THE JOURNAL OF Bmmcrca~ CHEMISTRY Vol. 2.54, No 21, Issue of November 10, pp. IloGi-11009, 1979 Printed m U.S.A.

Separation and Purification of a Potent Bactericidal/Permeability- increasing Protein and a Closely Associated Phospholipase A, from Rabbit Polymorphonuclear Leukocytes OBSERVATIONS ON THEIR RELATIONSHIP*

(Received for publication, May 7, 1979)

Peter Elsbach,$ Jerrold Weiss, Richard C. Franson, Susan Beckerdite-Quagliata, Aviva Schneider, and Lesley Harris

From the Department of Medicine, New York University School of Medicine, New York, New York

Two antibacterial proteins from rabbit polymorpho- nuclear leukocytes, a potent bactericidal cationic pro- tein that increases the envelope permeability of suscep- tible gram-negative bacteria and a phospholipase AZ, have been purified to near homogeneity by ion ex- change, gel filtration, and hydrophobic interaction chromatography. The apparently noncatalytic bacte- ricidal/permeability-increasing protein has an approx- imate molecular weight of 50,000 and is isoelectric at pH 9.5 to 10.0. The molecular properties, including amino acid composition, and the antibacterial potency and specificity of this rabbit leukocyte protein and of the bactericidal/permeability-increasing protein from human granulocytes that we have recently purified (J. Biol. Chem. 253, 2664-2672, 1978) are closely similar. Both proteins kill several strains of Escherichia coli and Salmonella typhimurium. Rough strains are more sensitive than smooth strains. All gram-positive bac- terial species tested are insensitive to high concentra- tions of either rabbit or human protein.

The phospholipase Aa, purified by hydrophobic inter- action chromatography on phenyl-Sepharose, ran as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 14,000 and had a specific enzymatic activity compa- rable to that of purified phospholipases A2 from other sources.

Separation of the phospholipase AZ from the bacte- ricidal/permeability-increasing protein has no notice- able effect on the bactericidal and permeability-in- creasing activities of the purified bactericidal protein, but removes the ability of the phospholipase AZ to hydrolyze the phospholipids of intact Escherichia coli. Upon recombination of the phospholipase AZ with the bactericidal/permeability-increasing protein, the phospholipase AZ regains its activity toward the phos- pholipids of intact E, coli suggesting that these two antibacterial leukocyte proteins act in concert.

We have previously reported the partial purification of a potent bactericidal protein fraction isolated from rabbit polymorphonuclear leukocytes that also possessed permeabil-

* This study was supported by United States Public Health Service Grant AM 05472. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 To whom correspondence regarding this article should be ad- dressed.

ity-increasing and phospholipase A, activities toward the en- velopes of susceptible Escherichia coli (l-3). We now report separation of the bactericidal/permeability-increasing protein from the phospholipase A2 and the further purification of the two proteins to near homogeneity. This separation results in the loss of the ability of the phospholipase A2 to hydrolyze the phospholipids of intact E. coli, but has no detectable effect on the bactericidal and permeability-increasing activities of the bactericidal protein. Recombination of the phospholipase A2 with the bactericidal/permeability-increasing protein re- stores the ability of the leukocyte phospholipase AZ to degrade the phospholipids of intact E. coli, suggesting that the two proteins act in concert as antimicrobial agents.

The molecular properties, the biological potency, and the antibacterial specificity of the rabbit leukocyte bactericidal/ permeability-increasing protein described herein and of the previously described bactericidal/permeability-increasing protein purified from human polymorphonuclear leukocytes (4) are closely similar.

