyophilized Naja naja naja (India) (Lot No. 52C-1250) venom was obtained from Sigma Chemical Co. (St. Louis, Mo.). The following lyophilized venoms were obtained from the Miami Serpentarium Laboratories (Miami, Fla.): Naja naja eeylonicus (Ceylon) (Lot No. NY 3.5 TL), Naja naja naja (Pakistan) (Lot No. NNPOL), Naja naja oxiana (Iran) (Lot No. NOOETL), Naja naja samarensis (Philippines) (Lot No. NEOETL), Naja naja siamensis (Thailand) (Lot No. NS1ETL). Sulphopropyl-Sephadex (C-25), Sephadex G-50 (fine) and the gel filtration protein standards aldolase, chymotrypsinogen A and ribonuclease A were obtained from Pharmacia Fine Chemicals (Piscataway, N.J.). Grade III yeast fl-nicotinamide adenine dinucleotide, reduced form (fl-NADH), disodium salt (Lot No. 103C-6380) and Type I rabbit muscle L-lactate-NAD oxidoreductase, EC 1.1.1.27 (crystalline suspension in 2.1 M (NH4)2SO4, spec. act., 80 units/mg protein) were obtained from Sigma Chemical Co. Grade A sodium pyruvate (Lot No. 100984) was obtained from Calbiochem (Richmond, Calif.). Heparin sulfate was obtained from ICN Nutritional Biochemicals Corporation (Cleveland, Ohio). Crystalline bovine serum albumin was purchased from Pentex, a division of Miles Research Laboratories (Kankakee, Ill.). RESULTS
Isolation and Characterization of Cytotoxins Sulphopropyl-Sephadex Chromatography. The elution profiles of venoms fractionated and the positions of the cytotoxic proteins isolated from each are shown in Fig. 1. Proteins eluting immediately before, between, or after these materials were tested and did not lyse SV101 cells. The proteins designated NNP-B (Fig. 1A) and NNSe-A (Fig. 1F) were cytotoxic but were not further purified nor characterized since they could not be obtained in sufficient quantity. Usually two additional ionexchange chromatographic steps, using the gradient system described for cytotoxins [18], were necessary to obtain homogeneous proteins. Moleeular Weights of the Cytotoxins. The molecular weights of NNI-A and NNI-B are between 7000 and 8000 [12, 14]. The elution volumes of other cytotoxins (Fig. 2) are similar to those observed for these two toxins, indicating that all of the cytotoxins isolated are within the same molecular weight range. No higher molecular weight species were observed upon gel filtration. For calculations, a molecular weight of 7000 was assumed for each toxin.
324 I0 2050405060708090100 ~02050405060708090100 (~ Nolonolonaja(Pakistan) (~ Nolonolo siomensis NNP-A ~ NNS-B
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Fig. 1. Sulphopropyl-Sephadex fractionation of Naja naja venoms. Lyophilized venoms (200 mg), dissolved in 5 ml of 0.05 M phosphate buffer, pH 6.0, were fractionated on 2.5 x 20 cm columns of sulphopropyl-Sephadex using a linear salt gradient [18]. Arrows mark the start of the gradient. Cytotoxic proteins are indicated in the figure by cross-hatching and are labeled with the abbreviations used for them in the text and in all figures and tables. The yields (percent protein recovered from whole venom) of each cytotoxin were: NNP-A, 9 ~ ; NNI-A, 13.5 %; NNI-B, 13 ~ ; NNC-A, 8 ~ ; NNC-B, 1 1 ~ ; NNS-B, 2 1 ~ ; NNO-C, 13%; NNSe-C, 10%.
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Fig. 2. Gel filtration of the cytotoxins. Cytotoxins (10--20 mg) were subjected to gel filtration on Sephadex G-50 (fine) (see Methods). 4-ml fractions were collected. Standard proteins used for column calibration and indicated in the figure were: aldolase (ALD), 158 000 real. wt; chymotrypsinogen A (CHYMO), 25000mol. wt; and ribonuclease A (RNase), 13700moi. wt. Symbols: L~--O, NNI-A; 0 - - - 0 , NNI-B; C]--C3, NNP-A; II--m, NNC-A; A - - A , NNC-B; ( ) - - ( ) , NNS-B; ~ - - / ~ , NNO-C; x - - ×, NNSe-C. The actual absorption of some of the toxins has been altered to simplify the figure.
325
Disc Gel Electrophoresis of Cytotoxins. The disc gel patterns obtained for whole venoms and the bands which correspond to the cytotoxins are shown in Fig. 3. The toxins, as seen, are the fastest cathodally-migrating major species of each venom. As also seen in the figure, the cytotoxins may be arranged in the following order of decreasing electrophoretic mobilities: C toxins > B toxins > A toxins. This difference in basicity was inferred by the elution order of different toxins upon ionexchange chromatography. + 6789
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Fig. 3. Disc gel electrophoresis of cytotoxins and their corresponding venoms. Disc gel electrophoresis of cytotoxins (20/*g) and venoms (100 yg) was conducted on 3-mm thick polyacrylamide gel slabs [18]. The reproduction shown is a composite of two gel slabs; common venoms and cytotoxins were present on each as references. Only major, well-stained protein venom components have been indicated. 1, NNP-A; 2, Noja naja naja (Pakistan), whole venom; 3, NNP-B; 4, NNI-A; 5, Naja naja naja (India), whole venom; 6, NNI-B; 7, NNC-A; 8, Naja naja ceylonictts, whole venom; 9, NNC-B; 10, NNS-B; 11, Najanajasiamensis, whole venom ; 12, NNO-C; 13, Najanalaoxiana, whole venom; 14, NNSe-C; 15, Naja naja sarnarensis, whole venom.
Amino Acid Analyses. The amino acid content of each of the cytotoxins isolated is shown in Table I. The amino acid contents presented in the table for N N I - A and N N I - B agree well with those previously reported [12, 14, 15]. None of the cytotoxins contain tryptophan, an amino acid commonly present in the venom neurotoxins. The A toxins contain one residue of glutamic acid and lack histidine and phenylalanine. The B toxins contain neither glutamic acid nor histidine but contain phenylalanine. The C toxins differ f r o m the other two types in that they contain a residue of histidine and fewer acidic amino acids. A comparison of the ratios of basic to acidic residues of each type explains the differences in basicities noted earlier. Release of Lactate Dehydrogenase from SVIO1 Cells by Cytotoxins Lactate dehydrogenase activity in SV101 cells was found to vary linearly with the number of cells in a given culture (Fig. 4). To verify that the oxidation of N A D H
326 TABLE I AMINO ACID COMPOSITION OF COBRA VENOM CYTOTOXINS Duplicate samples (approx. 50 nmol) of each cytotoxin were hydrolyzed and analyzed as described in Methods. Nanomoles corresponding to one residue of each of five amino acids (*) were calculated and an average value obtained. This average was used to determine the number of residues of each amino acid. The values presented in the table are averages of the two numbers obtained in this manner from duplicate analyses. The closest integral value(s) for each amino acid is given in parentheses. Amino acid
Type A
Type B
NNI-A
NNP-A
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NNI-B
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NNO-C
NNSe-C
Lys Arg* His Trp Asp* Glu* Thr Ser Pro Gly* Ala* '/z Cys Val Met Ile Leu Yyr Phe*
9.2 (9) 2.0 (2) 0 0 8.5 (8-9) 1.0 (1) 2.9 (3) 1.9 (2) 4.5 (4-5) 2.1 (2) 2.2 (2) 7.0 (8) 4.6 (5) 2.0 (2) 1.8 (2) 6.3 (6) 4.0 (4) 0
9.1 (9) 2.0 (2) 0 0 8.5 (8-9) 1.0 (1) 3.0 (3) 2.0 (2) 4.3 (4) 2.1 (2) 2.1 (2) 7.2 (8) 4.5 (4-5) 1.9 (2) 1.8 (2) 6.3 (6) 4.l (4) 0
9.0 (9) 2.1 (2) 0 0 8.5 (8-9) 1.0 (1) 3.0 (3) 2.0 (2) 4.3 (4) 2.1 (2) 2.1 (2) 6.7 (8) 4.1 (4) 2.1 (2) 1.7 (2) 6.3 (6) 4.2 (4) 0
8.9 (9) 2.3 (2) 0 0 6.7 (7) 0 3.1 (3) 2.0 (2) 5.3 (5) 2.1 (2) 2.1 (2) 6.3 (8) 5.4 (5) 2.0 (2) 0.8 (1) 6.3 (6) 3.9 (4) 1.0 (1)
9.5 2.1 0 0 6.8 0 3.2 2.1 6.7 2.2 2.1 6.9 6.1 2.2 0.9 6.3 4.1 0.9
9.0 2.2 0 0 7.8 0 2.8 1.9 4.6 2.1 2.7 6.3 4.4 1.9 1.4 6.1 2.8 1.1
10.4 (10) 1.0 (1) 1.0 (1) 0 5.2 (5) 0 1.9 (2) 2.7 (3) 5.3 (5) 2.1 (2) 3.1 (3) 7.0 (8) 6.4 (6) 2.0 (2) 1.3 (1) 6.1 (6) 1.9 (2) 1.9 (2)
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cells, cultured to different densities, were collected and counted; the supernatant fluids, obtained by sonication and centrifugation, were assayed for lactate dehydrogenase (see Methods). The total units (UT) of lactate dehydrogenase shown for each cell density represents the average of five determinations. The standard deviation from this average value is indicated.
