Purification and characterization of cytotoxin from the crude extract of Pseudomonas aeruginosa

Microbial Pathogenesis 1989 ; 6 : 103-112

Purification and characterization of cytotoxin from the crude extract of Pseudomonas aeruginosa Tetsuya Hayashi,* Yoshiyuki Kamiot and Yoshiro Terawaki Department of Bacteriology, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto 390, Japan. (Received October 4,1988 ; accepted in revised form November 11, 1988)

Hayashi, T. (Dept. of Bacteriology, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto 390, Japan), Y . Kamio and Y . Terawaki . Purification and characterization of cytotoxin from the crude extract of Pseudomonas aeruginosa. Microbial Pathogenesis 1989 ; 6 : 103-112. Pseudomonas aeruginosa cytotoxin is a protein toxin exhibiting cytotoxic effects on various eukaryotic cells . The toxin was purified from a crude extract of Pseudomonas aeruginosa 158 by trypsin treatment and serial chromatography . The purified cytotoxin was electrophoresed as a single protein band on polyacrylamide gel electrophoresis (PAGE) in the presence or absence of sodium dodecyl sulfate (SDS) . The molecular weight of the purified toxin was determined to be 29000 by SDS-PAGE and gel filtration chromatography in the presence of 6 M guanidine hydrochloride; the isoelectoric point was pH 6 .0 . The N-terminal amino acid sequence of the purified toxin was determined . The purified toxin showed the strongest cytotoxic effect on rabbit polymorphonuclear leukocytes and diverse degrees of cytotoxic effects on various eukaryotic cells including red blood cells and cultured cells . Key words : P. aeruginosa ; cytotoxin ; purification ; N-terminal amino acid sequence .

Introduction

Pseudomonas aeruginosa is a Gram negative bacterium, which is an opportunistic pathogen of importance in hospital-acquired infection, particularly in patients with reduced resistance.' Several components related to its virulence have been identified and characterized ." The cytotoxin, which was originally designated as leucocidin by Scharmann 4 and renamed cytotoxin by Lutz' is thought to be a virulence determinant, because this protein toxin has a cytotoxic action towards eukaryotic cells, especially leukocytes. This cytotoxin is also of considerable interest because of its unique mode of production : the toxin is produced as a cell-associated inactive precursor and converted into an active toxin by limited proteolysis during autolysis of the bacteria .' There are many reports on the purification and properties of the toxin prepared from the autolysate of P. aeruginosa 158 (PA158) . 5-12 However, little information is available of the structure of the cytotoxin itself . The cytotoxin purified by Scharmann 5 and Lutz 5 from the bacterial autolysate was not homogeneous . Their preparations contained several protein bands with different isoelectric points . In the present study, we ' Author to whom correspondence should be addressed . t Present address : Department of Agricultural Chemistry, Faculty of Agriculture, Tohoku University, Sendai 981, Japan . 0882-4010/89/020103+10$03 .00/0

© 1989 Academic Press Limited



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developed a new method for purification of the cytotoxin and succeeded in obtaining an active cytotoxin with electrophoretic homogeneity from a crude extract of PA158 . This report presents the purification procedures and some of the physicochemical and biological properties of the purified cytotoxin .

