Biological activities contaminating preparations of phospholipase C (α-toxin) from Clostridium perfringens

Toxicorr, 1973, Vol. 11, pp . 134-147. Pergamon Preu . Printed in Great Britain

BIOLOGICAL ACTIVITIES CONTAMINATING PREPARATIONS OF PHOSPHOLIPASE C (a-TOXIN) FROM CLOSTRIDIUM PERFRINGENS R. MBrs.BY, C:E. Nolzn and T. WAnsrxöM Department of Bacteriology and Department of Oral Microbiology, ICarolinska Institutet, 5-104 Ol, Stockholm 60, Sweden Departrnent of Bacteriology, National Bacteriological Laboratory, 5-105 21, Stockholm, Sweden (Accep~ed jor pubikation

15

AKquat 1972)

Abelract--Commential preparations of phospholipase C from Clostridium perfringena (wrlchil) were found to contain the following biological activities : a stxond haemolytic protein probably theta-haemolytin, protease, deoxyribonuclease, a-glut~sidase, endo-ß-N-aaetylgluoosaminidase, hyaluronate lyric acid phosphatase, sulphatase and ß-glucuronidase . Separation of phosphollpase C in density gradient isoelectric focusing in an extremely shallow pH gradient rostilted in two peaks sad removed most of the contaminating activities. Tile isoelectric points (pn of the phospholipase were 4"6 f 0"05, and S " 7 f 0"OS, haemolytin pI 6"8, deoxyri'bonuclease pI 4"6, ~do-~N,atxtylgh>cosaminidase pI S"S, a-ghlcosidase pI 4"7, hyaluronate lyric pI 4"7, acid phosphataae pI 4~7, protease pI 4" S and sulphatase, pI 4'S . The problem of interpreting prtwious biological studies on mammalian cell meanbranes by these rather crude preparations is discussed . INTRODUCTION Clostridium perjringens (previous welchü) produces a haemolytic toxin, a-toxin, which hydrolyzes lxithin to phosphorylcholine and diglyceride (MACFARr..ANI: and Kx1cHr,1941). This phospholipase C (phosphatidylcholine : choline phosphohydrolase, E.C. 3.1 .4.3) was later found to split also other phospholipids (MA"rsuato~ro, 1961 ; SArro and MUKOYAMA, 1968; SUGAHARA and OHSAIt.A, 1970) . However, PA3TAN et al. (1968) described a phospholipase specific for degradation of sphingomyelin which was stimulated by magnesium, while the enzyme specific for lecithin was activated by calcium (MACFARLANe, 1948). This phospholipase C is haemolytic, dermonecrotic, and lethal (SUGAIiARA and OIi3AICA, 1970) in contrast to that produced by Bacillus cereus (13oxvl~lTRB and JOHNSON, 1970) . During the last years, commercial preparations of Cl. perfringens phospholipase C have been used for a great variety of biological studies on cytotoxicity and membrane effects of sublytic doses (BLEt~t, 1965 ; LENAItn and SINtî13R, 1968; McI1.r wAnv and RAPPORT, 1971 ; PATRU\RCA et al., 1971 ; Ras>;rrrxAt and FAt]v, 1971 ; CUATRECASAS 1971a, b). The insuline-like e8'ect on glucose transport in fat cells was recently questioned, since an impurity in commercial preparations was found to stimulate glucose oxidation in brown fat cells (ROSENTHAL and FAIN, 1971). Reduction of infectivity of influenza virus, prevention of cholesterol esterification in human serum, and inhibition of impulse conduction in peripheral nerves and other biological effects have also been attributed to this enzyme (MIZUTANI and MIZUTANI, 1964; RowEr1, 1964; ROSSr>er~tc, 1970) . It is hard to interpret these observations because other biologically active substances are present in these commercial preparations. TOXICON 1973 Vä" Jl .

