Studies on sphingomyelinase of Bacillus cereus I. Purification and properties

247

Biochimica et Biophysics Acta, 528 (1978) 247-266 0 Elsevier/North-Holland Biomedical Press

BBA 57126

STUDIES ON SPHINGOMYELINASE OF BACILLUS CEREUS I. PURIFICATION AND PROPERTIES HIROH IKEZAWA, MAYUMI MORI, TETSUO OHYABU and RYO TAGUCHI Faculty ofPharmaceutical

Sciences, Nagoya City University, Mizuho-ku, Nagoya 467

(Jopod (Received July 21st, 1977)

summary A sphingomyelinase was purified 980.fold with recovery of 25.6% from the culture broth of Bacillus cereus, by (NH4)$04 precipitation and chromatography on CM-Sephadex, DEAEcellulose and Sephadex G-75. The purified preparation was free of lipase, protease and other phospholip~es. The enzyme specifically hydrolyzed sphingomyelin to ceramide and phosphorylcholine. Lysophosphatidylcholine was also attacked by the enzyme. The enzyme (J& = 24 000) was maximally active at pH 6-7. Other properties of the enzyme, including hemolytic activity and activation/inhibition studies, are reported.

Introduction Among various sphingomyelinases (EC 3.1.4.12, sphingomyelin cholinephosphohyd~l~), the enzyme from the culture filtrate of Staphylococcus aureus was the first to be characterised. The P-toxin of S. aureus, a typical ho&cold hemolysin which makes erythrocytes susceptible to hemolysis by cold shock after incubation with these cells at 37”C, was shown by Doery et al. [l] to be a phospholipase C which hydrolyzed sphingomyelin, yielding ceramide and phospho~Ichol~e. This enzyme has been purified and its properties investigated [ 2-4 1. Slein and Logan [ 51 demonstrated the presence of three different phospholipases C in the culture filtrate of ~ciZ~~s cereus; the so-called phospholipase C which hydrolyzes phosphatidylcholine and phosphatidylethanolamine, sphingomyefinase and phosphatidylinositol-specific phospholipase C. Recently, we have obtained sphingomyelinase of B. cereus in a sufficient purity. The purpose of the present report is to describe the purification procedures of this enzyme and some of its properties.

248

Materials and Methods Chemicals. A mixture of ceramides (Type III from bovine brain sphingomyelin) was purchased from Sigma Chemical Co. All other chemicals were of analytical reagent grade unless otherwise stated. Preparation of substrates. Phosphatidylcholine and phosphatidylethanolamine were prepared from egg yolk, and sphingomyelin was from bovine brain, by silicic acid column chromatography [ 61. Lysophosphatidylcholine was obtained by hydrolysis of phosphatidylcholine with phospholipase AZ from snake venom (Cro talus udaman teus) [ 71. Phosphatidylinositol was purified from the autolyzate of baker’s yeast by Trevelyan’s method [8] and column chromatography with silicic acid. Enzyme assays. Assays of sphingomyelinuse and otherphospholipuses C: Unless otherwise stated, the reaction mixtures for enzyme assays were incubated for 20 min at 37°C and contained 0.1 ml each 0.2 M borax/HCl (pH 7.0), 1% sodium deoxycholate, 10 mM MgC12, 10 mM substrate (sphingomyelin or phosphatidylcholine) and enzyme. The incubation mixture for phosphatidylinositol phospholipase C contained 0.2 ml enzyme and 0.1 ml each 0.2 M borax/HCl (pH 7.0), 1% sodium deoxycholate and 10 mM substrate. The reaction was stopped with chloroform/methanol/HCl (66 : 33 : 1, v/v), and the phosphate content measured [ 9,101. One unit of enzyme activity was defined as the amount which catalyzed the hydrolysis of 1 pmol of phospholipid per min at pH 7.0 and 37°C. To study substrate specificity, other phospholipids were added at 2 mM final concentration to the reaction mixture. Assay of lipuse and protease: Lipase activity was determined according to Taguchi and Ikezawa [ 111. Protease assay was carried out according to Kunitz’s method [12] with some modifications: The reaction mixture (at pH 6 or 8) containing equal volumes of enzyme solution and 2% casein (Hammarsten, Merk) was incubated at 30°C for 20 min and then stopped by the addition of 2 ml 0.4 M trichloroacetic acid. The reaction mixture was kept at 30°C for an additional 20 min, then filtered. To the filtrate, three volumes 0.4 M Na,CO, and one volume of Folin-Ciocalteu’s phenol reagent were added [13]. The mixture was kept at 30°C for 20-30 min, and the absorbance measured at 660 nm. Estimation of protein. Protein contents were estimated by measuring absorbance at 280 nm. Hydrolytic and hemoly tic actions on sheep ery throcytes. The action of the enzyme on sheep red cells was examined by using 10 mM MgClJ0.15 unit sphingomyelinase/5% erythrocyte suspension in phosphate-buffered saline (5 mM NaH,P0,/150 mM NaCl, pH 7.6), incubated at 37”C, with shaking. Aliquots were withdrawn from the reaction mixture after 0, 30, 45 and 75 min incubation. For measurement of hot-cold hemolysis, aliquots from the incubation mixture were immediately diluted to 10 volumes with phosphate-buffered saline precooled at O”C, cooled in an ice bath, and centrifuged. The resulting supernatants were measured spectrophotometrically at 550 nm. In the measurement of hot hemolysis, aliquots of the incubation mixture were centrifuged at room temperature. Then, the supernatant was diluted to 10 volumes with phos-