EXPERIMENTAL PROCEDURES

Materials-Carboxymethyl-Sephadex C-50, Sephadex G-50.super- fine, Sephadex G-100 medium, phenyl-Sepharose (CL 4B), and blue dextran 2ooO were purchased from Pharmacia Fine Chemicals AB. L-[l-‘%]Leucine (48 Ci/mol) was obtained from International Chem- ical and Nuclear Corp. and [l-‘4C]oleic acid (60 Ci/mol) from Amer- sham Searle Corp. Fatty acid poor bovine serum albumin, Fraction V, was bought from Pentex, Miles Research Products. Actinomycin D was sunnlied bv Merck Sham & Dohme. Acrvlamide. N.N’-meth-

__ I

ylenebisacrylamide, Tris, and bromphenol blue of electrophoretic purity grade were obtained from Bio-Rad Laboratories. Proteins used as standards for gel filtration and disc gel electrophoresis were bovine serum albumin (Miles Laboratories, Inc.), ovalbumin, chymotrypsin- ogen A, and ribonuclease A (Pharmacia Fine Chemicals). Sodium dodecyl sulfate (SDS,’ Sequanal grade) was obtained from Pierce Chemical Co. and sucrose (ultrapure, density gradient grade) from Schwarz/Mann. Ampholine-PAGplate (pH range 3.5 to 9.5) was obtained from LKB-Produkter AB and agarose universal electropho- resis film from Corning. Casamino acids were bought from Difco Laboratories and Hanks’ balanced salt solution from Microbiological Associates. Thin layer chromatography was performed on Silica Gel F254 plates supplied by Brinkmann Instruments. All other chemicals were of reagent grade and were obtained from the usual sources.

Polymorphonuclear Leukocytes-Polymorphonuclear leukocytes were obtained from overnight, sterile peritoneal exudates produced in rabbits, no more than once weekly, by injection of glycogen in physiological saline as described previously (5), except that no heparin was added to the collection flask. More than 95% of the cells were granulocytes as judged by differential cell counts.

Extraction of Bactericidal, Permeability-increasing, and Phos- pholipase A2 Actiuities-Leukocytes, sedimented by centrifugation

’ The abbreviation used is: SDS, sodium dodecyl sulfate.

11000

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Bactericidal Protein and Phospholipase AZ from Rabbit Leukocytes 11001

at 50 x g for 10 min, were resuspended in distilled water (3 x 10H cells/ml) and homogenized at 0°C in a glass homogenizer using a motor-driven Teflon pestle (1). Ice-cold 0.4 N HSO, was then added to a final concentration of 0.16 N HSO,. Extraction at 0°C for 30 min during periodic agitation and subsequent neutralization of the extract during prolonged dialysis against 1 mM Tris-HCl (pH 7.3) were carried out as recently described (1,6) Dense precipitates that formed during dialysis were removed by centrifugation at 23,OCO x g for 20 min. The remaining clear supernatant fluid in 1 ITIM Tris-HCl (pH 7.3) usually contains from 10 to 20% of the total leukocyte protein (Lowry method (7)) and exhibits at least as much bactericidal, permeability-increas- ing, and phospholipase Az activities per cell equivalent as do whole homogenates (1). These extracts stored at 4°C retain their full bio- logical activity for at least six months.

Chromatographic Methods-Ion exchange chromatography was performed at 4°C on carboxymethyl-Sephadex C-50 that had been swollen in 0.5 M Tris-HCl (pH 7.5) and equilibrated and degassed in 1 IIIM Tris-HCl (pH 7.3). Details are described in the legend to Fig. 1.

Gel filtration chromatography was performed on Sephadex G-50 (superfine) and Sephadex G-100 (medium) at 4°C. The Sephadex beads were prepared according to the instructions of the manufacturer and equilibrated in the elution buffer. The gel slurries were degassed before packing the columns. Both packing of and elution from col- umns were carried out under a hydrostatic pressure of 15 to 25 cm H,O. Details are given in the legends to Figs. 2 and 3.

Hydrophobic interaction chromatography was carried out on phenyl-Sepharose CL 4B at room temperature (approximately 20°C). The gel was washed with 10 volumes of each solvent used for elution before applying protein. Details are described in the legend to Fig. 7.

Concentration of Protein-The 0.8 M NaCl fraction obtained by carboxymethyl-Sephadex chromatography was dialyzed against dis- tilled water and concentrated by ultrafiltration at 4°C on Amicon Diaflo PM-30 membranes (Amicon Corp., Lexington, MA). The void volume fractions obtained by Sephadex G-50 chromatography were similarly concentrated on PM-IO membranes.