139

140

R. MÖLLBY, C. E. NORD and T. WADSTRÖM

Preliminary studies, started in this laboratory in order to compare substrate specificity and toxic properties of a staphylococcal phospholipase C with a specific action on sphingomyelin (ß-haemolysin ; WADS7RÖM and MÔLLBY, 1971 a, b) with commercial preparations of Cl. perfringens phospholipase C, revealed that the latter was contaminated with several enzymes. Work is now in progress to purify the perfringens enzyme, since no other highly purified phospholipase is available for these studies. Phospholipid splitting enzymes are also very valuable for structural studies of biological membranes (LENARD and SITIGER, 1968 ; COLEMAN et al., 1970 ; ZWAAL et al., 1971 ; RO1~.OFSEN et al., 1971). The data presented in this investigation show that all such data hitherto obtained must be interpreted with caution, since contaminating enzymes, such as neuraminidase, and other glycosidases and proteases may interfere with the breakdown of biological membranes. MATERIALS AND METHODS Materials. Partially purified phospholipase C from Clostridium perfringens was obtained from Worthington Biochem. Corp . (Freehold, N.J ., USA, code PHLC 9GB), Sigma Chem . Comp. (St. Louis, Mo., USA, lot nr . 61 C-6900), and Nutritional Biochemicals NBC, Cleveland, Ohio, U.S.A ., B grade, lot nr 901450). The preparations contained about 1-2 units per mg. Onc unit is defined as the amount of enzyme which liberates one pmole of water-soluble organic phosphorus from lecithin per minute, pH 7~2 at 37°C. Dithioerythritol and dithiothreitol, hyaluronic acid grade III-P, cholesterol, collagen, p-nitrophenylphosphate, elastinCongo red, L-leucyl-ß-Naphtyl-amide HCl (grade III-P), ribonucleic acid type XI and L-a-lecithin from soy bean (grade II-S) were also purchased from Sigma. The glucosidase substrates were obtained from Koch-Light, Colnbrook, Bucks, England. Columns (110 ml) for isoelectric focusing and carrier ampholytes (Ampholine) were purchased from LKB-Produkter, Stockholm-Bromma, Sweden and acrylamide from British Drug House, Poole, England. Anti-perfringens serum A (250 U/ml), B (100 U/ml), C (1000 U/ml), and D (280 U/ml) were purchased from l'Institut Pasteur, Paris, France. Highly purified deoxyribonucleic acid from calf thymus and ribonucleic acid from yeast were obtained from Worthington, Biochem. Corp . Casein, calcium chloride, zinc chloride, ethylene-diaminetetra-acetic acid (EDTA), and glycerol were purchased from Merck, Darmstadt, Germany and agarose from flndustrie Biologique Francaise, S.A ., Gennevilliers, Seine, France. All chemicals were of analytical grade. Eruyme and toxin assays . Phospholipase C activity of Cl. prefringens was determined by three different assays : (1) increase in the turbidity at 540 nm of an egg yolk suspension (07`rOLEN(3HI, 1969 ; KUSxNBt, 1957) ; (2) The egg yolk was replaced by L-a.-lecithin from soy bean and emulsified with 10 mM CaC12 and 0~ 1 M Tris HCl buffer, pH 7~2, by ultrasonic treatment (MSE Sonifier, amplitude 4-8 pm ; 1 min) . This suspension was stable for at least one hour at 37°C . The increase in turbidity was followed in a Bxkman DB-G spectrophotometer . The initial absorbance of the 2 ml was 0~2 and the amount of enzyme added in a suitable dilution was 0~1 ml . The activity was expressed as the increase in absorbance at 540 nm per 30 min (A Aaao 0/30 min) at 37°C . The rate of the increase in turbidity was linearly proportional to the enzyme concentration in the range 10-50 4~g. A specimen containing 1~5 pg gave an increase in absorbance at 540 nm of 001/min at 37°C (Aicm6ao~ . (3) Liberation of acid-soluble phosphorus from soy bean lecithin (O1'rOLENGHI, 1969). Haemolytic activity was assayed on sheep, rabbit, human and horse red blood cells 7~OXICON l973 Yd. ll.

. Clostridial phospholipase C

l4l

suspended in Tris HCl buf%red saline containing bovine serum albumin (0~1 per cent w/v) (WADSTRÖM ând MÖLLBY, 1971a) . Lipase activity was determined on tributyrine emulsified in sodium and calcium chloride as recently described (ARVtt~sox et al., 1971). Acid and alkaline phosphatase was determined usingp-nitrophenylphosphate (TORRIANI, 1968 ; Axvmsox et al., 1971). Caseinolytic and elastinolytic activities were determined on casein and elastin Congo red, as previously described (VssreRBSxc et al., 1967 ; AxvmsoN et al., 1971 ; Graze and Brxx, 1970). The enzyme assays for collagenase and hyaluronate lyase activity were both viswsimetric methods (SÖnIIt and Noxn, 1969). Deoxyribonuclease and ribonuclease activities were determined from the release of acid soluble nucleotides according to Ar.>rxAxnEx et al. (1961) and KALNITSKY et al. (1959) . Deoxyribonudease was also determined by a more sensitive viscosimetric assay (LUNDBLAD and Jox.~xssox, 1968). Endo-ß-N-acetylglucosaminidase activity was determined on cell wall murein prepared from Escherichia colt K-12 according to MAR~nN and KEMrnt (1970) and on whole cells of Micrococcus lysodeikticus (WADSTRÖM and HI3ATSUNE, 1970). a-D-glucoside glucohydrolase (E.C. 3.2 .1 .20), ß-glucoside glucohydrolase (E.C. 3.2.1 .22), a-n-galactohydrolase (E.C. 3.2.1 .22), ß-2acetamido-2-deoxy-n-glucoside acetamido-deoxy-glucohydrolase (E.C . 3.2.1 .30), ß-glucoronide glucuronohydrolase (E .C . 3.2 .1 :31) and sulphatase were assayed according to WEISSMANN et al. (1967) . Leucine aminopeptidase was assayed as described by GOLDBERG and RLrfENHllttî (1965). Protein estimation Protein estimation was carried out according to LowxY et dl. (1951) after thorough dialysis against 001 M sodium phosphate buffer, pH 7~0, for 24 hr .