327 m e a s u r e d was a result o f the a c t i o n o f this enzyme, o x a m i c acid, a competitive i n h i b i t o r o f the enzyme [24], was a d d e d to some o f the assay mixtures. W h e n present in e q u i m o l a r a m o u n t with substrate, o x a m i c acid decreased the rate o f N A D H o x i d a t i o n by a p p r o x . 33 ~ . T h e n u m b e r o f units o f lactate d e h y d r o g e n a s e p e r cell was calculated to be 2 . 5 . 1 0 - 4 - 3 • 10-4; 2.106 cells w o u l d c o n t a i n 500-600 t o t a l units o f enzyme. The average p r o t e i n c o n c e n t r a t i o n for the systems shown in the figure was 0.5 m g o f p r o t e i n p e r 2.106 cells. Therefore, the cultured cells contained 1000-1200 units o f lactate d e h y d r o g e n a s e p e r m g o f protein.
Ability of a Cytotoxin to Release the Theoretical Total Units of Lactate Dehydrogenasefrom SVIO1 Cells. A s seen in Fig. 5A, the t o t a l units o f enzyme released by the c y t o t o x i n f r o m b o t h high a n d low density cultures were a p p r o x i m a t e l y w h a t w o u l d be expected for the n u m b e r o f cells present in each system. The variability in the d e t e r m i n a t i o n o f enzyme units at any p o i n t was a p p r o x . ~-5 ~ at high levels o f enzyme ( > 100 units) and -L l0 ~o at low levels ( < 100 units).
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Fig. 5. (A) Release of lactate dehydrogenase (LDH) from SVI01 cells by cytotoxin NNI-B. Six cultures of SV101 cells were seeded, three to provide a final cell density of 1.5.106 cells and three of 3.106 cells. At the end of 48 h, one culture of each density was trypsinized, and the cells were recovered and counted (see Methods). The low density culture contained 1.4" l& cells and the high density culture contained 3.3" 106 cells. Cytotoxin NNI-B was added to the remaining two cultures of each density to a final concentration of 20 fig of toxin per ml of medium, and the systems were incubated at 37 °C. Supernatant media were assayed for lactate dehydrogenase (see Methods). Total enzyme units (Ur) in the low density cultures are indicated by ( A - - / ~ ) and those in the high density cultures by ( O - - O ) . (B) Percent of total lactate dehydrogenase (LDH) release by cytotoxin NNI-B versus time. The experimental systems and symbols for low and high density cultures are those described in A. The total enzyme units used for each time point is an average of the values obtained from the two cultures of each cell density; these units are expressed as percentages of the average total units released from each cell density at 220 min.
328 Fig. 5B shows the percent of total enzyme released by the cytotoxin at different times. Essentially linear rates (total units/t) of enzyme release were observed up to 50-70% release of the total enzyme present in the cells; after this point had been reached, rates decreased. The different time intervals necessary for the release of 50 of the total enzyme were proportional to the cell densities used.
The Release of Lactate Dehydrogenasefrom S VIOl Cells by Different Cytotoxins. All of the cytotoxins isolated were tested for their ability to lyse SVI01 cells (Fig. 6). The percent of enzyme released per time was calculated from the data shown. The times required for each cytotoxin to release 50 ~ of the total enzyme present were: NNSe-C, 45 min; NNO-C, 70 rain; NNS-B, 95 min; NNC-A, 110 min; NNC-B, 112 min; NNI-A and NNI-B, 117 rain; and NNP-A, 128 min. As seen, the Type C toxins released enzyme at faster rates than either the Type A or B toxins (for all practical purposes, Type A and Type B toxins were equally effective in releasing enzyme from the cells). i
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20 40 60 80 100 20 40 60 80 200 TIME. rain Fig. 6. Release of lactate dehydrogenase from SV 101 cells by Naja naja cytotoxins. Cytotoxins (10 #g of toxin per m] of medium) were added to cu]tures of SV]0] cells, and the systems were incubated at 37 °C. Aliquots of supernatant media were removed and assayed for lactate dehydrogcnase activity (see Methods). Symbols: © - - © , NNT-A; H , NNI-B; Vl--[D, NNP-A; i - - i , NNC-A; A - - A , NNC-B; (])--(]), NNS-B; A - - ~ , NNO-C; × - - × , NNSe-C.
Microscopic examination showed that cells appeared to be affected differently by the Type C (strong) toxins than by the Types A and B (moderate) toxins. With both strong and moderate cytotoxins, toxin-cell interaction was indicated by refractility of the cell membrane (localized phase dense areas appeared upon the surface of the otherwise transparent cells). With the moderate cytotoxins, the cells became increasingly refractile, contracted from the fibroblastic form, and detached individually from the culture vessel. Cultures lysed by the A- and B-type toxins contained much cellular debris and floating, discrete membrane fragments. With the strong, or C-
329 type toxin, however, initial refractility was followed by a rapid contraction of the cell, leaving anchoring processes still in place. As lysis progressed, cells lifted from the dish in sheets rather than individually as was the case with the moderate toxins. Type C-lysed cultures were characterized by floating sheets of cell membranes; nuclei were often observed in the flask. The observable differences in enzyme release and lysis by the A/ B and C cytotoxins may be an indication that the two classes are acting by different mechanisms or may simply be a result of the fact that the strong cytotoxins affect the cells so rapidly that they are unable to completely detach from one another and are removed in sheets rather than individually.
Effects of Various Factors on Cytotoxin-Induced Enzyme Release from SVIO1 Cells Preliminary experiments involving a moderate (NNI-B) and a strong (NNOC) cytotoxin were undertaken to determine the ability of these two types to release lactate dehydrogenase from SV101 cells under various conditions. The tissue culture system used contains factors (e.g. serum protein) which potentially could affect the action of the toxins and, therefore, only the most general inferences can be drawn from the following experiments. Effect of Cytotoxin Concentration. The rate of enzyme release by both cytotoxins NNI-B and NNO-C and the time required to release 50 ~ of the total enzyme present in the culture (Fig. 7) were proportional to the concentration of toxin. With both the strong and moderate cytotoxins there was a definite concentration below which enzyme release did not occur within the time interval of the experiment. As seen in Fig. 7, this concentration was lower for NNO-C (2/~g/ml) than for NNI-B (5 /~g/ml). The inability of NNI-B at a concentration of 2 #g of toxin per ml to release enzyme might be ascribed to non-specific binding of the toxin to serum protein, affinity of the toxin for the plastic culture flask, etc. ; however, NNO-C which should be influenced by the same factors was relatively active at this concentration, indicating, among other things, a possible difference in the mechanism of action of the two toxin types. Cytotoxin-treated cultures in which no enzyme release was observed were visually examined after 24 h. With both toxins, the cells appeared to be intact with relatively normal morphology except for isolated phase dense areas on membrane surfaces. Effect of Temperature. The action of both strong and moderate cytotoxins is very temperature dependent. During the first 60 min of exposure, there was virtually no enzyme release by either cytotoxin at temperatures below 30 °C (Fig. 8). At the end of this period, however, lactate dehydrogenase was released in all systems except that which contained NNI-B at 4 °C. The duration of the induced lag period appeared to be independent of temperature or cytotoxin used (Fig. 9). The greatest difference in lytic ability between NNO-C and NNI-B was observed at 4 °C. During the experimental time period, NNO-C induced a slow release of enzyme whereas NNI-B did not. Examination of both cultures at the end of 24 h at 4 °C showed that the strong cytotoxin had destroyed all of the cells originally present whereas the cells in the NNI-B system were still attached to the culture dish and, although highly refractile, appeared to be intact. Effect of Heparin Sulfate. Heparin sulfate has been reported to precipitate the basic proteins of cobra venom [1 ], and it was of interest to test the effect of this agent upon the rate of enzyme release from cytotoxin-treated cells.