Analytical Polyacrylamide Slab Gel Electrophoresis-Electro- phoresis in 12% polyacrylamide slab gels (l-mm thick) in the presence of SDS was performed according to the method of Neville (8). Dialyzed and lyophilized protein samples were resuspended in 40 ~1 of water containing 12.5 mrvr dithiothreitol and 1.5% SDS, incubated for 45 to 60 min at room temperature, and applied after addition of 5 d each of 67% sucrose and 0.1% bromphenol blue. Electrophoresis was carried out at 15 mA. The gels were fixed and stained in a solution consisting of 0.25% Coomassie brilliant blue in 25.7% methanol and 9.2% glacial acetic acid for 18 h at 37°C. The gels were destained by washing in a solution containing 35% ethanol and 5% glacial acetic acid.

Preparative Polyacrylamide Slab Gel Electrophoresis-Electra- phoresis was carried out exactly as described above except that thicker gels (2 or 3 mm) were used. All glassware used was acid washed. After electrophoresis, a thin vertical strip was taken and stained in a solution consisting of 0.4% Coomassie blue G-250 (Sigma Co.) in 3.5% perchloric acid at 37’C. Protein bands were visible within 30 min. Horizontal strips, corresponding to the location of the stained protein bands, were cut from the remainder of the slab gel which had been kept at 4°C. These strips were cut into small gel pieces (<2 mm3) and incubated with approximately 2 volumes of 0.1% SDS solution at 37°C. After 3 h, the supernatant was carefully removed and the above procedure was repeated three times. The combined 0.1% SDS eluate was treated with 3 volumes of chloroform/methanol (1:2) to selectively precipitate the protein, thereby eliminating acryl- amide, gel buffer constituents, and SDS which were not precipitated. The purity and approximate recovery of protein in this precipitate were determined by analytical SDS-polyacrylamide gel electropho- resis. Protein recovery was >75%. The protein precipitate was used directly for amino acid analysis (see Table III).

Estimation of Net Charge of Purified Protein-Isoelectric focusing was carried out on commercial (LKB, Ampholine PAGplate) poly- acrylamide slab gels, containing ampholytes in the pH range 3.5 to 9.5, according to the instructions of the manufacturer. Electrophoresis on 1% agarose gels (Corning, Universal Electrophoresis Film) was carried out in 0.05 M barbital buffer (pH 8.6) at 3 mA/lane.

Amino Acid Analysis-Analyses were performed in a Durrum D- 500 amino acid analyzer.

Bacteria-E. coli strains S15 and S17 (thii, leu-, thr-, lac-, mel-(X)) were kindly donated by Professor S. Nojima (Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan), E. coli

C600 (F-, thii’, thr-I, leu?, lacY1, tonA21, supE44, X-, lac Y-, 1acZ’) by Dr. Barbara J. Bachman (Curator, E. coli Genetic Stock Center, Department of Human Genetics, Yale University School of Medicine, New Haven, CT), E. coli ML308 225, and Listeria monocytogenes by Dr. Milton Salton and Dr. Joel Oppenheim (Department of Micro- biology, New York University School of Medicine, New York, NY). Salmonella typhimurium G-30 was the gift of Dr. Mary J. Osborn (Department of Microbiology, University of Connecticut Health Cen- ter, Farmington, CT) and S. typhimurium MS 395 and rough mutants derived from it (R5 and RlO) were given by Dr. Olle Stendhal (Department of Medical Microbiology, University of Linkoping, Lin- koping, Sweden) and other bacterial strains by Dr. Michael S. Sim- berkoff (Department of Microbiology, Veterans Administration Hos- pital, New York, NY).

Growth in culture was either in a triethanolamine-buffered (pH 7.75 to 7.9) minimal salts medium (9) (all E. coli strains except CSOO) or in nutrient broth (pH 7.3) (E. coli C600, S. typhimurium, Staphy- lococcus aureus, Bacillus subtilis, Micrococcus lysodeikticus, Strep- tococcus faecalis). Bacterial cultures grown overnight to stationary phase were transferred to fresh medium (diluted 1:lO) and the sub- cultures were incubated approximately 3 to 4 h at 37°C. All micro- organisms were harvested and used for assay of bactericidal and permeability-increasing activities during midlate logarithmic growth phase (6 to 10 x lO”/ml). Microbial concentrations were determined by measuring absorbance at 550 nm with a Coleman junior spectro- photometer. The microorganisms were sedimented by centrifugation at 6000 x g for 10 min and resuspended in sterile physiological saline in the desired concentration.