Isoelectric focusing in density gradients Carrier ampholytes (Ampholine 4-6, 40 per cent w/v) wen used to establish a pH gradient in a 110 ml column as earlier described (V1~1IItBERG et al., 1967). A column was first run for 36 hr at a final voltage of 600 V with 5 per cent (w/v) of an Ampholine gradient of glycerol (0-50 per cent v/v) without any protein sample. Fractions with a pH in the range 5~2-5~7 were pooled and used to create a new gradient in glycerol . The protein sample (20 mg of freeze-dried enzyme) was then dissolved in the less dense solution, and the electrofocusing was carried out at 4°C for 48 hr (600 V; 1-2 mA). Fractions of 2 ml were then collected and pH determined at 4°C (see below). Discelectrophoresis on polyacrylamide Analytical actylamide electrophoresis was carried out in a discontinuous ß-alaninoacetate buffer system and in a 2,6-lutidine/glycine-KOH/glycine system (FLATMARx, 1954) and proteins were stained with coomassie brilliant blue (WAns~rnÖM and Môt.LSY, 1971a, VF~TERBERG, 1971a).

lsoelectric focusing on polyacrylamide Gel slabs were prepared between glass plates as recently described (VE317~tHERC3, 1972). Gels containing Ampholine (2 per cent w/v) were photopolymerized and electrofocusing was carried out at 0~°C at a final voltage of 50 V/cm during 3 hr (SÖnt~tot.M et al., 1972). The gels were then stained with coomassie brilliant blue as recently described (VrsrExsmc, 1971a). Samples containing 50-400 ug of protein were applied on the gel on filter paper discs. Sperm whale myoglobin and human haemoglobin from fresh haemolysates were used as reference proteins and applied on the same gel. pH measurements were performed with a surface electrode (Ingold) (VF3fERBLRG, 1972). TUXlCON l97! Yd. ll .

14 2

R. MÖLLBY, C. E. NORD and T. WADSTRÖM

Immunodi,~usion and immunoelectrophoresis Immunodiffusion was carried out according to OUCHTERLANY (1958) and immunoelectrophoresis according to GRABAR and WILLIAMS (1953) . A sample of 20 Ftg phospholipase C was dissolved in the buffer (005 M veronal buffer, pH 8~6) and run for 1 hr at 4°C (100 V ; 12 mA) in a Beckman Microzone apparatus . A polyvalent Cl. perfringens antiserum (0~1 ml undiluted) was applied and plates were photographed after one day at 20°C and two days at 4°C. Spectrophotometry and pH determinations Spectrophotometry and pH determinations were performed as earlier described (V~RsERC et al., 196 . RESULTS

Separation of phospholipase C by isoelectric focusing. The commercial enzymes were separated in very shallow pH gradients, pH 5~2-5~7 (Fig. 1). Lecithinase which was haemo-

FIG . I . ISOELEGTRIC FOCUSING OF PHOSPHOIIPASE C FROM SIGMA (2O IRg DRY WIIGHT).

2 ml fractions were collected, pH measured at 4°C (~--~) and each fraction was assayed for lecithinase activity on a turbid suspension of lecithin at A,~, (o---(~ and for haemo lytic activity on at>cep n~ blood cells (O-O). The amount of protein content in each fraction (~~,) was estimated according to LOWRY et d. (1951).

lytic for rabbit, human, sheep and horse erythrocytes was separated in a narrow peak with an isoelectric point (pI) of 4~6 ~ 005. A broad shoulder of lecithinase activity was also found between pH 5~5-5~8 with a maximum at pH 5~7, and another peak containing haemolytic activity but low lecithinase activity focused at pH 6~8. The recovery of lecithinase activity was very low (4 and 8 per cent in two experiments) when assayed immediately after the draining procedure . Addition of 1 mM of calcium and zinc chloride (final concentration) to each fraction 30 min before the assay increased the recovered activity several fold (35 per cent total yield) . The lecithinase, pI 4~6 and pI 5~7, was stable upon storage at -20°C in this state for several weeks. Contaminating enzymes and toxins in phospholipase C. Addition of 1 mM calcium and zinc ions to the lecithinase and haemolytic assay system did not affect the activities before TOXICON 1973 Vol. Il .

Clostridial phospholipase C

143

TABLE I . INFLUENCE OF CALCIUM AND 71NC IONS, ErHYIENE DIAMINE TETAA-ACETIC ACID (EDTA), CHOLESTEROL AND DI77~IfOTREITOL (DTT) UPON LECITHINASE' AND HAEMOLYTICt ACrIV1iY AFTER L40EIECfR1C FOCUSING

pI 4~6

Added reagent

Control CaCI, and Z.nCI, 1 mM final tont . EDTA 1 mM final tont. Cholesterol# 0~1 mg/ml DTT 1 mM# final tont .