Results Purification of cytotoxin In the initial attempts to purify cytotoxin from PA158, we tried to obtain an autolysate by the method of Hirayama et al. 13 However, the yield of the lysate was too low to allow purification of the toxin . Therefore, we searched for cytotoxic activity in a crude extract of PA158, and found a high leukocytotoxic activity after treating it with trypsin . We used the crude extract as the starting material for purifying the cytotoxin . The crude extract prepared from a 40 I culture of PA158 was applied to a DEAESephadex A-50 column and eluted successively by buffer A (Fraction 1), buffer A with 0 .5 M NaCl (Fraction 2) and buffer A with 1 M NaCl (Fraction 3) . More than 90% of leukocytotoxic activity was recovered in Fraction 2 . Fraction 2 was concentrated on an Amicon PM10 membrane and treated with trypsin . In this operation, the cytotoxin that existed in a form of inactive precursor was converted into an active form with full leukocytotoxic activity . The toxin preparation was sequentially chromatographed on a DEAE-Sephadex A-50 column and FPLC using Superose TM12, Mono Q and Phenyl-Superose columns . Details of the chromatographic purification are shown in Fig . 1 . The purification procedures and the yields from each step are shown in Table 1 . Finally, 33 .5 mg of purified cytotoxin was obtained from a 40 I culture . In the purified preparation, we detected no proteolytic or phospholipase C activity . The toxin preparation did not form a line of precipitate against either antiexotoxin A, anti-elastase or anti-alkaline protease serum in gel diffusion tests . Purity and physiochemical properties of the purified cytotoxin The purified cytotoxin preparation showed a single band of protein on polyacrylamide gel electrophoresis (PAGE) in the presence or absence of sodium dodecyl sulfate (SDS) [Fig . 2 (a) and (b)] . Leukocytotoxic activity was found to co-migrate with the protein band on non-denaturing PAGE . The molecular weight of the purified toxin was 29000 as determined by SDS-PAGE . The value was the same as that obtained from gel filtration chromatography of the purified toxin in the presence of 6 M guanidine hydrochloride (data not shown) . Isoelectric focusing (IEF) of the purified toxin preparation on a polyacrylamide gel containing 8 M urea also showed a single protein band with an isoelectric point of pH 6 .0 [Fig . 2 (c)] . The amino acid composition of the purified toxin is presented in Table 2 . The molecular weight calculated from the amino acid composition was 29514 . No detectable carbohydrate was found in the purified preparation . The N-terminal sequence of 21 amino acid residues of the toxin was determined (Table 3) . The leukocytotoxic activity of the purified toxin was completely lost by heating at 90°C for 5 min . Biological activities of the purified cytotoxin Rapid and massive swelling of rabbit polymorphonuclear leukocytes (PMNs) without rupture of the cell membrane was observed after incubation with the purified toxin . The morphological change of the cells was the same as that described by Scharmann et al.' Similar morphological changes were observed after the same treatment of various species of leukocytes, including rabbit lymphocytes and alveolar macrophages and human PMNs and lymphocytes . Red blood cells (RBCs) from rabbit and human



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Fraction number Fig . 1 . Chromatographic purification of cytotoxin from P. aeruginosa 158 . Column chromatography profiles for each step of purification procedure are shown . (a) DEAE-Sephadex A-50 chromatography of trypsinized crude extract . (b) Gel filtration chromatography on Superose TM12 HR16/60 column . (c) Mono Q anion exchange chromatography . (d) Phenyl-Superose hydrophobic interaction chromatography . 0, leukocytotoxic activity ; absorbance at 280 nm ; A A, in (a) and -in (c), NaCl concen0 tration . Vertical arrows in (b) indicate the elution volume of each molecular weight standard, exclusion volume (Vo) and inclusion volume (Ve) .

showed liberation of hemoglobin and became ghost-like at a higher concentration of the toxin . This weak hemolytic activity was not due to contamination by other pseudomonal hemolytic components, such as heat-labile hemolysin (phospholipase C) or heat-stable hemolysin, because no phospholipase C activity was detected in the purified preparation, and its hemolytic activity was inactivated by heating the toxin at 90°C for 5 min . Furthermore, the leukocytotoxic and hemolytic activities were co-

Table 1

Purification of cytotoxin from crude extract of P.