Labeling of E. coli phospholipids-Bacterial phospholipids were labeled during growth in subculture. Aliquots of an overnight culture of E. coli (S15 or S17) grown in triethanolamine medium as described above were diluted 1:lO in fresh medium. After incubation for 75 min at 37’C, aliquots of the subculture were transferred to flasks contain- ing 0.2 pCi/ml of [1-“Cloleic acid complexed with 0.02% bovine serum albumin (fatty acid poor). After incubation for 75 min at 37’C, the bacteria were sedimented by centrifugation at 6090 x g for 10 min, resuspended in fresh triethanolamine medium, and reincubated for 30 min to permit the remaining unincorporated labeled precursor to be incorporated. The labeled bacteria were washed with 1% albumin, resuspended in saline, and used in one of two ways: 1) as live organisms (Fig. 5; Table VIII), to examine the effect of leukocyte protein fractions on the envelope phospholipids of intact E. coli, caused either by phospholipase activity in a given leukocyte fraction, and/or by activation of bacterial phospholipases (2, 3, 10, 11); 2) after autoclav- ing for 15 min at 120°C and 2.7 kg/cm’. This procedure inactivates heat-stable bacterial phospholipases (10) and renders the envelope phospholipids readily accessible to the action of added phospholipase A. We have found the autoclaved 14C-fatty acid labeled E. coli to be a convenient and economical mixed phospholipid substrate for the highly reproducible assay of phospholipases from numerous sources.

Assays for Permeability-increasing and Bactericidal Actiuities- Typical incubation mixtures contained 5 x 10” to 5 X 10” bacteria in a total volume of 0.4 ml of sterile physiological saline that also contained 10 pmol Tris-HCl buffer (pH 7.0), 25 ,td of Hanks’ solution (without phenol red), 250 pg of vitamin-free casamino acids, and leukocyte protein fraction in the indicated amount. Incubations were carried out at 37°C for 30 min.

Assay for Increased Bacterial Envelope Permeability-An effect of leukocyte fractions on the permeability of gram-negative bacteria was measured by determining the susceptibility of these bacteria to actinomycin D. The use of actinomycin D for assays of envelope permeability is based on the following observations. 1) Bactericidal concentrations of purified leukocyte fractions permit macromolecular synthesis by E. coli (and other gram-negative bacteria) to continue at normal or near normal levels for at least 2 h (l-3). 2) Gram-negative bacteria normally insensitive to the inhibitory effect of actinomycin D on RNA and protein synthesis because their envelope is imperme- able to the drug (12) become susceptible to its action within minutes after exposure to leukocyte fractions (1, 2, 6). Because the effect of bactericidal and permeability-increasing protein fractions on render- ing gram-negative bacteria susceptible to inhibition by actinomycin D is dose dependent, quantitative determination of permeability- increasing activity is possible (1). Assays were carried out in the incubation mixture described above by determining bacterial incor- poration of [“Clleucine (0.063 PCi, 0.13 mM) into cold trichloracetic acid-precipitable material, in the presence and absence of actinomycin D (12.5 pg) as previously described (1, 6). The same results are

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11002 Bactericidal Protein and Phospholipase AZ from Rabbit Leukocytes

obtained using [‘?]uracil as precursor (6). The permeability effect of a given leukocyte fraction is measured by calculating the inhibition of bacterial protein synthesis specifically attributable to actinomycin D using the following equation:

[

Bacterial [?Jleucine incorporation + leukocyte fraction + actinomycin D

l- 1 x 100 Bacterial [‘VJleucine incorporation

+ leukocyte fraction - actinomycin D

One arbitrary unit of permeability-increasing activity has ,been de- fined as a 50% reduction by actinomycin D of [“‘Clleucine incorpo- ration by 2.5 x 10” bacteria. E. coli S15 was used for determination of this biological activity of protein fractions obtained during purifica- tion.