Lecithinase activity

020

pI 5~7

Haemolytic activity

16

pI 6~8

Lecithinase activity

Haemolytic activity

8

Lecithinase activity -

Haemolytic activity

010

32

065

32

060

8

-

16

< ß"0S O~SS 055

16 4 1024

< 005 070 045

4 2 64

-

32 4 256

'~p~ ~ ~~~o nm Per ml Per rain on a lecithin suspension (see Materials and Methods) . tMeasured on sheep red blood cells after incubation at 37°C for 1 hr and 4°C for 2 hr. $Also containing 1 mM CaCI, and ZnCI,.

separation by iscelectric focusing, but had a pronounced activating effect on the purified enzyme (see above, and Table 1). After storage for 2 hr at 4°C with EDTA (1 mM final concentration) the lecithinase activity, but not the haemolytic activity of the purified enzyme (pI 4~5 and 5~7), was inhibited. A similar experiment with the haemolysin (pI 6~8) did not show inhibition or stimulation of the activity. Cholesterol (0" 1 mg/ml) inhibited more than 50 per cent of the haemolysin (pI 6"8) and the haemolytic activity of Ixithinase (pI 4~6 and 5~7) but had no effect on the lecithinase activity. Dithioerythritol and dithiothreitol increased the haemolytic activity of haemolysin (pI 6"8) 8-60 fold on erythrocytes from all four species but did not affect the lecithinase activity of peak pI 4" 6 and 5"7. Tht~ee fractions (pI 4~6, 5~7, and 6~8) showed similar haemolytic spectra on all four types of erythrocytes (sheep, rabbit, horse, and human 8 : 8 : 4 : 1). These results indicate that the three peak fractions contain theta-toxin or another haemolysin with similar properties (S1rtITH and Ho1.nEMAx, 1968) . TABLE 2 . IEOEI1crRIC PoIN~Ia (pI) oF THE DIFFEREIPI' ENZYMATIC AND TOXIC ACI'IVI1L~ IN A ODI10lERCIAL PRIiPARATIONt OF PHOSPHOLIPABB C FROM Cl perfr~lrgtnr Lecithinase C' I (a-toxin) ac II Hamolysin Protease' Acid phoaphatase Hyaluronate lyaae Dooxynôoauclease' a-Gluoosidase Endo-ß-N-acetylglucosaminidase Aryl sulphatase'

4~6 S "7 â8 4~5 4~7 4~7 4~6 4~7 S~S 4~5

'Precipitates in these fractions might interfere with the determination of the isoelectric points of this protein. fObtained from Sigma Chem. Comp., St. Louis, Mo., U.S.A . (see Materials and Methods) . T~OXICON 1973 Vol. !l.

144

R. MöLLBY, C. E. NORD and T. WADSTRÜTI

The iscelectric points of a contaminating deoxyribonuclease, a hyaluronate lyase, a protease, an a-glucosidase, an endo-ß-N-acetylglucosaminidase, a phosphatase and a sulphatase are shown in Table 2. Deoxyribonuclease was only detected by the sensitive viscosimetric assay. Protease activity was detected in all fractions in the pH range 3-55 with both assay systems. The maximum activity was found in three fractions pH 4~5-4~6 . This protease hydrolyses casein, haemoglobin, gelatin and several other substrates but not elastin (C.-E . NOxD, R. MÖLI,BY, and T. WansrxöM, to be published*). It is remarkable that 7 out of 10 enzyme activities in the commercial enzyme from Sigma focused in the narrow pH range pH 4~5-4~7. (Table 2.) The lecithinase, pI 5~7, was chosen for further purification and biological studies, since it is probably not contaminated by any of the activities for which we have assayed (R. MÖLI,BY and T. WADSTRÖM, unpublished data). The following activities were not detected in the commercial preparations : lipase, elastase, alkaline phosphatase, a-galactosidase, ß-galactosidase, ß-glucosidase, exo-ß-N-acetylglucosaminidase, ribonuclease and leucine aminopeptidase . Isoelectric focusing and discelectrophoresis on acrylamide . Analytical electrophoresis on acrylamide in one acidic and one basic buffer system revealed that 4-6 different protein bands were resolved after staining with coomassie brilliant blue (Fig. 2). The best separation was obtained in the alkaline buffer system indicating that the contaminants had a similar charge as phospholipase C (pI 4~6). This was confirmed by gel iscelectric focusing. (Fig. 3). Twelve to fourteen protein bands were separated in the commercial enzyme preparations when 400-SOO ltg was applied on the gel. Immwrodi;ffusion and immunoelectrophoresis. Commercial enzymes from Worthington and Sigma were analysed by gel immunodiffusion against diagnostic antisera for Cl. perfringens type A, B, C, and D (SMIrx and HoLn>~Inx, 1968). Several lines were detected against all four sera (Fig. 4). The results obtained after electrophoresis of the Sigma and Worthington enzymes in agarose gels followed by immunodiffusion are presented in Fig. 5. Four to six precipitin lines were visible after three days. DISCUSSION