Step number and process 1 . Initial crude extracts 2 . DEAE-Sephadex A-50 chromatography 3 . Trypsin treatment and DEAESephadex A-50 chromatography 4 . Superose TM 12 chromatography 5 . Mono Q chromatography 6 . Phenyl-Superose chromatography *Prepared from the cells of 40 1 culture .

aeruginosa 158

Total volume (ml)

Total activity (10 3 units)

Total protein (mg)

530

21 200

21 863

970

4340

19 530

10156

1923

92 .1

8571 23 881 63158 80000

49 .2 33 .6 25 .8 12 .7

870 89 45 .5 45

10440 7120 5460 2700

1218 298 .15 86 .45 33 .75

Specific activity (units/mg)

Yield (%) 100



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T . Hayashi et al. a

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Table 2 Amino acid composition of purified cytotoxin

Amino acid Lys His Trp Arg Asp Thr Ser Glu Pro Gly Ala Cys Val Met Ile Leu Tyr Phe

Relative molar quantity

Residues/ mol

9 .65 7 .98 8 .68 6 .95 28 .43 23 .93 23 .37 34 .27 8 .28 26 .47 16 .15

10 8 9 7 28 24 23 34 8 26 16

22 .86 2 .93 9 .41 19 .60 10 .82 7 .53

23 3 9 20 11 8

Total residues Calculated molecular weight

Table 3

267 29 514

N-terminal amino acid sequence of purified cytotoxin

Met Asn Asp Ile Asp Thr X X X Ala Trp Gly Arg Trp Lys Thr Ala Gin 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 X designates a residue which was not identified .

Tyr 19

Gly Thr 21 20



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x 0 50 60 70 80 90 100 Elution volume (ml) Fig . 3 . Gel filtration chromatography of the purified cytotoxin . Purified cytotoxin (150 ug) was loaded on a Superose TM12 HR 16/60 column and eluted with 20 mm sodium phosphated buffer (pH 7 .0) containing 0 .15 M NaCl at the flow rate of 0 .3 ml/min . Symbols : o-o, hemolytic activity ; o-o, leukocytotoxic activity ; absorbance at 280 nm . Vertical arrows indicate the elution volume of each molecular weight standard, exclusion volume (Vo) and inclusion volume (Ve) . 0 30 40

eluted on gel filtration chromatography of the purified toxin (Fig . 3) . The purified toxin also caused similar morphological changes on L cells and HeLa cells . The sensitivities of these cells to the purified toxin were compared by determining the values of CD 50 under standardized conditions (Table 4) . Rabbit PMNs were most sensitive to the toxin, the CD 50 being 6 .25 ng . Human RBCs were the most resistant, that is, 1200-fold more of cytotoxin (7500 ng) was required to destroy them . Immunological properties of the purified cytotoxin Immunodiffusion analysis using antiserum raised against the purified toxin showed a single line of precipitate against either the purified toxin, the bacterial crude extract, the trypsinized crude extract or the concentrated autolysate of PA158 (data not shown) . This antiserum did not form any line of precipitate against exotoxin A, alkaline Table 4 Comparison of cytotoxic activities of cytotoxin on various cells Cells (108/20 µl) Rabbit PMN leukocyte Lymphocyte Red blood cell Alveolar macrophage Human PMN leukocyte Lymphocyte Red blood cell Cultured cells HeLa cell L cell

CD5 O (ng/20 µl)

6 .25 200 468 .25 58 .6° 100 12 .5 7500 46 .8` 93 .75`

CD 50 values were determined by at least three independent experiments except for alveolar macrophages. Determined by a single experiment . `Determined by incubation of 1 .5-2 .0x10 5 cells with 40 pl of serial dilution of cytotoxin in LAB .TEKTM Tissue Culture Chamber/Slides (Miles, #4808) .



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protease or elastase of P . aeruginosa . The anti-serum neutralized not only the leukocytotoxic activity of the purified cytotoxin but also that of PA158 autolysate to the same extent (data not shown) .

