Studies on phosphatidylinositol phosphodiesterase (phospholipase C type) of Bacillus cereus

ARCHIVES OF BIOCHEMISTRY Vol. 190, No. 1, September,

AND BIOPHYSICS pp. 1-7, 1978

Studies on Phosphatidylinositol Phosphodiesterase Type) of Bacillus cereus II. In Viva and lmmunochemical TETSUO

OHYABU,

Faculty of Pharmaceutical

RYO

(Phospholipase

Studies of Phosphatase-Releasing TAGUCHI,

AND

HIROH

Sciences, Nagoya City University, M&ho-Ku,

Received

January

31, 1978; revised

April

Activity’.

C ’

IKEZAWA Nagoya 467, Japan

10, 1978

A phosphatidylinositol-specific phospholipase C (PIase), purified 447-fold from the culture broth of Bacillus cereus by ammonium sulfate precipitation and chromatography with CM-Sephadex and DEAE-cellulose, was analyzed in vitro and in vivo for its activity to induce the release of alkaline phosphatase from plasma membrane. By i.v. injection of the purified PIase into rata, alkaline phosphatase was released into blood stream quantitatively. Antiserum against PIase was prepared by immunization of rabbits and anti-PIase IgG was purified by ammonium sulfate precipitation and chromatography with DEAEcellulose to a homogeneous state as indicated by immundelectrophoresis. Nearly equivalent amounts of purified anti-PIase IgG completely neutralized both phosphatidylinositol-hydrolyzing and phosphatase-releasing activities of the purified PIase preparation, showing that PIase is responsible for phosphatase release from rat kidney slices. Also, anti-PIase IgG inhibited PIase-induced phosphatase release in vivo, i.e., the elevation of phosphatase level in the blood stream of rats. The liberated phosphatase had a molecular weight of lOO,OOO-110,000, and proved to be derived from the organs such as kidney and liver but not from intestine, as indicated by inhibition studies with L-phenylalanine and sodium deoxycholate. From in vivo and immunochemical studies, PIase was demonstrated to be the phosphatasemia factor originally proposed by M. W. Slein and G. F. Logan, Jr. [(1965) J.

Bacterial. 90, 69-811.

In the culture filtrate of Bacillus cereus, Slein and Logan (1) demonstrated the presence of three different phospholipases C, the so-called phospholipase C (EC 3.1.4.3) which hydrolyzes phosphatidylcholine and phosphatidylethanolamine, sphingomyelinase C (EC 3.1.4.12) and phosphatidylinositol-specific phospholipase C. They suggested the last enzyme to be associated with a phosphatasemia factor, which liberated alkaline phosphatase from animal tissues or cell homogenates. In their experiments, however, partially purified enzyme was used. In the foregoing study, we confirmed the phosphatase-releasing activity of highly purified PIase from B. cereus (2), and sim-

ilar results have recently been obtained with highly purified PIase3 from Clostridium novyi (3) and Staphylococcus aureus (4). However, there still remained the possibility that phosphatase-releasing action was due to minor contaminant(s) in the enzyme preparation. The purpose of the present report is to finally demonstrate identity of phosphatase-releasing and PIase activities by immunochemical method, and to describe in viva studies on the release of alkaline phosphatase by PIase, in order to clarify the physiological action of B. cereus PIase on plasma membrane. MATERIALS

AND

METHODS

All the chemicals used were reagent grade. Phosphatidylinositol was purified from the autolysate of

’ Part I is Ref. 2. *This study was supported in part by Scientific Research Grant from the Ministry of Education, Science and Culture of Japan.

3 Abbreviation used: PIase, specific phospholipase C. 1

phosphatidylinositol-

0003-9861/78/1901-0001$02.00/0 Copyright All rights

0 1978 by Academic Press, of reproduction in any form

Inc. reserved.