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Fig. 7. (A) Percent of total lactate dehydrogenase release by different concentrations of cytotoxin NNI-B versus time. Cytotoxin NNI-B was added to cultures of SV101 cells to give the following final concentrations of cytotoxin per ml of medium: /~--/~, 1/~g/ml; I1--11, 3#g/ml; 0 - - 0 , 5 ug/ml; C]--C], 10 #g/ml; and A - - A , 20 #g/ml. Systems were incubated at 37 °C and lactate dehydrogenase activity in supernatant media was monitored (see Methods). Reactions were allowed to proceed for 340 rain or until the level of enzyme became constant. The values at each time point represent percentages of the total units released upon complete lysis of each culture. For systems in which total lysis was not observed during the experimental period, the average of the total units found in completely lysed cultures was used as 100 ~ . (B) Percent of total lactate dehydrogenase release by different concentrations of cytotoxin NNO-C versus time. Cytotoxin NNO-C was added to cultures of SV 101 cells to give the following concentrations of cytotoxin per ml of medium; O--O, 0.1/~g/ml; I1--11, 1/~g/ml; ~x--~,, 2/~g/ml; ©--~), 10#g/ml; A - - A , 20/~g/ml; and [~--C3, 40/~g/ml. Experimental procedures and calculations were those described in A. I00
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Fig. 8. Effect of temperature upon lactate dehydrogenase (LDH) release by cytotoxins NNI-B and NNO-C. Cytotoxins NNI-B ( 0 - - 0 ) and NNO-C ( k - - A ) were added to temperature-equilibrated cultures of SV101 cells to final concentrations of 10/zg per ml of medium. Aliquots of supernatant media were periodically removed and assayed for lactate dehydrogenase (see Methods). The total enzyme units released by the cytotoxins after 60 rain at temperatures below 37 °C are expressed as percentages of the units released by the toxins at 37 °C during the same time interval.
331
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Fig. 9. Effect of temperature upon lactate dehydrogenase release by cytotoxins NNI-B and NNO-C. Cytotoxins NNI-B (A) and NNO-C (B) were added to temperature-equilibrated cultures of SV101 cells to final concentrations of 20 and 40/zg of cytotoxin per ml, respectively. Experimental conditions and protocol were those described in Fig. 8 and in Methods. The following incubation temperatures were used in both A and B: ~ - - A , 4 °C; 0 - - 0 , 15 °C; D--[~, 23 °C; &--A, 30 °C; 0 - - 0 , 37 °C. Fig. 10. Effect of heparin sulfate upon lactate dehydrogenase release from SV101 cells by cytotoxins NNI-B and NNO-C. Cytotoxins NNI-B (A) and NNO-C (B) were each added to three SV101 cultures at 20 and 40 pg of toxin per ml of growth medium, respectively. The systems were incubated at 37 °C. After 10 min of exposure, 2 mg of heparin sulfate in 2001d of distilled water was added to one flask of each set (A--A), and, after 40 min, the same amount of beparin was added to another flask ( D - - D ) . No heparin was added to either the third flask of a set ( O - - O ) or to a non-toxincontaining control ( × - - × ). The release of lactate dehydrogenase was monitored (see Methods).
C y t o t o x i n s N N I - B a n d N N O - C were each a d d e d to three cultures, the systems were i n c u b a t e d at 37 °C a n d h e p a r i n sulfate was a d d e d to specific cultures at different times (Fig. 10). As seen, the a d d i t i o n o f h e p a r i n resulted in a decrease in the rate o f enzyme release by b o t h cytotoxins and, eventually, to a cessation o f lysis. Visual e x a m i n a t i o n o f cultures after the a d d i t i o n o f h e p a r i n showed that as the rate o f lactate d e h y d r o g e n a s e release decreased, the cells b e c a m e less refractile a n d began to reassume a n o r m a l m o r p h o l o g y . A t the end o f the e x p e r i m e n t a l time period, the h e p a r i n - t r e a t e d cells were indistinguishable f r o m n o n - c y t o t o x i n - e x p o s e d control cultures. In o r d e r to allow longer c o n t a c t between c y t o t o x i n a n d cell, the experiment shown in Fig. I I was conducted. As seen in the figure, when the t e m p e r a t u r e o f the n o n - h e p a r i n - t r e a t e d , t o x i n - c o n t a i n i n g system was increased f r o m 4 to 37 °C, lactate d e h y d r o g e n a s e was released at a rate c o m p a r a b l e to t h a t observed with the system o r i g i n a l l y placed at 37 °C. The h e p a r i n - t r e a t e d system, when placed at 37 °C, initially released enzyme at a slow rate a n d then release stopped. Physical changes in
332
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Fig. 11. Effect of heparin sulfate and temperature upon the release of lactate dehydrogenase (LDH) from SV101 cells by cytotoxin NNI-B. Cytotoxin NNI-B was added to three SV101 cultures (20 ttg of toxin per ml of medium). Two of these cultures, plus a non-toxin-containing control, were immediately placed at 4 °C; the remaining culture ( © - - © ) was incubated at 37 °C. At the end of 130 min (arrow), 2 mg of heparin sulfate in 200 #1 of distilled water was added to one of the toxin systems at 4 °C ([E--[B) and this, together with the non-heparin-treated, toxin-containing system ( A - - A ) and control ( × - - × ) , was placed at 37 °C. Lactate deh),drogenase activity in supernatant media was monitored (see Methods).