249

phate-buffered saline preincubated at 37”C, and the absorbance at 550 nm was determined. For the purpose of determination of phospholipids, aliquots from the mixture were extracted with chloroform/methanol [ 141. The extracted phospholipids were separated into sphingomyelin and other phospholipids by silica gel H thinlayer chromatography with chloroform/methanol/acetic acid/water (50 : 30 : 8 : 4, v/v). The spots were scraped off and further extracted with chloroform/ methanol/formic acid (1 : 1 : 2, v/v) and then with chloroform/methanol (2 : 1, v/v). After evaporation of the solvents of combined extracts, the residues were subjected to phosphate analysis [ 9,101. Assay of phosphatase-releasing activity. According to the method described by Ikezawa et al. [ 151, the enzyme solution containing purified sphingomyelinase and/or phosphatidylinositol phospholipase C of B. cereus was analyzed for phosphatase-releasing activity. Cultured method. B. cereus IAM 1208 was used. Routine culture medium contained 10 g polypeptone, 10 g yeast extract, 5 g NaCl and 0.4 g Na2HP04 per 1; the pH of the medium was adjusted to 7.0 with 1 M NaOH. 40 1 medium was inoculated with 2.5% (v/v) of the preculture of the organism and incubated at 37°C for 5 h in a KR-S Jar Fermenter (Kant0 Rika Co. Ltd., aeration: 10 l/ min, pH 7.0). Isoelectric focusing of phospholipases. A crude enzyme preparation which was obtained by precipitation of the cell-free culture broth with (NH.+)#O., at 80% saturation, was dialyzed against 0.02 M Tris * HCl (pH 8.5), adsorbed batchwise to DEAE-cellulose and eluted with 0.3 M NaCl. The eluate was concentrated and applied to isoelectric focusing apparatus (Ampholite, pH 5-8, in a gradient of O-50% glycerol, v/v) according to Vesterberg and Svensson [ 161. The sample (75 mg protein) was placed in the center of a 110 ml column (LKB-Produkter, Stockholm, Sweden) and electrolysis was carried out at 4°C for 26 h at 900 V. After the electrolysis, 2-ml fractions were collected and subjected to pH measurement at room temperature. After determination of the pH, each fraction was subjected to phospholipase assays with phosphatidylcholine, sphingomyelin and phosphatidylinositol as substrates. Results Isoelectric points of phospholipase C. As shown in Fig. 1, the treatment of isoelectric focusing revealed the presence of two forms of phosphatidylcholinehydrolyzing phospholipase C having p1 values of 6.8 + 0.1 and 7.5 ? 0.1. Zwaal and Roelofsen [17] suggested that the isoelectric point of B. cereus phospholipase C be the value within the range between 7.2 and 8.5. Apparently, one of the p1 values obtained, 7.5, corresponds to the value that they proposed. However, the proportions of two forms of the enzyme varied with the preparations applied to the isoelectric focusing, suggesting that these two fractions be not the isozymes but the different molecular forms of the same enzyme. On the other hand, sphingomyelinase or phosphatidylinositol-hydrolyzing phospholipase C was obtained in a single form. The p1 value of sphingomyelinase was 5.6 rf.0.1 and that of phosphatidylinositol phospholipase C, 5.4 t 0.1.