2

OHYABU,

TAGUCHI,

baker’s yeast by Trevelyan’s method (5) and by column chromatography with silicic acid. The purity was assured by Silica Gel H thin-layer chromatography with chloroform/methanol/acetic acid/water (8515: l&4). Assay of phospholipases C (PIase, phosphatidylcholine-hydrolyzing phospholipase C, and sphingomyelinase C) were performed according to the method described previously (2). Protein contents were estimated by the method of Lowry et al. (6). Procedures for the purification of PIase andphos-

phatidylcholine-hydrolyzingphospholipase

C. Bacil-

lus cereus IAM 1208 was used as the source of enzymes. Culture method for this organism was as described previously (2). However, the method for purification of PIase was slightly modified: The culture broth was precipitated by ammonium sulfate at 80% saturation [80% (NH&S04ppt] at 4°C. After dialysis of ammonium sulfate precipitate against 0.005 M Tris-maleate (pH 6.5), the enzyme solution obtained was applied onto a column of CM-Sephadex (4 x 19 cm), equilibrated with the same buffer. The active fraction (CM-Sephadex C-50 breakthrough fraction) was concentrated, dialyzed against 0.02 M Tris-HCl, pH 8.5, and applied onto a column of DEAE-cellulose (4 x 19 cm) which had been equilibrated with the same buffer. The active fraction thus obtained as DEAE-cellulose eluate, was concentrated and used as purified PIase in the experiments throughout the study. The details of purification procedures are as described in the previous report on B. cereus sphingomyelinase (7). Highly purified phosphatidylcholine-hydrolyzing phospholipase C preparation was obtained from the column of CM-Sephadex in the PIase purification by eluting with 0.5 M NaCl, and used in the comparative study with PIase. Production of antiserum and purification of antiPlase-immunoglobulin G (IgG). A half milliliter of purified PIase (DEAE-cellulose eluate in Table I) was mixed with an equal volume of Freund’s complete adjuvant (Iatron, Tokyo) and injected into back skin of two rabbits subcutaneously. The injection was repeated four times at intervals of 10 days. Approximately 0.5 mg of protein per rabbit was administrated at each injection. Seven days after final injection, the animals were bled. Then, antiserum obtained was precipitated with ammonium sulfate at 50% saturation at 4°C. Then the resulting precipitate was dissolved in 0.9% NaCl and treated again with ammonium sulfate at 38% saturation. The precipitate thus obtained was collected by centrifugation and dissolved in 0.001 M phosphate buffer, pH 8.0. Finally, anti-PIase IgG fraction was isolated as breakthrough effluent by column chromatography on DEAE-cellulose equilibrated with the same buffer. Immunoprecipitation. Two-tenths milliiter of antiPIase IgG solutions was mixed with 0.2 ml of enzyme solution (final composition: 0.01 M Tris-HCl buffer

AND

IKEZAWA

containing 75 mM NaCl, pH 8.0). The mixtures were incubated for 90 min at 37°C and then allowed to stand overnight at 4°C. The precipitate was removed by centrifugation at 3000 rpm for 10 min, and the enzyme activity in the supernatant was determined. Assays of phosphatase-releasing activity in vitro and in viva. The in vitro assay using rat kidney slices was as reported previously (2). The incubation mixtures containing various amounts of rat kidney slices and PIase in 0.25 M sucrose were incubated at 37°C for 90 min. Slices were removed by centrifugation and alkaline phosphatase activity in the resulting supernatants was assayed according to Engstrom’s method (a), by incubation of the reaction mixture at O’C for 12 mm (substrate: p-nitrophenylphosphate). Two-tenths milliiiter of purified PIase solutions was injected into the tail veins of male albino Wistar rata weighing 200-300 g. Then at definite intervals, 0.2 ml aliquots of blood samples were withdrawn from the right jugular vein, according to the method described by Upton (9), and mixed with 0.8 ml of 85 mu citrate60 IIIM NaCl in 10 mM phosphate, pH 7.6. After centrifugation, alkaline phosphatase in plasma was determined by incubation at 37’C for 30 min according to Engstrom’s method (8). Three rats were used per dose of injected PIase, and mean values of blood alkaline phosphatase were calculated at every dose and interval, to get the plots of time course curves. Inhibition studies on alkaline phosphatase from kidney, liver, intestine, and blood were performed as follows. The slices from rat kidney, liver, and intestine (each 0.5 g, wet weight) were suspended in 4.8 ml of 0.25 M sucrose and incubated with 0.2 ml of PIase solution (40 milliunits in 0.02 M Tris-HCl buffer, pH 8.5) at 37°C for 60 min. Released alkaline phosphatase in the supernatant was diluted to 1.2 milliunits for kidney and intestine (about 100 times) and 3.0 milliunits for liver (about 2 times). Then, the activity of released alkaline phosphatase was measured at 37°C for 30 min with or without the inhibitor such as Lphenylalanine or sodium deoxycholate in the assay mixture. RESULTS