Discussion The present study described a new purification method of the cytotoxin of P. aeruginosa . Our 29 kDa toxin purified from the trypsin-treated crude extract of PA158 showed a single band on either SDS-PAGE or PAGE or IEF gel electrophoresis in contrast to the results of Scharmann s and Lutz .' Their cytotoxin purified from autolysates of PA158 showed a single band (27 .5 kDa and 25 .1 kDa, respectively) on SDS-PAGE but several bands on native PAGE or IEF . In the procedures described here, an active cytotoxin was obtained by trypsinization of the crude extract and purified by a series of chromatographic steps without ammonium sulfate precipitation . This method resulted not only in a high yield of cytotoxin but also in a highly homogeneous toxin preparation . Our purified toxin is derived from the same precursor of the cytotoxin purified by Scharmann and Lutz, on the following grounds . (i) Antiserum against our 29 kDa cytotoxin neutralized the leukocytotoxic activity in an autolysate of PA158 . (ii) The amino acid composition of the 29 kDa toxin showed a marked similarity to that of Lutz .' Recently, Baltch et al . detected a 29 kDa cytotoxin in trypsinized crude extracts from various clinical isolates of P . aeruginosa including PA158 . 14 They described that the purified cytotoxin of Lutz also migrated at the 29 kDa position . The leukocidin purified by Hirayama et al ." from an autolysate of PA158 showed a similar value of CD 50 against rabbit PMNs to that of ours . The leukocidin, however, seems to be different from our 29 kDa toxin because of large differences in the molecular weight (42 .5 kDa) and in the amino acid composition . Further studies are needed to clarify the relationships between these pseudomonal toxins . The purified 29 kDa toxin showed cytotoxic actions against various eukaryotic cells with diverse sensitivities . The most sensitive were rabbit PMNs and the most resistant were human RBCs . Rabbit PMNs were 30-fold more sensitive than lymphocytes, whereas human PMNs were 10-fold more resistant than lymphocytes . Other problems to be clarified are the structure and location of the inactive precursor and the mechanism of conversion of this precursor to the active form . Our attempts to purify this inactive precursor have not yet been successful . Cloning of the cytotoxin gene would be very helpful in revealing the primary structure of the precursor and the mechanism of its conversion into the active form at the molecular level . The Nterminal amino acid sequence, described in this study, will be useful in synthesizing oligonucleotide probes to screen for the gene on the PA158 chromosome . This study is in progress in our laboratory .

Materials and methods Bacterial strain and culture condition . Pseudomonas aeruginosa 158, kindly supplied by Dr Hirayama, Institute of Medical Science, University of Tokyo, was shown to produce cytotoxin by Scharmann 4 and was the same strain that Scharmann s and Lutz 5 used for the purification of cytotoxin . Medium for liquid cultures of the cells was tryptic soy broth containing 0 .5% glucose .' PA158 was inoculated from a fresh preculture into a 3 I flask containing a 500 ml medium . Cells were grown aerobically by shaking at 30'C and harvested at an early stationary phase by continuous centrifugation of 20000 g and washed twice with saline containing 0 .8 mm MgCIZ . Cell pellets were stored at -80'C until preparation of a crude extract .