P~sr~x et al. (1968) and DIx>~t (1970) purified phospholipase C from Cl. perfringens (welchü) by ion exchange chromatography, salt fractionation and Sephadex chromatography . Both preparations possessed lecithinase C activity, but the enzyme of C/. perfringens, ATCC 10543, was most active on sphingomyelin (P~sr~x et al., 1968). Recently SUGAHAItA and Oxs~tu (1971) resolved two molecular forms of Cl. perfringens a-toxin by isoelectric

focusing . Both forms degraded lecithin and sphingomyelin and were associated with lethal and haemolytic activity. BERNIiE111i©t et al. (1968) found two subcomponents (pI 5~2 and 5~5) in iscelectric focusing and SMYrx and Axsurxxo~-r (1969) found three components (pI 520, 527 and 539) . No data on the recovery were reported, but the latter authors found that the carrier ampholytes inhibited enzyme and toxic activity . The results of this investigation show that phospholipase C of Cl. perfringens can be separated by iscelectric focusing with a rather good recovery if calcium and zinc ions are supplemented after the separation. These divalent ions are necessary for enzymatic activity (ISPOLATOVSKAYA, 1970). Removal of ions during electrofocusing and the chelating properties of the carrier ampholytes (VE4TERBERG, 1971b) give a yield of less than 10 per cent. *Beta-glucuronidase activity was detected in the commercial preparation but it was not tested for after isoelectric focusing due to lack of wmmercially available substrate. TOXICON 1973 Vd. ll.

FIG . Z . DISCELECTROPHORFBIS ON POLYACRYLAMIDE OF THREE DIFFERENT COMMERCIAL PREPARATIONS OF PHOSPHOLiPASE C (O'1S mg}.

From left to right : phospholipase C from Sigma at pH 4'S on 7'S per ant (wJv) acrylamide ; at pH 8'3 on 10 per cent ; at pH 8'3 on 7'S per cent . Phospholipase C from Worthington at pH 4'S on 7'S per cent acrylamide ; at pH 8ß on 10 perant ; at pH 8'3 on 7~5 per cent . Phosphopace C from Nutritional Biochemicals at pH 4'S on 7~S per cent acrylamide ; at pH 8'3 on 7~5 per cent .

TO .YICO~Y 1973 I~~1. JI .

FP 144

FIG. 3. ISOELEC7RIC FOCI)31NG ON POLYACRYLAMIDE OF PHOSPHOLIPASE C.

From left to right: phospholipase C from Nutritional Biochemicals (050 mg dry weight) ; (2) Worthington (050 mg); (3) Sigma Chem . Comp . (050 mg); (4) Nutritional Hiochemicals (040 mg); (5) Worthington (040 mg); (6) Sigma (040 mg) ; (7) Haemoglobin from a fresh human haemolysate .

TOXICON J973 Vol. JJ

FIG. 4 . IMMUNODIFFUSION OF PHOSPHOLIPASE C AGAINST DIFFERENT DIAGNOSI7C ANTISERA FOR

ci. pEl'f!'lIIgQIIS .

The centre basin contains phospholipase C (20 ug) from Sigma (Fig. 4a, s), from Worthington (Fig. 4b, w) and from NBC (Fig . 4c, n). Block letters in the peripheral basins indicate serological specificity of clostridial antiserum . Each basin contains about 10 111 of undiluted antiserum .

TOXICON 1973 Vol. ll.

FIG . S . IMMUNOELEC'rROPHORESIS IN O'OSM SODIUM VERONAL BUFFER (pH S'6) OF PHOSPHOLIPASE C AGAINST DIAGNOSTIC ANTISERA FOR Cl . Perjrillgells . The upper basin (s) contains 20 ~Ig of phospholipase C from Sigma and lower basin (w) 20 ug

of phospholipase C from Worthington . The anode to the right . Antiserum (0'1S ml) against Cl . perjrinRells type A (Fig . Sa), type ß (Fig . Sb), type C (Fig . Sc) and type D (Fig . Sd) . roxlco .v

is+-.1 n;,l .

m.