Purification of Plase. In the previous report, we showed purification procedures of PIase, getting the enzyme in a high purity. However, the method did not give the enzyme in a sufficient quantity. Thus in the present work, we adopted procedures similar to those used for purification of sphingomyelinase (71, as a modified method for purification of PIase. Table I summarizes the purification thus achieved by these procedures (see under Materials and Methods). Phosphatidylcholine-hy-

PHOSPHATIDYLINOSITOL

PHOSPHOLIPASE TABLE

PURIFICATION Purification Culture broth 80% WIdzS04 CM-Sephadex fraction DEAE-cellulose n The

PIase

OF PHOSPHATIDYLINOSITOL step

Protein

(mg)

Total

eluate activity

3,250 550 88

in each purification

activity (units)

cereus

OF

Bacillus cereusd

C (PIAsE) Specific activity (units/mg)

Purification

Re;ry 0

8,748 8,560

0.054

4,150

7.55

140

47.4

2.100

23.9

447

24.0

step, was determined

drolyzing phospholipase C was removed by adsorption to CM-Sephadex and sphingomyelinase was separated from PIase by DEAE-cellulose chromatography, as reported in the foregoing work with sphingomyelinase (7). The enzyme preparation finally obtained was 447-fold as active as the culture broth and the yield of the enzyme activity was 24.0% (Table I). Both the yield and specific activity in the final enzyme preparation were improved as compared with those in the previous report (2), where recovery was 8.4% and specific activity was 13.74 units/mg protein. Neutralization of phosphatidylinositolhydrolyzing and phosphatase-releasing activities by antibody. Anti-PIase IgG, purified from rabbit antiserum by ammonium sulfate precipitation and DEAE-cellulose column chromatography, was analyzed for its purity by immunoelectrophoresis. As shown in Fig. la, a single precipitin band was observed between purified rabbit anti?Iase IgG and goat anti-(rabbit IgG) serum or goat anti-(rabbit serum) serum. Thus purified rabbit IgG proved to be homogeneous by immunoelectrophoresis. Specificity of the purified IgG was then tested by immunodiffusion (Fig. lb) and enzyme neutralization (Fig. 2). In Fig. lb, a single precipitin line appeared between purified IgG and PIase, but this IgG did not react with phosphatidylcholine - phospholipurified pase C. Therefore, purified IgG was shown to be specific for PIase. In the neutralization experiments, the enzymes were mixed with varying amounts of purified anti-PIase IgG. The resulting immunoprecipitates were removed by centrifugation and the activities remaining in the supernatants were determined (Fig. 2). Both phosphatidylinositol-hydrolyzing and phosphatase-

3

B.

I

PHOSPHOLIPASE

162,000 ppt C-50 breakthrough

C OF

2.63

according

to the method

1 49

100 97.9

in the text.

releasing activities of purified PIase (40 milliunits) were completely neutralized by about 6 pg of anti-PIase IgG, whereas phosphatidylcholine-hydrolyzing activity (7 milliunits) of purified phospholipase C separated by CM-Sephadex chromatography was not precipitated by anti-PIase IgG (9 pg). Thus it follows that PIase is responsible for the release of alkaline phosphatase from rat kidney slices. In some preparations of PIase, a minor activity of phosphatidylcholine-hydrolyzing phospholipase C still remained. However, anti-PIase IgG was not able to neutralize phosphatidylcholine-hydrolyzing activity (17 milliunits) in the purified PIase preparation, showing that this activity is due to a minor contaminant different from PIase but not to PIase itself. Also a minor phosphatidylinositol-hydrolyzing activity present in purified phosphatidylcholine-hydrolyzing phospholipase C (CM-Sephadex eluate), was completely neutralized by anti-PIase IgG. The molecular weight of alkaline phosphatase released from rat kidney slices. The molecular weight of kidney alkaline phosphatase liberated by PIase was estimated to be lOO,OOO-110,000 by gel filtration on a Sephadex G-200 column (1.8 X 65 cm) equilibrated with 0.01 M Tris-HCl buffer (pH 7.5) according to the method described by Andrews (lo), with reference proteins such as catalase, aldolase, bovine serum albumin, and cytochrome c (Fig. 3). Release of alkalinephosphatase in vivo. The enzyme solutions containing varying units of PIase were injected into the tail veins of rats. After administration of PIase, the activity of alkaline phosphatase in rat blood plasma increased linearly with time, in proportion to the enzyme units injected (Fig. 4). When 1.50 units of PIase was ad-