250

Fraction

number

Fig. 1. Distribution of three phospholipases C after isoelectric focusing of crude enzyme preparation (DEAEcellulose batch eluata) from B. cereus. -0, phosphatidyliaositol Phospholipase C activity; A-----+ sphingomyelinase activity; ‘J-, pho~hatidylcho~e-hy~olyz~g phosphoiipase. C activity; +. . . . -, PH.

Preparation of crude enzyme. After cultivation, the cells of B. cereus were removed by centri~gation and the culture broth was pre~ipi~ted with (NH,)$04 at 80% saturation. The resulting precipitate was collected by continuous centrifugation at 25 000 X g, dissolved in distilled water and dialyzed against 0.005 M Trislmaleate (pH 6.5). This preparation was used as starting material for the ~hromato~aphic purification of the enzyme. Chromatography on CM-Sephadex C-50 column. 50 ml of the dialyzed solution was placed on a column (4 X 19 cm) of CM-Sephadex C-50 equilibrated with 0.005 M Trislmaleate (pH 6.5); 2-ml fractions were collected (flow rate, 54 ml/h). The fractions containing both sphingomyelinase and phosphatidylinositol phospholipase C activities were obtained by washing the column with the same buffer, concentrated and dialyzed against 0.02 M Tris - HCl buffer (pH 8.5). Phosphatidyl~holine-hydrolyzing phospholipase C activity was obtained by elution with 0.5 M NaCl/0.005 M Tris/maleate buffer (pH 6.5). Chromatography on DEAE-cellulose column. 25 ml of the dialyzed solution was applied to a DEAE-cellulose column (Brown, 4 X 19 cm), which had previously been equilibrated with 0.02 M Tris * HCl (pH 8.5). The proteins were eluted with a linear gradient of O-O.3 M NaCl in the buffer. Sphingomyelinase activity was completely separated from the activities of phosphatidylinositol-specific and residual phosphatidylcholine-hydrolyzing phospholipase C (Fig. 2). The fractions con~ning sph~gomyelin~e activity were pooled and concentrated. Gel filtration on Sephadex G-75. The concentrated eluate from the DEAEcellulose column was applied to a Sephadex G-75 column (2 X 30 cm) equ~ibrated with 0.01 M Tris - HCl (pH 7.5) and eluted with the same buffer. Sphingomyelinase activity appeared as a single protein peak (Fig. 3), distinct from the major peak of other protein(s). The specific activity increased about

251

r

I

I

ID

- 0.3 E

I

k $ +-a2 :

5 b P

0.5

- 0.1

Fraction number

Fig. 2. Chromatography of sphingomyelinase of B. cereus on a DEAE-cellulose column. The enzyme solution (CM-Sephadex eluate) containing 550 mg of protein was applied to a column (4 X 19 cm). The conditions of chromatography are shown in the text; 19-ml fractions were collected at a flow rate of 54 ml/h. -, NaCl gradient;X- - - - - -X,A289nm; a---a, sphingomyelinase activity; O----O, phosphatidylinositol phospholipase C activity; q0, phosphatidylcholine-hydrolyzing phospholipase C activity.

980-fold and the recovery of sphingomyelinase activity was 25.6% (compared to precipitate at 80% (NH4)$04 saturation). A summary of the purification procedure is given in Table I. Unfortunately, the protein concentration of purified enzyme was not enough to perform polyacrylamide gel electrophoresis. The purified enzyme was free from phosphatidylcholineand phosphatidylinositol-hydrolyzing activities, and did not contain protease or lipase activity to an appreciable extent.

2

-

20

”

..