form and refractility of these latter cells were those observed when heparin was added during a period of active enzyme release. DISCUSSION
Although the rate at which each toxin induces enzyme release and the manner in which lysis is achieved appears to be related to the basicity of the toxin, the difference in activity observed between strong and moderate cytotoxins may also be accounted for by different primary or tertiary structures or by different receptors for each. It is assumed that the initial step in the lytic process is an electrostatic attachment of the toxin to negatively charged receptor groups on the cell membrane. However, were the neutralization of a portion of the membrane surface charge the principal event in lysis, any very basic protein could mimic the action of the cytotoxins. We (DiMari, S. J. and Chatman, V. B., unpublished) have exposed SV101 cells to cobrotoxin (pI 9.4), the principal neurotoxin from the venom of N. naja atra [25], and to venom proteins which are similar to the toxins in molecular size and behavior on ion-exchange chromatography and upon disc gel electrophoresis; no measureable release of enzyme was observed. As shown, the ability to lyse tumor cells is the property of specific proteins in the venoms studied and these proteins appear to form an homologous series based upon their amino acid contents. The tertiary structure of the cytotoxin appears to be essential for its action
333 since reduced toxins have been reported to be unable to lyse Yoshida cells [12, 15]. In view of its high disulfide content, the cytotoxin, like the analogous neurotoxin, must be a rigid molecule with its polar residues oriented in a specific manner on the molecular surface. The principal polar residue in these proteins is lysine, and it is assumed that lysines play an active role in the lytic process as opposed to a passive one (e.g. present only to solubilize the toxin in aqueous media). The regular spacing of lysine residues throughout the primary sequences of NNI-A and NNI-B [12, 14] would appear to be sufficient to allow a specific number of these residues to be within a prescribed area of the molecular surface, making multipoint electrostatic contact of the cytotoxin with charged membrane receptors possible. An equilibrium binding of toxin to cell membrane could explain the ability of heparin sulfate to stop the lytic process. Heparin may be removing unbound cytotoxin from solution and reversing the balance of equilibrium, causing partially bound cytotoxin to dissociate from the membrane. If heparin were removing only unbound toxin and reducing the level of toxin in the system to sub-lytic concentrations, one would expect the remaining cells in the culture to be refractile as was observed when such doses were applied. This, however, was not the case; after heparin treatment, infected cells returned to a completely normal appearance. The addition of heparin did not result in an immediate cessation of cell destruction. Slow enzyme release continued for an hour or longer until it stopped, indicating that perhaps some toxin molecules were more firmly bound than others or that they had penetrated into the membrane or cytosol. Experiments designed to determine whether cytotoxin P6 acted primarily on the cell membrane or in the cytosol were inconclusive [7]. The effect of temperature upon the action of the cytotoxins resembles that observed for the intermixing of cell surface antigens throughout fused membranes of mouse-human heterokaryons [26]. This phenomenon was ascribed to a variation in cell membrane fluidity with temperature and to the effect which the state of the membrane has upon the ability of molecules to diffuse through it. Therefore, one possible explanation for the temperature effect observed is that the toxin, in order to act, must bind to a specific number of non-localized membrane receptors and that the ability of receptors and of partially bound toxin molecules to diffuse to within binding distance of one another is decreased by a temperature-dependent alteration of membrane fluidity. Temperatures below 30 °C may be breaking down the lytic process into its component steps and exposing a cooperativity effect which is obscured by the rapidity of the overall process at 37 °C. There are several possible explanations which would account for the data presented here and by others; some of these have been discussed above. With the cultured tumor cells, the cytotoxin's action may involve a complicated series of events including: (i) interaction with cell membrane components, altering membrane structure and generating local areas of increased permeability; (2) direct or indirect interference with vital cell membrane transport processes ; (3) penetration of the toxin into the cytosol either by its own action or by pinocytosis; (4) interference with metabolic processes within the cell. As yet, insufficient experimentation has been conducted with these materials in one test system to allow a definite mechanism of action to be proposed.
334 ACKNOWLEDGMENTS This w o r k was s u p p o r te d by N a t i o n a l Institutes o f E n v i r o n m e n t a l H e a l t h Sciences G r a n t N o . ES 00267. T h e authors wish to t h a n k Miss Lila Cl ack for the quantitative a m i n o acid analyses. 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
Condrea, E., De Vries, A. and Mager, ~I. (1964) Biochim. Biophys. Acta 84, 60-73 Condrea, E., Mammon, Z., Aloof, S. and De Vries, A. (1964) Biochim. Biophys. Acta 84, 365-375 Wolff, J., Salabe, H., Ambrose, M. and Larsen, P. R. (1968) J. Biol. Chem. 243, 1290-1296 Larsen, P. R. and Wolff, J. (1968) J. Biol. Chem. 243, 1283-1289 Larsen, P. R. and Wolff, J. (1967) Science 155, 335-336 Braganca, B. M., Badrinath, P. G. and Ambrose, E. J. (1965) Nature 207, 534-535 Patel, T. N., Braganca, B. M. and Bellare, R. A. (1969) Exp. Cell Res. 57, 289-297 Braganca, B. M. and Khandeparker, V. G. (1966) Life Sci. 5, 1911-1920 Braganca, B. M., Patel, N. T. and Badrinath, P. G. (1967) Biochim. Biophys. Acta 136, 508-520 Larsen, P. R. and Wolff, J. (1968) Biochem. Pharmacol. 17, 503-510 Takechi, M., Sasaki, T. and Hayashi, K. (1971) Naturwissenschaften 58, 323-324 Hayashi, K., Takechi, M. and Sasaki, T. (1971) Biochem. Biophys. Res. Commun. 45, 1357-1362 Sharon, N. and Lis, H. (1972) Science 177, 949-959 Takechi, M., Hayashi, K. and Sasaki, T. (1972) Mol. Pharmacol. 8, 446~51 Takechi, M. and Hayashi, K. (1972) Biochem. Biophys. Res. Commun. 49, 584-590 Lee, C. Y., Lin, J. S. and Wei, J. W. (1970) in Toxins of Animal and Plant Origin (De Vries, A. and Kochva, E., eds), pp. 307-317, Gordon and Breach, New York Narita, K. and Lee, C. Y. (1970) Biochem. Biophys. Res. Commun. 41, 339-343 Chatman, V. B. and DiMari, S. J. (1974) Toxicon 12, 405-414 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 Lui, T.-Y. and Chang, Y. H. (1971) J. Biol. Chem. 246, 2842-2848 Todaro, G. J., Habel, K. and Green, H. (1965) Virology 27, 179-185 Smith, J. D., Freeman, G., Vogt, M. and Dulbecco, R. (1960) Virology 12, 185-196 Kornberg, A. (1953) in Methods in Enzymology (Colowick, S. P. and Kaplan, N. O., eds), Vol. 1, pp. 441~143, Academic Press, New York Papaconstantinou, J. and Colowick, S. P. (1961) J. Biol. Chem. 236, 278-284 Yang, C. C. (1965) J. Biol. Chem. 240, 1616-1618 Frye, L. D. and Edidin, M. (1970) J. Cell Sci. 7, 319-335 Tu, A. T. (1973) Annu. Rev. Biochem. 42, 235-258
SAMUEL J. DIMARI, KENNETH J. LEMBACH and VIRGINIA B. CHATMAN Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, Tenn. 37232 (U.S.A.) (Received October 14th, 1974)
SUMMARY Eight basic proteins which lyse virus-transformed mouse fibroblasts in culture have been isolated from the venoms of six Asian Naja naja subspecies. These cytotoxins appear to represent an homologous series of proteins, all within the molecular weight range of 7000-8000. They have been divided into three arbitrary types on the basis of amino acid composition, electrophoretic mobitities and elution order upon ion-exchange chromatography. The rate at which the toxins effect cell lysis: (1) appears to be a function of the basicity of each toxin; (2) is dependent upon toxin concentration; (3) is temperature dependent; and (4) is inhibited by heparin sulfate. In view of the physical changes, which the cell undergoes during lysis and of the various factors which affect the action of these proteins, it is proposed that interaction of membrane receptors with the toxin, leading to alteration of cell membrane structure, is the principal event which ultimately leads to the disruption of the cell.
INTRODUCTION Various workers have isolated basic protein fractions from the venom of the Indian cobra Naja naja naja and have named them according to the physiological action which they elicit in different test systems. These fractions have been called direct lytic factor (acts synergistically with phospholipase A in the lysis of erythrocytes [i, 2]), cobramines A and B (affect the permeability of thyroid slices to ions and Abbreviations: NNI-A and NNI-B (Type A and B cytotoxins from Naja naja naja (India) (NNI)); NNP-A and NNP-B (Type A and B cytotoxins from Naja naja naja (Pakistan) (NNP)); NNC-A and NNC-B (Type A and B cytotoxins from Naja naja ceylonicus (NNC)); NNS-B (major cytoxin from Naja naja siamensis (NNS)) ; NNO-C (major cytotoxin from Naja naja oxiana (NNO)); NNSe-C (major cytotoxin from Naja naja samarens& (NNSe)). Type A, B, and C have been used for the cytotoxins rather than Type I, II, and III in order to differentiate them from Type I and II venom neurotoxins. The different types of cytotoxin contain approximately the same number of amino acid residues and disulfides whereas the two types of neurotoxin differ from one another both in the number of amino acid residues and in the number of disulfides in each [27].