4

OHYABU,

TAGUCHI,

AND

IKEZAWA

b

FIG. 1. Immunochemical analysis of purified rabbit anti-PIase IgG. (a) Precipitin reactions in agar gel after electrophoresis and diffusion. Thirty microliters of rabbit anti-PIase IgG (0.5 mg/ml), puritied by ammonium sulfate precipitation and DEAE-cellulose chromatography, was placed on a middle well, and electrophoresed in Toyo immunoelectrophoresis apparatus (Toyo Industrial Corp., Tokyo) at 3 mA for 50 min, and then allowed to diffuse overnight at room temperature against goat anti-(rabbit IgG) serum (Medical Biology Laboratory, Nagoya) in the upper trough, and against goat anti-(rabbit serum) serum (Medical Biology Laboratory, Nagoya) in the lower trough. (b) Precipitin reactions observed by immunodiffusion in agar gel, using 30-4 aliquots. The upper well contained anti-PIase IgG (0.5 mg/ml). Purified PIase (1 mg/ml) and purified phosphatidylcholine-hydrolyzing phospholipase C (CM-Sephadex eluate, 1 mg/ml), were placed in the left and right well, respectively. The slide was then allowed to stand overnight at room temperature and stained with amido black 1OB.

ministrated, the increase in blood alkaline phosphatase reached in plateau at 0.045 unit/ml after 60 min. Neutralization of the release of alkaline phosphatase in vivo by antibody. As shown in Fig. 5, the increase in blood alkaline phosphatase was suppressed, when purified PIase was injected after preincubation with anti-PIase IgG. When the enzyme was injected simultaneously with anti-PIase IgG,

the increase in blood alkaline phosphatase became approx. half of that observed in the absence of antibody. Origin of alkaline phosphatase in blood stream. Table 11 shows the effects of Lphenylalanine and sodium deoxycholate of kidney, liver and intestinal alkaline phosphatase isozymes. At 15 mu, L-phenylalanine inhibited the intestinal enzyme by 67% and the kidney and liver isozymes by 46%,

PHOSPHATIDYLINOSITOL

PHOSPHOLIPASE

C OF I

I

1.0 Anti-&se

IgG(pg)

FIG. 2. Neutralization of phospholipase C activities and phosphatase-releasing activity with anti-PIase IgG. The phospholipase C fractions were mixed with varying amounts of purified, rabbit anti-PIase IgG, and the activities remaining in the supernatants were measured after centrifugation. The purified PIase fractions treated with anti-PIase IgG were mixed with 0.16 g of rat kidney slices, then the mixtures were incubated and alkaline phosphatase-releasing activity was determined as described in the text. W---O, The activity of alkaline phosphatase, liberated by the purified PIase (DEAE-cellulose eluate). Assay was duplicated; M, phosphatidylinositol-hydrolyzing activity in the purified PIase preparation (DEAE-cellulose eluate); A-A, phosphatidylcholine-hydrolyzing activity in the partially purified PIase preparation (contaminated fractions of DEAE-cellulose eluate); w phosphatidylcholine-hydrolyzing activity in the purified phospholipase C preparation (CM-Sephadex eluate).

respectively. On the other hand, 10 mu sodium deoxycholate inhibited the intestinal enzyme by 42% and the isozymes of kidney and liver by 69 and 67%. At higher concentrations of L-phenylalanine or sodium deoxycholate, the degree of inhibition was not further increased significantly. Time courses of the changes in susceptibility of liberated alkaline phosphatase with 15 mM L-phenylalanine or 10 mru sodium deoxycholate were followed. Before injection of 1.50 units of PIase (at zero time), blood alkaline phosphatase was inhibited by L-phenylalanine by 56% and sodium deoxycholate by 31%. Thus blood phosphatase at zero time was mainly of the intestinal type. With increase in blood phosphatase by administration of PIase, the degree of inhibition by 15 mM L-phenylalanine decreased, in contrast to the increase in the