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Purification of cytotoxin . All purification procedures were performed at 4°C except that chromatographic procedures on FPLC were carried out at room temperature . (1) Preparation of crude extract : the frozen cell pellets derived from a 40 I culture was suspended in 500 ml of 20 mm sodium phosphate buffer, pH 7 .0 (buffer A) containing 0 .1 mm NaCI and 1 mm EDTA . The cells were broken in a French pressure cell at 1000 kg/cm 2, and centrifuged at 15000 g for 20 min to remove unbroken cells . Subsequently the crude extract was clarified by centrifugation at 205700 g for 2 h to remove the cell debris and dialysed against buffer A . (2) DEAE-Sephadex A-50 chromatography of the crude extract : the dialysate was applied to a DEAE-Sephadex A-50 column (8x24 cm) pre-equilibrated with buffer A . The column was washed with 2000 ml of buffer A and then eluted with 2000 ml of buffer A containing 0 .5 M NaCI and 1000 ml of buffer A containing 1 M NaCl . Since approximately 40% of potent leukocytotoxic activity was found in the washed fraction, this fraction was again applied to the column and washed and eluted in the same manner . The fractions eluted by 0 .5 M NaCI gradient of the 1st and 2nd chromatography were pooled . (3) Trypsin treatment : the pooled fraction was concentrated on an Amicon PM-10 membrane and the preparation (7630 mg) was incubated with 200 mg of trypsin and 1 mm of CaCl 2 at 37°C for 2 h with gentle shaking . The trypsin-treated preparation was dialyzed against buffer A . (4) DEAE-Sephadex A-50 chromatography of trypsin-treated preparation : the dialysate was clarified by centrifugation and was applied to a DEAE-Sephadex A-50 column (6 .4x38 cm) equilibrated with buffer A . The column was washed with 4000 ml of buffer A and then eluted with a 4000 ml linear gradient of 0-0 .5 M NaCl in buffer A at a flow rate of 50 ml/h . Fractions with leukocytotoxic activity were pooled and concentrated on an Amicon filter PM-10 . (5) Superose TM12 gel filtration chromatography on FPLC : gel filtration chromatography using Superose TM12 HR 16/60 column was performed using a Pharmacia FPLC system . The preparation (2 ml in each time) was applied to the column equilibrated with 20 mm Tris-HCI, pH 7 .5 (buffer B) containing 0.15 M NaCl, and eluted with the same buffer at a flow rate of 0 .3 ml/min . Fractions with leukocytotoxic activity were pooled and dialysed against buffer B . (6) Mono Q anion exchange chromatography on FPLC : the dialysate was applied to a Mono Q HR 5/5 column equilibrated with buffer B . For the best resolution, 1 ml of the preparation (3 .35 mg protein) was applied in each time . After washing the column with 10 ml of buffer B, proteins were eluted with a linear gradient of 0 .1-0 .2 M NaCI in buffer B at a flow rate of 1 ml/min . Fractions with leukocytotoxic activity were collected and dialyzed against 50 mm sodium phosphate buffer (pH 7 .0) containing 100 mm ammonium sulfate . (7) Phenyl-Superose hydrophobic interaction chromatography on FPLC : the dialysate was centrifuged at 20000 g for 20 min to remove aggregated substances and then applied to a Phenyl-Superose HR 10/10 column pre-equilibrated with 50 mm sodium phosphate buffer (pH 7 .0) containing 100 mm ammonium sulfate . For the best resolution, 1 ml of the dialysate (2 mg protein) was applied in each time . Column was washed with 8 ml of the equilibrated buffer and eluted with 100 mm sodium phosphate buffer (pH 7 .0) at a flow rate of 1 ml/min . Toxincontaining fractions were pooled and concentrated on an Amicon filter PM-10 . The final material was dialysed against 0 .1 M sodium phosphate buffer (pH 7 .0) containing 20% (w/v) glycerol and stored at -20°C . The cytotoxin could be stored in this manner for at least one year without detectable loss of the activity . Leukocytotoxic activity assay . In the course of purification, each preparation was monitored for its leukocytotoxic activity. The leukocytotoxic activity was assayed by the slide adhesion method using rabbit PMNs as the target cells ." Rabbit PMNs were partially purified from peripheral blood by sedimentation in Dulbecco's phosphate-buffered saline (PBS) containing 0 .8% polyvinyl alcohol . The cells were washed twice with saline and suspended in PBS (1 x 105 leukocytes/mm 3) . An aliquot (20 µl) of the cell suspension was placed on a glass slide and kept for 30 min at room temperature in a moist chamber to make PMNs adhere to the surface of the glass and then gently washed with PBS to remove RBCs and lymphocytes . About 70% of the cells on the glass slide were PMNs (approximately 106 PMNs/spot) . Excess buffer was removed with tissue paper and a 20 it of serial dilutions of the toxin preparation in PBS containing 0 .5% gelatin as a stabilizing agent were added to the spot . After incubation for 1 h at 37°C in a moist chamber, morphological changes of PMNs were observed using a phase contrast microscopy . An end point was determined as the highest dilution of the toxin preparation that caused morphological changes in 50% of PMNs in the preparation . The leukocytotoxic activity was expressed in units-the number of units in a toxin preparation being