321 metabolites [3-5]), cytotoxin P6 (shows selective lysis of tumor cells over other cell types [6] and was reported to be a phospholipase C [7-9]), cardiotoxins (depolarize smooth muscle cells by inducing loss of K + [10]), and cytotoxins I and II (selectively lyse Yoshida sarcoma and ascites hepatoma cells [11, 12]). All of these various activities are assumed [11] to be different manifestations of the same two proteins present in the venom, with cytotoxin P6 a mixture of these plus perhaps a contaminating phospholipase. These proteins apparently induce alterations in membrane permeability or in membrane structural organization. They not only affect different membrane systems to different extents, but their action also appears to be sensitive to slight changes in membrane component content or to surface group configuration within different strains of the same cell type [2, 7]. In this respect, they resemble the plant lectins (e.g. concanavalin A), molecular probes used to detect membrane surface changes in erythrocytes and tumor cells [13]. These toxins would, therefore, appear to be not only potential antitumor agents, but are also potentially useful molecular probes for elucidating membrane ultrastructure and its role in biological functions of the cell. Three cytotoxins, the term used here for these proteins, have been isolated and sequenced: cytotoxins I and II (referred to in this paper as NNI-A and NNI-B, respectively) from the venom of the Indian cobra [12, 14, 15] and the cytotoxic [16] cardiotoxin from the venom of the Formosan cobra Naja naja atra [17]. The present study was undertaken to determine whether such membrane-active proteins are common components of Asian cobra venoms and if so, whether they vary in their abilities to lyse virus-transformed mouse fibroblasts in culture. METHODS
Isolation of Cytotoxins Cytotoxins were isolated from lyophilized whole venoms by repeated chromatography on the cation exchange resin sulphopropyl-Sephadex (C-25) followed by gel filtration on Sephadex G-50 (fine) as previously described [18]. Samples of cytotoxins used in experiments were either individual or combined fractions from the final sulphopropyl-Sephadex chromatography or from gel filtration. Homogeneity of isolated toxins was determined by both disc gel electrophoresis and amino acid analysis. Protein concentrations were determined by the method of Lowry et al. [19] with bovine serum albumin as standard and by amino acid analyses.
Characterization of Cytotoxins Molecular Weight Determination. Molecular weights of the cytotoxins were estimated by gel filtration on a calibrated 2.5 x 70 cm column of Sephadex G-50 (fine) eluting with 0.05 M sodium phosphate buffer, pH 6.0. Flow rate varied from 19 to 22 ml/h. Disc gel Electrophoresis. The apparatus, gel percentages, conditions for electrophoresis, gel staining and destaining procedures were those previously described [18]. Amino Acid Analysis. Aliquots of combined fractions obtained from gel filtration and to which norleucine had been added as a standard were lyophilized
322 to dryness in combustion tubes and were subjected to 24 h methanesulfonic acid hydrolysis [20] in vacuo at 115 °C. Amino acid contents were determined using a Beckman amino acid analyzer, Model 120C updated to a Model 121 and equipped with an automatic sample applicator. Assay for Cytotoxicity: Growth of SVIO1 Cells. A simian virus 40 (SV40)transformed derivative of mouse 3T3 cells, designated SV101 [21], was used to assay cytotoxicity. This cell line was kindly provided by Dr Howard Green, Department of Biology, Massachusetts Institute of Technology. Cells were maintained in Dulbecco's medium [22] supplemented with 5 ~ calf serum. Experimental cultures (25 cm 2) were initiated from trypsinized cell suspensions and were incubated at 37 °C for 48 h before use. Approx. 2 • 106-4 106 cells in 5 ml of medium were utilized for each experiment unless otherwise stated. •
Lactate Dehydrogenase Assays General Procedure. The release of the cytoplasmic marker enzyme lactate dehydrogenase was used to monitor lysis of SV101 cells. Units of enzyme released were determined spectrophotometrically by measuring the rate of N A D H oxidation (pyruvate substrate) using the reagents and experimental procedures described by Kornberg [23]. Reaction was initiated by the addition of the enzyme and was followed for 3 min using a Model 240 Gilford spectrophotometer (0.1 absorbance unit full scale) fitted with automatic sample changer and a Honeywell Model 6040 recorder. Standard curves correlating the rate of N A D H oxidation with units of lactate dehydrogenase were prepared using serial dilutions of commercial rabbit muscle enzyme. The rate of reaction was linear within the range of enzyme concentration present in the cultured cells. One unit of lactate dehydrogenase activity is defined as that quantity of enzyme necessary for the oxidation of 1 nmol of N A D H per rain at non-limiting substrate and N A D H concentrations. Determination of Lactate Dehydrogenase Content of SVlO1 Cells. SV101 cells were subcultured at different densities using the above procedure. At the end of the growth period, medium was removed, the cells adhering to the culture flasks were washed twice with 37 °C phosphate-buffered saline, pH 7.2, and were removed from the flasks by treatment with 5 ml of 0.25 ~ trypsin in phosphate-buffered saline for 5 min at 37 °C. Cells were collected by centrifugation at 1500 rev./min using a Sorvall Model RC-3 refrigerated centrifuge. The packed cells were washed twice by centrifugation with 5 ml of phosphate-buffered saline and were then suspended in 1-3 ml of phosphate-buffered saline to give approximately the same number of cells per ml in the different systems. The total number of cells in each suspension was determined using a Coulter Counter. Each cell suspension was sonicated for 30 s at 4 °C with a Sonifier Cell Disrupter, Model WI85D (Heat Systems-Ultrasonics, Inc., Plainview, L.I., N.Y.) fitted with a microprobe. The supernatant solutions obtained after centrifugation (5000 rev./min for 30 min) were assayed for lactate dehydrogenase activity by the above method. Protein content of each supernatant was determined by the method of Lowry et al. [19].
Determination of Lactate Dehydrogenase Released .from SVIO1 Cells by Cytotoxins. A 0.2-ml aliquot of the serum-containing medium in which the cells had been cultured was withdrawn from each system to serve as a "zero time" sample. Flasks were inverted to remove medium from direct contact with cells, and the
323 cytotoxin was added to the medium. The cytotoxin-cell systems were equilibrated at the indicated temperatures for 5 to 10 min and were then inverted to bring the cytotoxin-containing medium into contact with the cells ("zero time point"). All of the experiments presented in each figure were conducted on the same day with cells grown and treated in the same manner. At various time intervals, 0.2-ml aliquots of supernatant media were withdrawn from each system, and samples (50-1130/A) were assayed for lactate dehydrogenase. The total units of enzyme shown in the figures have been corrected for volume changes during the experiment, for units of enzyme removed with each aliquot and for the units of enzyme present in the system at time "zero". MATERIALS Lyophilized Naja naja naja (India) (Lot No. 52C-1250) venom was obtained from Sigma Chemical Co. (St. Louis, Mo.). The following lyophilized venoms were obtained from the Miami Serpentarium Laboratories (Miami, Fla.): Naja naja eeylonicus (Ceylon) (Lot No. NY 3.5 TL), Naja naja naja (Pakistan) (Lot No. NNPOL), Naja naja oxiana (Iran) (Lot No. NOOETL), Naja naja samarensis (Philippines) (Lot No. NEOETL), Naja naja siamensis (Thailand) (Lot No. NS1ETL). Sulphopropyl-Sephadex (C-25), Sephadex G-50 (fine) and the gel filtration protein standards aldolase, chymotrypsinogen A and ribonuclease A were obtained from Pharmacia Fine Chemicals (Piscataway, N.J.). Grade III yeast fl-nicotinamide adenine dinucleotide, reduced form (fl-NADH), disodium salt (Lot No. 103C-6380) and Type I rabbit muscle L-lactate-NAD oxidoreductase, EC 1.1.1.27 (crystalline suspension in 2.1 M (NH4)2SO4, spec. act., 80 units/mg protein) were obtained from Sigma Chemical Co. Grade A sodium pyruvate (Lot No. 100984) was obtained from Calbiochem (Richmond, Calif.). Heparin sulfate was obtained from ICN Nutritional Biochemicals Corporation (Cleveland, Ohio). Crystalline bovine serum albumin was purchased from Pentex, a division of Miles Research Laboratories (Kankakee, Ill.). RESULTS
Isolation and Characterization of Cytotoxins Sulphopropyl-Sephadex Chromatography. The elution profiles of venoms fractionated and the positions of the cytotoxic proteins isolated from each are shown in Fig. 1. Proteins eluting immediately before, between, or after these materials were tested and did not lyse SV101 cells. The proteins designated NNP-B (Fig. 1A) and NNSe-A (Fig. 1F) were cytotoxic but were not further purified nor characterized since they could not be obtained in sufficient quantity. Usually two additional ionexchange chromatographic steps, using the gradient system described for cytotoxins [18], were necessary to obtain homogeneous proteins. Moleeular Weights of the Cytotoxins. The molecular weights of NNI-A and NNI-B are between 7000 and 8000 [12, 14]. The elution volumes of other cytotoxins (Fig. 2) are similar to those observed for these two toxins, indicating that all of the cytotoxins isolated are within the same molecular weight range. No higher molecular weight species were observed upon gel filtration. For calculations, a molecular weight of 7000 was assumed for each toxin.