5

B. cereus

1.5

20

“E/b

2.5

I 1

FIG. 3. Determination of molecular weight by gel filtration of alkaline phosphatase released from rat kidney slices by the action of PIase. In the gel filtration of l-ml samples on Sephadex G-206 column, 2-ml fractions were collected at a flow rate of 40 ml/hr. Reference proteins described in the text were dissolved in 0.01 M Tris-HCl (pH 7.5) at 1 mg/ml, prior to application onto the column. Other experimental details are described in the text.

Time(min) FIG. 4. Release of alkaline phosphatase in vivo by PIase. Each unit (0,0.36,0.75, and 1.50) of PIase was used for injection as described in the text. The mean values were calculated from the increase in the activity of alkaline phosphatase obtained by three rata per dose, and were plotted against time after injection. o--O, 1.50 units; A-A, 0.75 unit; W, 0.36 unit; O---O, control (0 unit, i.e., injection of solvent, 0.02 M Tris-HCl, pH 8.5); A-A, PIase preheated at 100°C for 10 min.

degree of inhibition by 10 mu sodium deoxycholate. At 60 min after PIase injection, the level of blood alkaline phosphatase reached twice that before injection and the degrees of inhibition of alkaline phosphatase by both inhibitors became almost equal (44%). These results indicate that the

6

OHYABLJ,

TAGUCHI,

Time( min) FIG. 5. Neutralization of alkaline phosphatase-releasing activity in vivo with rabbit anti-PIase IgG. Each 0.75 unit of PIase was injected: 1, without IgG (O--O); 2, simultaneously with 25 pg anti-PIase IgG (A-A); and 3, after preincubation with 25 pg antiPIase IgG at 37°C for 90 min (U--U). The alkaline phosphatase activity in blood was measured as described in the text. The mean values of the increase in the activity of alkaline phosphatase were calculated as shown in Fig. 4, and were plotted against time after injection. TABLE

II

INHIBITORY EFFECTS OF VARYING CONCENTRATIONS OF L-PHENYLALANINE OR SODIUM DEOXYCHOLATE ON ALKALINE PHOSPHATASE ISOZYMES” Enzyme source

Inhibition

(%) by

L-Phenylalanine

Kidney Liver Intestine

(5 mM)

(10 md

(15 mM)

(5 t-f)

(10 mM)

8 19 23

36 40 57

46 42 67

65 62 40

69 67 42

D Isozymes released from tissue slices of rat kidney, liver, and intestine by the action of B. cereus PIase. Alkaline phosphatase assay was performed with or without the inhibitor at 37°C for 30 min. The percentage inhibition by L-phenylalanine or sodium deoxycholate was calculated by use of controls without these inhibitors. The activity of phosphatase isozymes used were 1.2 milIiunits (kidney), 3.0 rnilliunits (liver), and 1.2 milliunits (intestine), respectively.

liberated alkaline phosphatase was similar to that present in kidney or liver. DISCUSSION

Purified preparations of PIase from B. cereus (1,2), C. nouyi (3), and S. aureus (4)