40

-

J

“-

60

60 Fraction

100

120

140

160

number

Fig. 3. Gel filtration of sphlngomyelinase on a Sephadex G-75 column. The enzyme solution (DEAEcellulose eluate) containing 13.5 mg of protein was applied to a column (2 X 30 cm). The conditions of gel fiiation are shown in the text; 1.5~ml fractions were collected at a flow rate of 20 ml/h. X-X, sphingomyelinase activity. 4289nm; A--d,

252 TABLE I PURIFICATION The enzyme in the text.

OF SPHINGOMYELINASE

activity

was

Step

80% (NHq)2S04 CM-Sephadex DEAEcekiose Sephadex G-75

FROM B. CEREUS

IAM 1208

determined with sphingomyelin as substrate. according to the method described

precipitate

Total activity (units)

Protein

1031 687 596

3250 550 13.5 0.84

264

(mg)

Specific activity (unitslmg) 0.32 1.25 44.2 314

Recovery (96)

100 66.7 37.8 25.6

Ptification

1 3.9 138 981

Identification of the reaction products. The products resulting from hydrolysis of sphingomyelin by the purified enzyme were identified, after 60 min incubation. The amount of enzyme used was 3.65 - 10e2 units per reaction mixture. The upper and lower phases (see Materials and Methods) were analyzed by paper and thin-layer chromatography, respectively. The paper chromatogram was developed with chloroform/formic acid/water (80 : 13 : 7, V/V) on a strip of Toyo Roshi No. 50 paper. The spots were visualized with Hanes-Isherwood’s reagent [ 181, and the product was identified as phosphorylcholine (Rf = 0.63) by comparing with a standard sample (Tokyo Kasei Kogyo Co. Ltd., Japan). Silica gel G thin-layer chromatography confirmed the presence of ceramides and unreacted sphingomyelin in the chloroform fraction, using chloroform/methanol/lO% NH40H (60 : 35 : 8, v/v). The components were stained with 0.6% anisaldehyde/2% HzS04 in acetic acid by heating at 120°C for 15 min. Sphingomyelin and a mixture of ceramides from bovine brain were used as reference lipids. Molecular weight. The molecular weight of this enzyme was estimated by gel filtration-. on a Sephadex G-75 column (2.6 X 100 cm) 1191 in 0.01 M Tris * HCl buffer (pH 7.5). Reference proteins (bovine serum albumin, ovalbumin, chymotrypsinogen A and cytochrome c) were applied to the same column and the molecular weight of sphingomyelinase was calculated to be approx. 24 000. Effect of PH. Activity measurements were carried out after 20 min incubation at 37°C with buffers ranging from pH 3.5 to 11 (pH 3.5-5.6, acetate; pH 5.8-8.5, Na2B40,/HC1; pH 9.1-11.0, NaZB40,/NaOH). The optimal pH for the degradation of sphingomyelin was in the range, pH 6-7. Incubation of the purified enzyme for 24 h at 4°C in various buffers (pH 5.110) prior to addition of the substrate, indicated that this sphingomyelinase was stable between pH 6.0 and 7.5. The routine assay mixture and conditions (incubation for 20 min at 37”C, pH 7.0) were used for the activity measurements of the treated enzyme. Influence of several effecters. The enzyme was highly activated by Mg2’; in contrast with other phospholipases, however, Ca2’ was inhibitory (Fig. 4). The enzyme was completely inhibited in the presence of 5 mM CaCl,. Zn2+ was essentially without effect (unpublished data). The hydrolysis of sphingomyelin by this enzyme was stimulated more than 3-fold at concentrations of deoxycholate above 0.1%. Other effecters were incubated with the enzyme in the absence of substrate

253

o= 0

Metal

100

50 ion

COnCentrOtiOn

(mM)

Fig. 4. Effect of C!a2+ and Mg2+ on the activity of sphingomyelinase. The incubation system and assay method are the same as those described in the text except that C&l2 was added at various concentra0, MgCl2. tions to the incubation mixture. u, C&l2 ; O-

at 5°C for 2 h, then enzyme activity was measured. 0.25 mM EDTA completely inhibited sphingomyelinase activity, whereas o-phenanthroline was without effect at the same concentration. Sulfhydryl agents such as monoiodoacetic acid, pchloromercuribenzoate, and glutathione did not influence the enzyme activity at 0.04 mM, suggesting that sulfhydryl group(s) were not involved in