324 I0 2050405060708090100 ~02050405060708090100 (~ Nolonolonaja(Pakistan) (~ Nolonolo siomensis NNP-A ~ NNS-B
Nolonolo (~)
nolo (india)
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Nolo nolo oxiono
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NolohaloceylonicuSNNC. A Nolonolo I @ NNC-B ~ samorensis
I0 2030405060708090100 102030405060708090100 FRACTION
NUMBER
Fig. 1. Sulphopropyl-Sephadex fractionation of Naja naja venoms. Lyophilized venoms (200 mg), dissolved in 5 ml of 0.05 M phosphate buffer, pH 6.0, were fractionated on 2.5 x 20 cm columns of sulphopropyl-Sephadex using a linear salt gradient [18]. Arrows mark the start of the gradient. Cytotoxic proteins are indicated in the figure by cross-hatching and are labeled with the abbreviations used for them in the text and in all figures and tables. The yields (percent protein recovered from whole venom) of each cytotoxin were: NNP-A, 9 ~ ; NNI-A, 13.5 %; NNI-B, 13 ~ ; NNC-A, 8 ~ ; NNC-B, 1 1 ~ ; NNS-B, 2 1 ~ ; NNO-C, 13%; NNSe-C, 10%.
I
I
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Fig. 2. Gel filtration of the cytotoxins. Cytotoxins (10--20 mg) were subjected to gel filtration on Sephadex G-50 (fine) (see Methods). 4-ml fractions were collected. Standard proteins used for column calibration and indicated in the figure were: aldolase (ALD), 158 000 real. wt; chymotrypsinogen A (CHYMO), 25000mol. wt; and ribonuclease A (RNase), 13700moi. wt. Symbols: L~--O, NNI-A; 0 - - - 0 , NNI-B; C]--C3, NNP-A; II--m, NNC-A; A - - A , NNC-B; ( ) - - ( ) , NNS-B; ~ - - / ~ , NNO-C; x - - ×, NNSe-C. The actual absorption of some of the toxins has been altered to simplify the figure.
325
Disc Gel Electrophoresis of Cytotoxins. The disc gel patterns obtained for whole venoms and the bands which correspond to the cytotoxins are shown in Fig. 3. The toxins, as seen, are the fastest cathodally-migrating major species of each venom. As also seen in the figure, the cytotoxins may be arranged in the following order of decreasing electrophoretic mobilities: C toxins > B toxins > A toxins. This difference in basicity was inferred by the elution order of different toxins upon ionexchange chromatography. + 6789
123
I0 II
12113 14
15
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Fig. 3. Disc gel electrophoresis of cytotoxins and their corresponding venoms. Disc gel electrophoresis of cytotoxins (20/*g) and venoms (100 yg) was conducted on 3-mm thick polyacrylamide gel slabs [18]. The reproduction shown is a composite of two gel slabs; common venoms and cytotoxins were present on each as references. Only major, well-stained protein venom components have been indicated. 1, NNP-A; 2, Noja naja naja (Pakistan), whole venom; 3, NNP-B; 4, NNI-A; 5, Naja naja naja (India), whole venom; 6, NNI-B; 7, NNC-A; 8, Naja naja ceylonictts, whole venom; 9, NNC-B; 10, NNS-B; 11, Najanajasiamensis, whole venom ; 12, NNO-C; 13, Najanalaoxiana, whole venom; 14, NNSe-C; 15, Naja naja sarnarensis, whole venom.
Amino Acid Analyses. The amino acid content of each of the cytotoxins isolated is shown in Table I. The amino acid contents presented in the table for N N I - A and N N I - B agree well with those previously reported [12, 14, 15]. None of the cytotoxins contain tryptophan, an amino acid commonly present in the venom neurotoxins. The A toxins contain one residue of glutamic acid and lack histidine and phenylalanine. The B toxins contain neither glutamic acid nor histidine but contain phenylalanine. The C toxins differ f r o m the other two types in that they contain a residue of histidine and fewer acidic amino acids. A comparison of the ratios of basic to acidic residues of each type explains the differences in basicities noted earlier. Release of Lactate Dehydrogenase from SVIO1 Cells by Cytotoxins Lactate dehydrogenase activity in SV101 cells was found to vary linearly with the number of cells in a given culture (Fig. 4). To verify that the oxidation of N A D H
326 TABLE I AMINO ACID COMPOSITION OF COBRA VENOM CYTOTOXINS Duplicate samples (approx. 50 nmol) of each cytotoxin were hydrolyzed and analyzed as described in Methods. Nanomoles corresponding to one residue of each of five amino acids (*) were calculated and an average value obtained. This average was used to determine the number of residues of each amino acid. The values presented in the table are averages of the two numbers obtained in this manner from duplicate analyses. The closest integral value(s) for each amino acid is given in parentheses. Amino acid
Type A
Type B
NNI-A
NNP-A
NNC-A
NNI-B
NNC-B
NNS-B
NNO-C
NNSe-C
Lys Arg* His Trp Asp* Glu* Thr Ser Pro Gly* Ala* '/z Cys Val Met Ile Leu Yyr Phe*
9.2 (9) 2.0 (2) 0 0 8.5 (8-9) 1.0 (1) 2.9 (3) 1.9 (2) 4.5 (4-5) 2.1 (2) 2.2 (2) 7.0 (8) 4.6 (5) 2.0 (2) 1.8 (2) 6.3 (6) 4.0 (4) 0
9.1 (9) 2.0 (2) 0 0 8.5 (8-9) 1.0 (1) 3.0 (3) 2.0 (2) 4.3 (4) 2.1 (2) 2.1 (2) 7.2 (8) 4.5 (4-5) 1.9 (2) 1.8 (2) 6.3 (6) 4.l (4) 0
9.0 (9) 2.1 (2) 0 0 8.5 (8-9) 1.0 (1) 3.0 (3) 2.0 (2) 4.3 (4) 2.1 (2) 2.1 (2) 6.7 (8) 4.1 (4) 2.1 (2) 1.7 (2) 6.3 (6) 4.2 (4) 0
8.9 (9) 2.3 (2) 0 0 6.7 (7) 0 3.1 (3) 2.0 (2) 5.3 (5) 2.1 (2) 2.1 (2) 6.3 (8) 5.4 (5) 2.0 (2) 0.8 (1) 6.3 (6) 3.9 (4) 1.0 (1)
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10.4 (10) 1.0 (1) 1.0 (1) 0 5.2 (5) 0 1.9 (2) 2.7 (3) 5.3 (5) 2.1 (2) 3.1 (3) 7.0 (8) 6.4 (6) 2.0 (2) 1.3 (1) 6.1 (6) 1.9 (2) 1.9 (2)
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cells, cultured to different densities, were collected and counted; the supernatant fluids, obtained by sonication and centrifugation, were assayed for lactate dehydrogenase (see Methods). The total units (UT) of lactate dehydrogenase shown for each cell density represents the average of five determinations. The standard deviation from this average value is indicated.
327 m e a s u r e d was a result o f the a c t i o n o f this enzyme, o x a m i c acid, a competitive i n h i b i t o r o f the enzyme [24], was a d d e d to some o f the assay mixtures. W h e n present in e q u i m o l a r a m o u n t with substrate, o x a m i c acid decreased the rate o f N A D H o x i d a t i o n by a p p r o x . 33 ~ . T h e n u m b e r o f units o f lactate d e h y d r o g e n a s e p e r cell was calculated to be 2 . 5 . 1 0 - 4 - 3 • 10-4; 2.106 cells w o u l d c o n t a i n 500-600 t o t a l units o f enzyme. The average p r o t e i n c o n c e n t r a t i o n for the systems shown in the figure was 0.5 m g o f p r o t e i n p e r 2.106 cells. Therefore, the cultured cells contained 1000-1200 units o f lactate d e h y d r o g e n a s e p e r m g o f protein.