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numerically equal to the dilution (in ml) at the end point . Since the cytotoxic activity in the crude extract was quite weak without trypsin treatment, the potent activity in such a preparation was determined after a small portion of the preparation was treated with trypsin (40 µg/mg sample protein) at 37 ° C for 2 h . In this case, trypsin inhibitor was not used because incubation of PMNs with trypsin (100 pg/ml) caused no morphological changes at least within 1 h, and no detectable change was observed in the leukocytotoxic activity with or without inhibitor . Measurement of cytotoxic activities of the purified cytotoxin . PMNs and lymphocytes of rabbit and human were prepared from venous blood on isokinetic Ficoll gradient ." Rabbit alveolar macrophages were prepared by the method of Myrvik et al." Purity and viability of the prepared cells were at least more than 90% as determined by Wright-Giemsa or esterase staining and trypan blue dye exclusion test . RBCs were prepared from heparinized venous blood . Cells were washed twice with saline and suspended in PBS at a concentration of 5x104 cells/mm 3 . The cell suspension containing 10 6 cells was placed on a glass slide, and incubated with equal volume of serial dilution of purified toxin, and cytotoxic activity was determined in the same manner as described in leukocytotoxic activity assay . The CD 50 value was determined as the smallest amount of the toxin required to cause the morphological changes in 50% of the cells . HeLa and L cells were grown on a tissue culture chamber/slide (Miles #4808) containing Eagle's Minimum Essential Medium supplemented with 10% fetal calf serum . To examine a cytotoxic effect of the toxin, the culture medium was replaced by PBS and cells (1 .52 .0x105 /chamber) were incubated with 40 µl of serial dilution of the purified toxin at 37°C for 1 h . Gel filtration chromatography of the purified cytotoxin . The purified toxin (150 ug) was loaded on a Superose TM12 HR 16/60 column equilibrated with 20 mm Tris-HCI (pH 7 .5) containing 0 .15 M NaCl, and eluted with the same buffer at the flow rate of 0 .3 ml/min . The leukocytotoxic and hemolytic activities of column fractions (1 ml) were determined . For the gel filtration chromatography in a denatured condition, the purified toxin (150 µg) was dialysed against 1 M Tris-HCI (pH 8 .5) containing 6 .5 M guanidine hydrochloride . The toxin was, then, reduced by the incubation with 50 mm DTT for 1 h at room temperature, followed by incubation with 100 mm monoiode acetoamide for 1 h . The toxin was applied to the column equilibrated with 6 M guanidine hydrochloride and eluted with the same solution at 0 .3 ml/min . Polyacrylamide gel electrophoresis. SDS-PAGE was performed as described by Laemmli 17 on a 12% acrylamide separating slab gel . PAGE in the absence of SDS was performed on a 9% acrylamide slab gel in an identical manner and proteins were not boiled before being loaded . Gels were stained with Coomassie brilliant blue R 250 . With a non-denaturing PAGE of the purified toxin, the gel, run in parallel, was cut into 2 mm width and the leukocytotoxic activity of each section was measured . Isoelectric focusing . Isoelectric focusing of the purified toxin was performed on a slab gel according to the method of Ames and Nikaido . 78 Before staining the gel, pH gradient was determined using a surface grass electrode (LKB 2117-111) at a room temperature . Amino acid analysis. The amino acid composition of the purified toxin was determined by Hitachi type-835 analyzer using samples hydrolysed in 4 M methanesulphonic acid at 110°C for 24 h in evacuated sealed tubes . 17 N-terminal sequence determination . The N-terminal amino acid sequence was determined by automated Edman degradation using a gas phase protein sequencer (model 470A, ABI Co .), equipped with on-line PTH amino acid analyzer (model 120A PTH analyzer, ABI Co .) . Determination of protein and carbohydrate. Protein was determined by the method of Lowry et al. 20 with bovine serum albumin as the standard . Carbohydrate content was determined by gas chromatography according to the method of Clamp et al 21 Measurement of enzymatic activities . Proteolytic activity was measured using denatured casein as substrate . 22 Activity of phospholipase C was assayed using p-nitrophenylphosphorylcholine as substrate .23,24 Preparation of antiserum . A rabbit was injected intradermally with 10 µg of the purified



Pseudomonal cytotoxin

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cytotoxin mixed with an equal volume of Freund's complete adjuvant (Difco) . After a 4 week interval, 50 ug of the toxin mixed with an equal volume of Freund's complete ajuvant was administered two times . Two weeks after the last immunization, the rabbit was sacrificed and its serum was separated .