Metal

ion

COnCentPatiOn

(t?TM)

Fig. 6. Effect of the concentration of divalent cations on the reactivation of EDTA-inhibited sphingomyelinase. The mixtures containing 0.05 ml of the enzyme solution (3.6 * lo-‘ units). 0.06 ml of 6 mM EDTA and 0.1 ml of 0.2 M borax/IX1 (PH 6.5). were kept at 6OC for 2 h. Then, to each mixture was added 0.1 ml of metal chloride solution at 5, 2.5 or 1.25 mM. After being allowed to stand for 1 h at S’C. the mixture was subjected to sPhingomyelinase assay, by incubating with 0.1 ml each of 1% sodium deoxycholate and 10 mM sphingomyelin at 37“C for 30 min and by subsequent nnalysis of resulting organic phosphate (PhosPhorylcholine) according to the procedures described in.the text. o---o, bfgc12; *-, cac12 ; xX, ZnC12.

254

TABLE

II

HYDROLYSIS OF PHOSPHOLIPIDS IN INTACT SHEEP ERYTHROCYTES ASE IN RELATION TO THE HEMOLYSIS INDUCED BY ENZYME The change in the amounts of sphingomyelin and other analyzed as described in the method section of the text.

phospholipids

BY

SPHINGOMYELIN-

with concomitant

hemolysis

were

Time (min)

Phospholipid

hydrolysis

Sphingomyelin Others Hemolysis

0

30

45

15

0 0

13 6

28 2

96 4

4 4

10 1

31 12

69 41

(%)

(%)

Hot-cold Iysis Hot Iysis

the activity of the enzyme. Zn” and Ca2’ partly restored the EDTA-inhibited enzyme activity (Fig. 5). However, Mg’+, not only reversed the EDTA inhibition, but also caused the activation of the enzyme beyond the basal activity before EDTA treatment. Even at 0.25 mM, Mg2+ stimulated the EDTAinhibited enzyme by 1.5-fold of basal activity. Substrate specificity. The enzyme showed high specificity for sphingomyelin; under the conditions examined, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol were not hydrolyzed by the purified enzyme to any appreciable extent (less than 0.1% of sphingomyelin-hydrolyzing activity). TABLE

III

THE RELEASE OF ALKALINE PHOSPHATASE PHOLIPASE C AND SPHINGOMYELINASE

INDUCED

BY PHOSPHATIDYLINOSITOL

PHOS-

0.16 g (wet weight) of kidney slices from albino Wistar rat, were incubated at 37’C for 90 min with the enzyme solutions described below. The total volume of reaction mixture was 4 ml. After incubation, the slices were removed by centrifugation and the liberated alkaline phosphatase in the supernatant was determined as described previously [201. Experiment

(1) (2) (3) (4) (5) (6) (7) (8) (9)

Release of alkaline phosphatase (munitslml)

No.

Phosphatidylinositol phospholipase Phosphatidylinositol phospholipase (5 munits) PhosphatidyIinositoI phospholipase (10 munits) Phosphatidylinositol phospholipase (20 munits) Phosphatidylinositol phospholipase (40 munits) Spingomyelinase (5 munits) Sphingomyelinase (10 munits) Sphingomyelinase (20 munits) Sphingomyelinase (40 munits)

Experiment A

Experiment B

C (10 units) C (10 munits)

+ sphingomyelinase

26 20

24 25

C (10 munits)

+ sphingomyelinase

24

25

C (10 munits)

+ sphingomyclinase

25

21

C (10 munits)

+ sphingomyelinase

20

17

0

5 2 0

255

On the other hand, when 0.365 unit of the enzyme was incubated with lysophosphatidylcholine in the presence of 2 mM Mg2+ and 0.2% sodium deoxycholate for 50 min at 37”C, an increase of monoglyceride was detected in the chloroform fraction. This has been shown likewise with staphylococcal p-toxin

1201.