Ability of a Cytotoxin to Release the Theoretical Total Units of Lactate Dehydrogenasefrom SVIO1 Cells. A s seen in Fig. 5A, the t o t a l units o f enzyme released by the c y t o t o x i n f r o m b o t h high a n d low density cultures were a p p r o x i m a t e l y w h a t w o u l d be expected for the n u m b e r o f cells present in each system. The variability in the d e t e r m i n a t i o n o f enzyme units at any p o i n t was a p p r o x . ~-5 ~ at high levels o f enzyme ( > 100 units) and -L l0 ~o at low levels ( < 100 units).
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Fig. 5. (A) Release of lactate dehydrogenase (LDH) from SVI01 cells by cytotoxin NNI-B. Six cultures of SV101 cells were seeded, three to provide a final cell density of 1.5.106 cells and three of 3.106 cells. At the end of 48 h, one culture of each density was trypsinized, and the cells were recovered and counted (see Methods). The low density culture contained 1.4" l& cells and the high density culture contained 3.3" 106 cells. Cytotoxin NNI-B was added to the remaining two cultures of each density to a final concentration of 20 fig of toxin per ml of medium, and the systems were incubated at 37 °C. Supernatant media were assayed for lactate dehydrogenase (see Methods). Total enzyme units (Ur) in the low density cultures are indicated by ( A - - / ~ ) and those in the high density cultures by ( O - - O ) . (B) Percent of total lactate dehydrogenase (LDH) release by cytotoxin NNI-B versus time. The experimental systems and symbols for low and high density cultures are those described in A. The total enzyme units used for each time point is an average of the values obtained from the two cultures of each cell density; these units are expressed as percentages of the average total units released from each cell density at 220 min.
328 Fig. 5B shows the percent of total enzyme released by the cytotoxin at different times. Essentially linear rates (total units/t) of enzyme release were observed up to 50-70% release of the total enzyme present in the cells; after this point had been reached, rates decreased. The different time intervals necessary for the release of 50 of the total enzyme were proportional to the cell densities used.
The Release of Lactate Dehydrogenasefrom S VIOl Cells by Different Cytotoxins. All of the cytotoxins isolated were tested for their ability to lyse SVI01 cells (Fig. 6). The percent of enzyme released per time was calculated from the data shown. The times required for each cytotoxin to release 50 ~ of the total enzyme present were: NNSe-C, 45 min; NNO-C, 70 rain; NNS-B, 95 min; NNC-A, 110 min; NNC-B, 112 min; NNI-A and NNI-B, 117 rain; and NNP-A, 128 min. As seen, the Type C toxins released enzyme at faster rates than either the Type A or B toxins (for all practical purposes, Type A and Type B toxins were equally effective in releasing enzyme from the cells). i
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20 40 60 80 100 20 40 60 80 200 TIME. rain Fig. 6. Release of lactate dehydrogenase from SV 101 cells by Naja naja cytotoxins. Cytotoxins (10 #g of toxin per m] of medium) were added to cu]tures of SV]0] cells, and the systems were incubated at 37 °C. Aliquots of supernatant media were removed and assayed for lactate dehydrogcnase activity (see Methods). Symbols: © - - © , NNT-A; H , NNI-B; Vl--[D, NNP-A; i - - i , NNC-A; A - - A , NNC-B; (])--(]), NNS-B; A - - ~ , NNO-C; × - - × , NNSe-C.
Microscopic examination showed that cells appeared to be affected differently by the Type C (strong) toxins than by the Types A and B (moderate) toxins. With both strong and moderate cytotoxins, toxin-cell interaction was indicated by refractility of the cell membrane (localized phase dense areas appeared upon the surface of the otherwise transparent cells). With the moderate cytotoxins, the cells became increasingly refractile, contracted from the fibroblastic form, and detached individually from the culture vessel. Cultures lysed by the A- and B-type toxins contained much cellular debris and floating, discrete membrane fragments. With the strong, or C-
329 type toxin, however, initial refractility was followed by a rapid contraction of the cell, leaving anchoring processes still in place. As lysis progressed, cells lifted from the dish in sheets rather than individually as was the case with the moderate toxins. Type C-lysed cultures were characterized by floating sheets of cell membranes; nuclei were often observed in the flask. The observable differences in enzyme release and lysis by the A/ B and C cytotoxins may be an indication that the two classes are acting by different mechanisms or may simply be a result of the fact that the strong cytotoxins affect the cells so rapidly that they are unable to completely detach from one another and are removed in sheets rather than individually.
Effects of Various Factors on Cytotoxin-Induced Enzyme Release from SVIO1 Cells Preliminary experiments involving a moderate (NNI-B) and a strong (NNOC) cytotoxin were undertaken to determine the ability of these two types to release lactate dehydrogenase from SV101 cells under various conditions. The tissue culture system used contains factors (e.g. serum protein) which potentially could affect the action of the toxins and, therefore, only the most general inferences can be drawn from the following experiments. Effect of Cytotoxin Concentration. The rate of enzyme release by both cytotoxins NNI-B and NNO-C and the time required to release 50 ~ of the total enzyme present in the culture (Fig. 7) were proportional to the concentration of toxin. With both the strong and moderate cytotoxins there was a definite concentration below which enzyme release did not occur within the time interval of the experiment. As seen in Fig. 7, this concentration was lower for NNO-C (2/~g/ml) than for NNI-B (5 /~g/ml). The inability of NNI-B at a concentration of 2 #g of toxin per ml to release enzyme might be ascribed to non-specific binding of the toxin to serum protein, affinity of the toxin for the plastic culture flask, etc. ; however, NNO-C which should be influenced by the same factors was relatively active at this concentration, indicating, among other things, a possible difference in the mechanism of action of the two toxin types. Cytotoxin-treated cultures in which no enzyme release was observed were visually examined after 24 h. With both toxins, the cells appeared to be intact with relatively normal morphology except for isolated phase dense areas on membrane surfaces. Effect of Temperature. The action of both strong and moderate cytotoxins is very temperature dependent. During the first 60 min of exposure, there was virtually no enzyme release by either cytotoxin at temperatures below 30 °C (Fig. 8). At the end of this period, however, lactate dehydrogenase was released in all systems except that which contained NNI-B at 4 °C. The duration of the induced lag period appeared to be independent of temperature or cytotoxin used (Fig. 9). The greatest difference in lytic ability between NNO-C and NNI-B was observed at 4 °C. During the experimental time period, NNO-C induced a slow release of enzyme whereas NNI-B did not. Examination of both cultures at the end of 24 h at 4 °C showed that the strong cytotoxin had destroyed all of the cells originally present whereas the cells in the NNI-B system were still attached to the culture dish and, although highly refractile, appeared to be intact. Effect of Heparin Sulfate. Heparin sulfate has been reported to precipitate the basic proteins of cobra venom [1 ], and it was of interest to test the effect of this agent upon the rate of enzyme release from cytotoxin-treated cells.