AND

IKEZAWA

have been reported to exhibit phosphatasereleasing activity in vitro. On the other hand, both phosphatidylcholine-hydrolyzing phospholipase C (2) and sphingomyelinase (7) did not release alkaline phosphatase from rat kidney slices. Therefore, there exists the possibility that phosphatase-releasing activity is one of the common, specific properties of bacterial PIases. In the present study, we showed by use of purified preparations of enzyme and IgG that the equivalent amount of anti-PIase IgG required for neutralization of phosphatase-releasing activity was consistent with that needed for neutralization of phosphatidylinositol-hydrolyzing activity. Thus, according to an immunochemical criterion, we demonstrated that these two activities were due to the same protein. Finally, the possibility of contribution of minor contaminant(s) in the enzyme preparation to phosphatase-releasing activity was excluded. Furthermore, purified PIase induced the increase in the level of blood alkaline phosphatase, when injected into rats, and this phosphatase-releasing activity in uiuo was also suppressed by anti-PIase IgG. From these results obtained in vitro and in uiuo, B. cereus PIase proved to be a phosphatasemia factor found by Slein and Logan (1). Probably, other PIases of bacterial sources such as C. nouyi and S. aureus must also possess phosphatasemic activity in uiuo as well as well as in uitro, as suggested by Taguchi and Ikezawa (3) and Low and Finean (4). In the present study, the molecular weight of alkaline phosphatase, which was released from rat kidney slices by B. cereus PIase, was estimated to be lOO,OOO-110,000. This value almost agrees with that of peak A in isoelectric focusing profiles, obtained by Nose (11)) of alkaline phosphatase from rat kidney. Therefore, it appears that a kidney alkaline phosphatase is released by PIase in the form free from other membrane constituents. Several inhibitors such as L-phenylalanine (12-14), bile acid (15), L-homoarginine (16), and L-tryptophan (17), have been reported to be organospecific for isozymes of mammalian alkaline phosphatase. Fishman

PHOSPHATIDYLINOSITOL

PHOSPHOLIPASE

et al. (12, 13) found that L-phenylalanine inhibited rat and human intestinal alkaline phosphatase isozymes but had little or no effect on isozymes from other organs. Also, Bodansky (15) found that bile salts specifically inhibited alkaline phosphatase isozymes of human bone and liver. In view of these findings we studied the effects of Lphenylalanine and sodium deoxycholate on alkaline phosphatase isozymes which were released from tissue slices of rat kidney, liver and intestine by B. cereus PIase. The behaviors of isozymes of intestinal alkaline phosphatase towards these two inhibitors were the reversal of those of isozymes of kidney and liver. On the basis of this result, we followed time course of changes which occurred in the degrees of inhibition by these inhibitors of blood alkaline phosphatase, after i.v. injection of PIase into rats, in order to characterize phosphatase liberated in Go. As the result, B. cereus PIase proved to release alkaline phosphatase from the organs such as kidney and liver, and not from intestine. Since alkaline phosphatase originally present in normal plasma derived mainly from intestine, PIase-induced release of alkaline phosphatase in uiuo must be a direct reflection of organotropic action of PIase on plasma membranes of the organs such as kidney or liver, but not of intestine.

C OF

7

B. cereus

REFERENCES

1. SLEIN, M. W., AND LOGAN, Bacterial. 90, 69-81.

JR., G. F. (1965)

J.

2. IKEZAWA, H., YAMANEGI, M., TAGUCHI, R., MIYASHITA, T., AND OHYABU, T. (1976) Biochim. Biophys. Actu 450, 154-164. 3. TAGUCHI, R., AND IKEZAWA, H. (1978) Arch. Biothem. Biophys. 186.196-201. 4. Low, M. G., AND FINEAN, J. B. (1977) Biochem. J. 167.281-284. 5. TREVELYAN, W. E. (1966) J. Lipid. Res. 7, 445-447. 6. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 7. IKEZAWA, H., MORI, M., OHYABU, T., AND TAGUCHI, R. (1978) Biochim. Biophys. Acta 528, 247-256. L. (1964) Biochim. Biophys. Acta 92, 8. ENGSTRBM, 71-78. 9. UPTON, R. A. (1975) J. Pharm. Sci. 64, 112-114. 10. ANDREWS, P. (1965) Biochem. J. 96, 595-606. Il. NOSE, K. (1976) J. Biochem. 79.283-288. W. H., GREEN, S., AND INGLIS, N. R. 12. FISHMAN, (1962) Biochim. Biophys. Acta 62.363-375. W. H., GREEN, S., AND INGLIS, N. R. 13. FISHMAN, (1963) Nature 198,685-686. W. H. (1968) Arch. 14. GHOSH, N. K., AND FISHMAN, Biochem. Biophys. 126,700-706. 15. BODANSKY, 0. (1937) J. Biol. Chem. 118.341-362. 16. LIN,

C. W., AND FISHMAN,

W. H. (1972)

J. Biol.

Chem. 247,3082-3087. 17. LIN, C. W., SIE, G. H., AND FISHMAN, Biochem. J. 124,509-516.

W. H. (1971)