Hemolytic activity in relation to hydrolysis of phospholipids on sheep ery throcy te membrane. Purified sphingomyelinase hemolyzed sheep red cells (Table II). Throughout the hemolytic process, the enzyme did not act on other phospholipids significantly, whereas sphingomyelin was hydrolyzed almost completely (96%), with 41% of hot lysis after 75 min incubation. Phosphatase-releasing activity of sphingomyeiinase and phosphatidylinositol phospholipase C. Sphingomyelinase did not cause appreciable release of alkaline phosphatase from slices of rat kidney, even at 40 munits/ml sphingomyelinase concentration (Table III). Also the phosphatase release induced by phosphatidylinositol phospholipase C (10 munits/ml) was not enhanced in the presence of 5-40 munits/ml sphingomyelinase. Discussion In the present study, sphingomyelinase of B. cereus was purified from other phospholipases and characterized as one of the C-type phospholipases. In the previous study on phosphatidylinositol phospholipase C [ 151 we used column chromatography on DEAE-cellulose, as an early step, followed CMSephadex. However, the method was not ideal since sphingomyelinase or phosphatidylinositol phospholipase C was not fully separated from contaminating phosphatidylcholine-hydrolyzing phospholipase C. In the previous study, we, therefore, sacrificed the yield of phosphatidylinositol phospholipase C in the eluate from the DEAE-cellulose column, in order to get this enzyme sufficiently pure. Isoelectric focusing revealed that phosphatidylcholinehydrolyzing phospholipase C had two pI values, 6.8 and 7.5. Thus in the present study, we used column chromatography on CM-Sephadex prior to DEAE-cellulose, since the major part of phosphatidylcholine-hydrolyzing phospholipase C was adsorbed to the column of CM-Sephadex at pH 6.5. DEAEcellulose chromatography then enabled separation of sphingomyelinase from phosphatidylinositol phospholipase C. We were thus able to obtain sphingomyelinase without contamination from the two phospholipases C, after gel filtration on Sephadex G-75. The results show that the properties of the enzyme resemble those of sphingomyelinase (&toxin) of S. aureus, especially with respect to substrate specificity and activation by Mg2+. Although the concentration of Mg2+ for maximal activation was relatively high (25-50 mM), the reactivation by Mg2+ of EDTA-inhibited enzyme suggests the possibility that Mg’+ be involved in the catalytic center of the enzyme molecule. On the other hand, our foregoing study [2l]revealed that the specific action of Clostridium nouyi phospholipase C (y-toxin) on sphingomyelin was stimulated in the presence of Mg2+, but no such effect was observed with other phospholipids. Mg*+ may alter the physical state of the sphingomyelin micelles to be favorable for the attack of enzymes such as sphingomyelinases of S. aureus and B. cereus, and phospho-

256

lipase C of Cl. novyi. The p-toxin of S. aureus hydrolyzes both sphingomyelin and lysophosphatidylcholine, but does not attack other phospholipids [20] The purified sphingomyelinase of B. cereus also hydrolyzes lysophosphatidylcholine, but does not act on phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol. Thus these two enzymes must have similar substrate specificities, although differing in their molecular properties. WadstrSm and Mijllby [2] showed that sphingomyelinase of S. aureus had a pl of 9.4 i 0.1 and a molecular weight of 38 000, whereas, we have shown that B. cereus sphingomyelinase has a pT= 5.6 i 0.1 and a molecular weight of 24 000. It has been shown that sphingomyelinase and phosphatidylcholinehydrolyzing phospholipase C do not release alkaline phosphatase from rat kidney slices nor influence phosphatase-releasing activity of phosphatidylinositol phospholip~e C. The purified sphingomyelinase of B. ceretis was shown to be a hot-cold hemolysin in the present report (as shown with the enzyme from S. aureus [ 2 J ). During prolonged incubation, however, the enzyme of 3. cerem hemolyzed sheep erythrocytes without cold shock. The mode of lytic action on sheep erythrocytes of this enzyme was now in progress in our laboratory. Addendum In the foregoing work with B. cerem phosphatidylinositol phospholipase [15], a error due to mistyping in the draft was found in the strain number B. cereus: correct, B. cereus IAM 1208; incorrect, B. cereus IAM 1205.

C of

Acknowledgement This work was supported Education in Japan.

in part by the Scientific

Grant from the Ministry of

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

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