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Fig. 7. (A) Percent of total lactate dehydrogenase release by different concentrations of cytotoxin NNI-B versus time. Cytotoxin NNI-B was added to cultures of SV101 cells to give the following final concentrations of cytotoxin per ml of medium: /~--/~, 1/~g/ml; I1--11, 3#g/ml; 0 - - 0 , 5 ug/ml; C]--C], 10 #g/ml; and A - - A , 20 #g/ml. Systems were incubated at 37 °C and lactate dehydrogenase activity in supernatant media was monitored (see Methods). Reactions were allowed to proceed for 340 rain or until the level of enzyme became constant. The values at each time point represent percentages of the total units released upon complete lysis of each culture. For systems in which total lysis was not observed during the experimental period, the average of the total units found in completely lysed cultures was used as 100 ~ . (B) Percent of total lactate dehydrogenase release by different concentrations of cytotoxin NNO-C versus time. Cytotoxin NNO-C was added to cultures of SV 101 cells to give the following concentrations of cytotoxin per ml of medium; O--O, 0.1/~g/ml; I1--11, 1/~g/ml; ~x--~,, 2/~g/ml; ©--~), 10#g/ml; A - - A , 20/~g/ml; and [~--C3, 40/~g/ml. Experimental procedures and calculations were those described in A. I00
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Fig. 9. Effect of temperature upon lactate dehydrogenase release by cytotoxins NNI-B and NNO-C. Cytotoxins NNI-B (A) and NNO-C (B) were added to temperature-equilibrated cultures of SV101 cells to final concentrations of 20 and 40/zg of cytotoxin per ml, respectively. Experimental conditions and protocol were those described in Fig. 8 and in Methods. The following incubation temperatures were used in both A and B: ~ - - A , 4 °C; 0 - - 0 , 15 °C; D--[~, 23 °C; &--A, 30 °C; 0 - - 0 , 37 °C. Fig. 10. Effect of heparin sulfate upon lactate dehydrogenase release from SV101 cells by cytotoxins NNI-B and NNO-C. Cytotoxins NNI-B (A) and NNO-C (B) were each added to three SV101 cultures at 20 and 40 pg of toxin per ml of growth medium, respectively. The systems were incubated at 37 °C. After 10 min of exposure, 2 mg of heparin sulfate in 2001d of distilled water was added to one flask of each set (A--A), and, after 40 min, the same amount of beparin was added to another flask ( D - - D ) . No heparin was added to either the third flask of a set ( O - - O ) or to a non-toxincontaining control ( × - - × ). The release of lactate dehydrogenase was monitored (see Methods).
C y t o t o x i n s N N I - B a n d N N O - C were each a d d e d to three cultures, the systems were i n c u b a t e d at 37 °C a n d h e p a r i n sulfate was a d d e d to specific cultures at different times (Fig. 10). As seen, the a d d i t i o n o f h e p a r i n resulted in a decrease in the rate o f enzyme release by b o t h cytotoxins and, eventually, to a cessation o f lysis. Visual e x a m i n a t i o n o f cultures after the a d d i t i o n o f h e p a r i n showed that as the rate o f lactate d e h y d r o g e n a s e release decreased, the cells b e c a m e less refractile a n d began to reassume a n o r m a l m o r p h o l o g y . A t the end o f the e x p e r i m e n t a l time period, the h e p a r i n - t r e a t e d cells were indistinguishable f r o m n o n - c y t o t o x i n - e x p o s e d control cultures. In o r d e r to allow longer c o n t a c t between c y t o t o x i n a n d cell, the experiment shown in Fig. I I was conducted. As seen in the figure, when the t e m p e r a t u r e o f the n o n - h e p a r i n - t r e a t e d , t o x i n - c o n t a i n i n g system was increased f r o m 4 to 37 °C, lactate d e h y d r o g e n a s e was released at a rate c o m p a r a b l e to t h a t observed with the system o r i g i n a l l y placed at 37 °C. The h e p a r i n - t r e a t e d system, when placed at 37 °C, initially released enzyme at a slow rate a n d then release stopped. Physical changes in
332
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Fig. 11. Effect of heparin sulfate and temperature upon the release of lactate dehydrogenase (LDH) from SV101 cells by cytotoxin NNI-B. Cytotoxin NNI-B was added to three SV101 cultures (20 ttg of toxin per ml of medium). Two of these cultures, plus a non-toxin-containing control, were immediately placed at 4 °C; the remaining culture ( © - - © ) was incubated at 37 °C. At the end of 130 min (arrow), 2 mg of heparin sulfate in 200 #1 of distilled water was added to one of the toxin systems at 4 °C ([E--[B) and this, together with the non-heparin-treated, toxin-containing system ( A - - A ) and control ( × - - × ) , was placed at 37 °C. Lactate deh),drogenase activity in supernatant media was monitored (see Methods).
form and refractility of these latter cells were those observed when heparin was added during a period of active enzyme release. DISCUSSION
Although the rate at which each toxin induces enzyme release and the manner in which lysis is achieved appears to be related to the basicity of the toxin, the difference in activity observed between strong and moderate cytotoxins may also be accounted for by different primary or tertiary structures or by different receptors for each. It is assumed that the initial step in the lytic process is an electrostatic attachment of the toxin to negatively charged receptor groups on the cell membrane. However, were the neutralization of a portion of the membrane surface charge the principal event in lysis, any very basic protein could mimic the action of the cytotoxins. We (DiMari, S. J. and Chatman, V. B., unpublished) have exposed SV101 cells to cobrotoxin (pI 9.4), the principal neurotoxin from the venom of N. naja atra [25], and to venom proteins which are similar to the toxins in molecular size and behavior on ion-exchange chromatography and upon disc gel electrophoresis; no measureable release of enzyme was observed. As shown, the ability to lyse tumor cells is the property of specific proteins in the venoms studied and these proteins appear to form an homologous series based upon their amino acid contents. The tertiary structure of the cytotoxin appears to be essential for its action
333 since reduced toxins have been reported to be unable to lyse Yoshida cells [12, 15]. In view of its high disulfide content, the cytotoxin, like the analogous neurotoxin, must be a rigid molecule with its polar residues oriented in a specific manner on the molecular surface. The principal polar residue in these proteins is lysine, and it is assumed that lysines play an active role in the lytic process as opposed to a passive one (e.g. present only to solubilize the toxin in aqueous media). The regular spacing of lysine residues throughout the primary sequences of NNI-A and NNI-B [12, 14] would appear to be sufficient to allow a specific number of these residues to be within a prescribed area of the molecular surface, making multipoint electrostatic contact of the cytotoxin with charged membrane receptors possible. An equilibrium binding of toxin to cell membrane could explain the ability of heparin sulfate to stop the lytic process. Heparin may be removing unbound cytotoxin from solution and reversing the balance of equilibrium, causing partially bound cytotoxin to dissociate from the membrane. If heparin were removing only unbound toxin and reducing the level of toxin in the system to sub-lytic concentrations, one would expect the remaining cells in the culture to be refractile as was observed when such doses were applied. This, however, was not the case; after heparin treatment, infected cells returned to a completely normal appearance. The addition of heparin did not result in an immediate cessation of cell destruction. Slow enzyme release continued for an hour or longer until it stopped, indicating that perhaps some toxin molecules were more firmly bound than others or that they had penetrated into the membrane or cytosol. Experiments designed to determine whether cytotoxin P6 acted primarily on the cell membrane or in the cytosol were inconclusive [7]. The effect of temperature upon the action of the cytotoxins resembles that observed for the intermixing of cell surface antigens throughout fused membranes of mouse-human heterokaryons [26]. This phenomenon was ascribed to a variation in cell membrane fluidity with temperature and to the effect which the state of the membrane has upon the ability of molecules to diffuse through it. Therefore, one possible explanation for the temperature effect observed is that the toxin, in order to act, must bind to a specific number of non-localized membrane receptors and that the ability of receptors and of partially bound toxin molecules to diffuse to within binding distance of one another is decreased by a temperature-dependent alteration of membrane fluidity. Temperatures below 30 °C may be breaking down the lytic process into its component steps and exposing a cooperativity effect which is obscured by the rapidity of the overall process at 37 °C. There are several possible explanations which would account for the data presented here and by others; some of these have been discussed above. With the cultured tumor cells, the cytotoxin's action may involve a complicated series of events including: (i) interaction with cell membrane components, altering membrane structure and generating local areas of increased permeability; (2) direct or indirect interference with vital cell membrane transport processes ; (3) penetration of the toxin into the cytosol either by its own action or by pinocytosis; (4) interference with metabolic processes within the cell. As yet, insufficient experimentation has been conducted with these materials in one test system to allow a definite mechanism of action to be proposed.
334 ACKNOWLEDGMENTS This w o r k was s u p p o r te d by N a t i o n a l Institutes o f E n v i r o n m e n t a l H e a l t h Sciences G r a n t N o . ES 00267. T h e authors wish to t h a n k Miss Lila Cl ack for the quantitative a m i n o acid analyses. 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
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