Immunodiffusion test . Double immunodiffusion tests were performed according to the method of Ouchterlony."

Preparation of the bacterial autolysate . The autolysate of PA158 was prepared according to the method of Hirayama et aI. 13 and concentrated on an Amicon filter PM-10 . It contained 1000 units/ml of leukocytotoxic activity . Materials. All chromatographic materials were obtained from Pharmacia . Trypsin (TPCK treated Type XIII) was obtained from Sigma . Molecular weight standard for SDS-PAGE and gel filtration chromatography was purchased from BDH Ltd, UK . It contained lactate dehydrogenase (M,=145900 . M r of subunits = 36 500), ovotransferrin (78 000), ovalbumin (45000), carbonic anhydrase (30000), myoglobin (17200) and cytochrome C (12300) . Purified elastase and alkaline protease of P. aeruginosa were obtained from Nagase Biochemical Co . Ltd, Japan . Purified exotoxin A, anti-exotoxin A, anti-elastase and anti-alkaline protease serum were kindly provided by Dr Y . Homma, the Kitasato Institute . All other chemicals were analytical reagent grade .

The authors thank Dr F . Sakiyama, Institute for Protein Research, Osaka University for amino acid analyses and Dr T. Hirayama, Institute of Medical Science, University of Tokyo and Dr I . Kato, School of Medicine, Chiba University, for advice on leukocytotoxic activity assay . We are very grateful to Drs Y . Yamada and T . Baba for cell culture and Drs H . Matsumoto and Y . Itoh for valuable suggestions . We also thank Mr A . Rahaman, Tohoku University, for his critical reading of the manuscript . This research is supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan .

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15 . Pretlow TG, Luberoff DE . A new method for separating lymphocytes and granulocytes from human peripheral blood using programmed gradient sedimentation in an isokinetic gradient. Immunology 1973;24 :85-92 . 16 . Myrvik QN, Leake BS, Fariss B . Studies on pulmonaly alveolar macrophages from the normal rabbit : a technique to procure them in a high state of purity . J Immunol 1961 ; 86 :128-32 . 17 . Laemmli UK . Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 1970 ; 227 : 680-5 . 18 . Ames GFL, Nikaido K . Two dimensional gel electrophoresis of membrane proteins . Biochemistry 1976 ; 15 :616-23 . 19 . Simpson RJ, Neuberger MR, Lie TY . Complete amino acid analysis of proteins from a single hydrolysate . J Biol Chem 1976 ; 251 : 1936-40. 20 . Lowry OH, Rosebrough NJ, Farr AL, Randall RJ . Protein measurement with the Folin phenol reagent . J Biol Chem 1951 ; 193 : 265-75 . 21 . Clamp JR, Dawson G, Hough L . The simultaneous estimation of 6-deoxy-L-galactose (L-fucose), Dmannose, D-galactose, 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine) and N-acetylneuraminic acid (sialic acid) in glycopeptides and glycoproteins . Biochim Biophys Acta 1967 ; 148 : 342-9 . 22 . Wretlind B, Wadstrom T . Purification and properties of a protease with elastase activity from Pseudomonas aeruginosa . J Gen Microbiol 1979 ; 103 : 319-27 . 23 . Berka RM, Gray GL, Vasil ML . Studies of phospholipase C (heat-labile hemolysin) in Pseudomonas aeruginosa . Infect Immun 1981 ; 34 : 1071-4 . 24 . Kurioka K, Matsuda M . Phospholipase assay using p-nitrophenylphosphorylcholine together with sorbital and its application to studying the metal and detergent requirement of the enzyme . Anal Biochem 1976 ; 75: 281-289 . 25 . Ouchterlony 0 . Antigen-antibody reactions in gel . Acta Pathol Microbiol Scand 1949 ; 26 : 132-142 .