TOXICON 1973 Vol. 11

Clostridial phospholipase C

]43

et al. (1970) also recently reported that human liver alkaline phosphatase was recovered after electrofocusing only upon addition of divalent rations. The results obtained in acrylamide gel electrophoresis and electrofocusing reveal that commercial preparations of clostridial phospholipase C are contaminated with several proteins of similar charge (Fig. 2 and 3). This was also confirmed by immuncelectrophoresis in agarose (Fig. 5). Separation by iscelectric focusing in extremely shallow pH gradients (pH 5~2-5~7) as reported in this paper, is a method with a very high resolving power for separation of proteins according to charge (V13sreRS>?RC, 1971b). Experiments are now in progress to combine this method with salt fractionations and molecular sieve chromatography for purification of clostridial phospholipase C with a high degree of purity (W~osTRöM, Noxn, and IVIÖI,LBY, to bepublished). Acrylamide electrophoresis ingels of di& scent pore sins and agarose immuncelectrophoresis will then be used to reveal impurities of the final product. However, since the enzyme will be used for biological studies in comparison with highly purified sphingomyelinase C from S. aureus (w~n~rRSM and Müt.l.sY, 1971a, b) all other toxin or enzyme contaminants found in commercial preparations of the clostridial enzyme must be eliminated before studies on different mammalian cell membranes are started. Cv~~rRl:ces~s (1971b) reported that clostridia) phospholipase C and phospholipase A from Vipers russelli unmasked insulin receptors in fat cell membranes and stimulated glucose uptake . This stimulating effect was observed several years ago by Hl~cll~lt (1965) and Ronslz.l. (1964) . However, tryptic digestion of the fat cells lead to a selective and profound fall in the aüinity of the membranes for insulin (Cv~7~c~s~s,1971a). RosF.rlrx~L and F~lx (1971) found that three different commercial preparations of clostridiel phospholipase C were contaminated with neuraminidase and an insulin-like activity, which was not inhibited by EDTA or stimulated by calcium as was phospholipase . These and other biological studies on erythrocytes (LENARD and SINGI~I, 1968), leucocyte membranes, and metabolism (P~~RCA et al., 1971) mitochondria and submitochondrial particles (t)rrol .l=.rrcFU and Howau x, 1970; Bulesrelly et aL, 1971 a, b), purified myelin (McL,l.welx and RAPPORT, 1971), etc. must be interpreted with caution until highly purified phospholipase C becomes available . It is also possible that both a lecithinase C and a sphingomyelinase can be separated, as indicated in the work by PASTAx and collaborators (1968) . It is also very interesting that another bacterial phospholipase C from Bacillus cereus is not haemolytic in purified state, in spite of its broad substrate specificity (ZWAAL et aL, 1971). A lecithinase C from Aeromonas hydrophila is not cytolytic (WAnsTltöie and Wle>:Tt .lrm unpublished data), while another phospholipase from a gram negative bacterium, Acinetobacter calcoaceticulyses rabbit erythrocytes (L1~IMAxrt, 1971) . It is obvious that much more work must be done on extracellular toxins and enzymes of Cl.perfringens with modem separation techniques before final conclusions on the different biological activities can be drawn. LATNER

Ackaowledgm~ents-We are gratdul to Dr. B. Ron.o~, Dr . R. F. A. Zw~w, aad Dr. C. S~rix for stimulating criticism and valuable advice . The skilful technical asaiataace of Miss M. K~eu .aa®v, Miss 1. KüHtv, and Mr. P. Aurarwat is giatefully acknowledged . This investigation was supported by a giant from the Swedish Medical Research Council (16X-2362). Addadwn ARer thin paper was submitted for publication we became aware of a recent paper by Cnso er d. (1971) in which acrylamide electrophoresis was used on a preparative scale for extensive publication of C perms fringeris phoapholipase C. Commercial preparations gave up to eleven bands in analytical acrylamide 710XlCON J973 Yol. !!.

146

R. MÖLLBY, C. E. NORD and T. WADSTRÜM

electrophoresis which agrees very well with the results of the analysis by disc electrophoresis and gel electrofocusing reported in this paper. Recently dostridial enterotoxin type A was purified (HAUSC~nin and Hr13F1IIMER, 1971). Enterotoxicity in a ligated intestinal loop showed that two of the commercial preparations were contaminated with enterotoxin with a pI of 4~4 which is in good agreement with the isoelectric point reported by these authors. REFERENCES Ar.ExANUea, M., Ht:ersr., L. A. and Hutewrrz, J. (1%1) The purification and properties of microooccal nuclease . J. biol. Chem. 236, 3014 . Atevn~soN, S., Hoca~, T. and WAnsrxöM, T. (1971) Influence of cultivation conditions on the production of extracellular proteins by Staphylococcus aortas . Acts path. mfcrobiol. stand. Sect. B79, 399. B~ er ,~ , M. (1%S) Phospholipase C and nuchanisms of action of insulin and cortisol on glucose entry into free adipose cells. Biochem. biophys. Res. Commux . 21, 202. Bsnra~a, A. W., GausAO~, P. and AvtoAn, L. S. (1%8) Isoelectric analysis of cytolytic bacterial proteins. J. Bact. 9S, 2439 . Borrvstvixe, P. F. and JoHtvsort, C. E. (1970) Bacillus cereus toxin. In : Microbial Toxins, Vol. III, p. 415, (lUi, S. R., KAnrs, S. and MoNr~, T. C., Eds.). New York-London: Academic Press. Atrxsmtv, C., Lorrna, A. and RACtcrat, E. (1971a) Effect of phospholipaae on the structure and function of mitochondria . J. biol. Chem. 246, 4075 . Baaslw, C., KANDACH, A. and Rwctcery E. (1971b) Effect of phospholipase and lipases on submitochondrial particles. J. biol. Chem . 246, 4083 . CAau, A., PALA, V., MONACELLI, R. and NANrn, G. (1971) Phospholipase C from Clostridium perjrilrgens : purification by electrophoresis on acrylamide-agar gel. ltd. J. Biochem. 20, 166. Cot~MAN, R., FtNFrw, J. H., KNVrroN, S. and LtMHtuctc, A. R. (1970) A structural study in the modification of erythrocyte ghosts by phoapholipase C. Biochim. biophys. Acts 219, 81 . CuA~txacAS~s, P. (1971a) Pcrtubation of the insulin receptor of isolated fat cells with proteolytic enzymes. Direct measurement of insulin-rooeptor interactions . J. biol. Chem . 246, 6522. Cvxneec~ass, P. (1971b) Unmasking of insulin receptors in fat cells and fat cell membranes. Pertubation of membrane lipids. J. biol. Chem. 246, 6532. Dnvea, B. A. (1970) Purification and properties of Clostridium welchü phoapholipase C. Biochim. blophys. Acts 198, 514. FLAnrAxtc, T. (1964) On the heterogeneity of beef heart cytochrome c. I. Separation and isolation of subfractions by discelectrophoresis and column chromatography. Acts them . stand. 18, 1936 . GotDSEteo, I. A. and Rurt:NSBtea, A. M. (1963) The colorimetric determination of kucine aminopeptidase in urine and serum of normal subjects and patients with cancer and other diseases . Caltcer 11, 283. GreAeAx, P. and Wn * 7"..~ C. A. (1953) Méthode penrtettant l'étude coßjugée des propriétés ékdrophoretiques et immunachimiques d'un mélan~ de protéines. Application su sérum sanguin. Biochim. biophys. Acts 10, 193. HAtlacrrtt~, A. H. W. and Hrtst~x, R. (1971) Purification and characteristics of the enterotoxin of Clostridium perjrirtgens type A. Cmr. J. Microbial. 17, 1425 . I90POUTOVSRAYA, M. V. (1970) Rok of metals in the activity of Clostrdhlm perjdpgens lecithinase. Biodttmtrtry (Russian Bfokhimiyo) 35, 434. KAravrrsxv, G., HvMr~r., J. P, and D~nxs, C. (1959) Some fedora which affect the enzymatic digestion of ribonucleic acid . J. blot. Chem. T.34, 1512 . Ktlatrtv~e, D. J. (1957) An evaluation of the egg-yolk reaction as a test for lecithinase activity . J. Bact. 73, 297. LArtvea, A. L., PAxsoNS, M. E. and Sxn.t~t, A. W. (1970) Isoekctric focusing of human liver alkaline phosphatase. Blochem. J. 118, 299. LEHMANN, V. (1971) Phospholipase activity of Acinetobacter calrnacYticus. Adapath . microbiol. steed. Sect . 79B, 372. LetvAren, J. and Sn~aOe, S. J. (1968) Structure of membranes' reaction of red blood all membranes with phospholipase C. Scienee 159, 738. Lowxv, O. H., R0.4®RUUCII'I, N. J., FAren, A. L. and RANDALL, R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem . 193, 265. LvNner.An, G. and JOHANS~ON, B. (1968) Proteinase and deozyribonuckase activity in the sea urchin sperm. Eezymologla 35, 345. MACFAawaE, M. G. and Krnar~rr, B. C. J. G. (1941) The biochemistry of bacterial toxins. I. Lechhinase activity of Cl. wrlchü toxins . Biochem . J. 35, 884. MACFAtuaNe, M. G. (1948) The biochemistry of bacterial toxins . 2. The enzymic specificity of Clostridium welchü lecithinase. Biochem. J. 42, 587. 710XICON 1973 Yol. ll.

Clostridial phospholipase C

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McIta.w~nv, D. L. and Rerpoxr, M. M. (1971) The effects of phospholipase C (Clostridium perfringens) on purified myelin . BiochLn. bioplrys. Acts 239, 71 . Mwx~tv~t, H. H. and Kn~t:a, S. (1970) F.ndo-ß-N-acetylglucosaminidase from Clostriäum perjringara, lyric for cell wall murein of Gram negative bacteria . l. Bact.102, 347. MeastnNOro M. (1%1) Studies on phospholipids II. Phospholipase activity of Clostridium perjrixgena toxin. l. Biodrem., Tokyo 49, 23 . Mrzarwivt, H. and Mizvrrntvt, H. (1964) Action of phospholipase C oa infiueaza virus. Nature, Load 204, 781 . Onot~vorn, A. C. (1969) Phospholipase C. Meth. Eezym.14,188. Orro»tn, A. C. and Howt~x, M. H. (1970) Membrane structure: morpholigical and chemical elterations in phospholipasaC-treated mitochondria and red cell ghosts . l. Mtmbr. B1oI. 2,180. Outer, d. (1938) Diffusion-in-gel methods fair immunological analysis .lrogr. Allergy S, 1. Pearerr, L, Mwo~e, V. and Kw~rtw, R. (1968) A phospholipase specdflc for sphingomyelin from Clartr~ dium PQJ~ttts. ,l. btol. C.ham . 243, 3730. Peziwxc~, P., C~t~t, R., M~trssz, M., MorrcAr vo, S. sad Ross, F. (1971) Phospholipid splitting and metabolic stimulation in polymorphonuclear kucocytea. l. Reticuloetrdothd. Soc. 10, 251. Ro~na>a.t, M. (1964) Metabolism of isolated fat cells. J. blot. Ghent. 239, 375. Ro~.ot+sat~, B., Zw~., R. F. A., Cotu+axtus, P., Woo®wNen, C. B. and vex D~a, L. L. M. (1971) Action of pure phospholipese A, and phospholipase C on human erytluocytea cell ghosts . Bioehirn. biophya. Acts 241, 925. Roc+, P. (1970) Ptimction of phospholipids in axons: depletion of membrane phosphorus by tt+eatmmt with phospholipase C. Toxkon 8, 235. Roasrnxu, J, w. and Fens, J. H. (1971) Insulin-h7re effect of clostridial phospholipase G neuraminidaae, and other bacterial factor on brown fat odls. l. äol. Chsm . 246, 3888 . Roww, R. (1964) Prevention of cholesterol esterification in human serum by en~ymic degradation of phoapholipids. Bioehim. biopltya. Acts 84, 761. Serro, K. sad Mvtcrnrtiu, K. (1968) F~zymatic hydrolysis of phoapholipids by Clostridium pafrbtgata phospholipase C. Biodtim. btophya. Acts 164, 596. Sam, L. S. and Hoz~tv, L. V. (1968) 7hapathogenic anoerobk bacteria. Springfield: Thomas. SMrtx, C. J. and Anavrrnvorr, J. P. (1969) Isoelectrlc focusing of ClartrldGrm perjrlttgena a sad 0-toxins . 1. gar. Micnoblol. 59, v (abstract). Suawtutu, T. sad ~AaA1CA, A. (1970) Two molecular farms of Clostridium pafrbrgena a-toxin aasodated with lethal, hemolytic and enzymatic activities . ,lap. I mad. Sd. Blol. T3, 61 . SbnHRaotat, J., AIlFSrMI, P. and Wenstn8tit, T. (1972) Arapid method for isoelectricfocusing in polystaylamide gel" Fabs Letters 24, 89. S8®an, P.-Ö. sad NOAn, C:& (1969) Determination of hysluronidsse activity is dental plaque material. 1. p~Odortt. Res. 47, 208. Toxtwrn, A. (1968) Alkaline phosphatase of Fadterlchia ooU. Meth. Ettym.1?, 212. Wenstnät, T. sad Hraeratrt~, K. (1970) Bacteriolytic enzymes from Stgp!{yloeoccus aureus. P~uification of an endo-ß_N-acetylglucoaamiaidase . Bioehem.1.120, 725. Wenatx8tr, T, and Mbra.Hr, R. (1971a) Studies on extracelular proteins from Stapl{ylococcua aureus . VI. Production and purification of ß-haemolysin in large scale. Biochlm. btopJtya. Acta 242, 288. wenatxbet, T. and MöLt~r, R (1971b) Studies on extracellular proteins from Staphybcocats aunats VII. Studies oa ß-haemolysin. Btochfm. btophya. Acta 24?, 308. W~xw, B., Rownv, G., 1~t~nsa","., J. A. and Fn~tnuct, D. (1%7) Mammalian a-acetylglucosaminidase . F~zymic pmpertiea, tissue distribution and intracellular lotion. Biodtendatry 6, 207. Vt=sr~eameo, O. (1971a) Staining of pmteia zones after isoekcMc fowaing is polyacrylamide gds. Blochim. biophya. Acts Z4i, 345. Vt~asao, O. (1971b) Isoelearic focusing of proteins . Meth . Enzym. 22, 389. Via, o. (1972) Isoelectric focusing of Proteins is polyaQylamide gds. Blochlm. bfopltya. Acts 257, 11 . via, o., wenaraata, T., via, K., svt~msoN, H. area Metsroxsrt, B. (1967) studies on exhacellular proteins from Stapltyloeoccus aweus. Blochim. biopJrys. Acts 133, 435. Zww~., R. F. A., RO~AI+aBN, B., COI~UAItta, P. and vw Dtaa~t, L. L. M. (1971) Complete purification and some properties of phospholipase C from Bacillus cer+etta. BfochLn. biophya. Acta 233. 474.

7nYlCON1973 Yal. ll.