Assay for Bactericiclal A&u&y-After 30-min incubation, lo-p1 samples were taken from the incubation mixtures, serially diluted in sterile isotonic saline, and plated on either nutrient agar (E. coli, S. typhimurium, B. subtilis) or brain heart infusion agar (all strains). Sensitivity to the microbicidal activity of the purified fraction ob- tained in this study was not affected by the agar medium used for colony formation. The number of colony-forming units on the plates was determined after incubation at 37°C overnight (all bacterial strains except M. lysodeikticus) or 2 to 3 days at room temperature (M. lysodeikticus)

Assay for Leukocyte Phospholipase Al Activity (1,13)-Leukocyte fractions were incubated with 2.5 x 10” autoclaved [‘4C]oleic acid- labeled E. coli (S15) (approximately 5 nmol of phospholipid) in a total volume of 0.5 ml also containing 40 prnol Tris maleate buffer (pH 7.5) and 5 pmol of CaC12. Incubations were carried out at 37°C for time periods of up to 15 min. Phospholipid hydrolysis increases approximately linearly with increasing time and/or protein fraction added until hydrolysis reaches 20 to 25%. Hydrolysis by leukocyte fractions, starting with neutralized acid extracts, is maximal between pH 7.0 and 8.0; activity falls sharply below pH 6.0 and above pH 8.0. The pH optimum and kinetics (14) of hydrolysis by the leukocyte phospholipase A2 remain essentially the same during progressive purification. The labeled product of hydrolysis is almost exclusively “‘C-free fatty acid, consistent with the AZ specificity of leukocyte phospholipase A (3, 13). One arbitrary unit of phospholipase activity has been defined as 1% hydrolysis/h.

Lipid Extraction and Fractionation-Lipids were extracted ac- cording to the procedure of Bligh and Dyer (15). The removed aqueous methanolic upper phase was washed once with 0.5 volume of CHCl:, to optimize recovery of monoacylphosphatides. The combined CHCL extracts were dried under a nitrogen stream, redissolved in 0.1 ml CHCL/CH:IOH (2:1), and transferred to commercial Silica Gel F254 plates (Brinkmann Instruments, Westbury, NY). Monoacyl- phosphatides, diacylphosphatides, and fatty acids were separated in a solvent system consisting of chloroform/methanol/distilled water/ glacial acetic acid (65:25:4:1, v/v). Under experimental conditions giving rise to “C-labeled free fatty acids as the only labeled product of hydrolysis, chromatography was carried out in a solvent system consisting of petroleum ether/diethyl ether/glacial acetic acid (8O:ZO: 1, v/v) which separates phospholipids and free fatty acids (13). Lipid species were identified by comparison of RF with that of authentic standards after visualization following exposure of the plates to iodine vapors. Liquid scintillation counting of thin layer fractions scraped off the plates into counting vials was carried out as described previ- ously (13).

RESULTS

Purification of Bactericidal/Permeability-increasing Protein

Ion Exchange Chromatography-Carboxymethyl-Sepha- dex chromatography of acid extracts of polymorphonuclear leukocytes is an effective step in the purification of a protein fraction with bactericidal, permeability-increasing, and phos pholipase Al activities toward several gram-negative bacterial species (1). In the present study the elution procedure was modified by using a stepwise increase in NaCl concentration in place of a linear NaCl gradient. This modification has rendered this chromatographic step reproducible regardless of the protein load applied.

Although the cytoplasmic granules contain the bulk of the bactericidal, permeability-increasing, and phospholipase AZ activities of the polymorphonuclear leukocytes (l), we have elected to use as starting material acid extracts of whole homogenates because these yield approximately three times more of the three activities than do extracts of isolated gran- ules. The protein fraction eluted with 0.8 M NaCl (Fig. 1; Fraction G) contains almost all bactericidal/permeability-in- creasing and phospholipase Az activities recovered in the whole eluate. The specific activities of the Fraction G derived from whole homogenate and from isolated granules are com- parable, indicating that no major components derived from

0 0

06 08 20

1,500 ,” LD z- 2. 3

750 2

1

0

Fraction Number

FIG. 1. Sephadex chromatography of acid extract of rabbit polymorphonuclear leukocytes. Acid extract of rabbit leukocyte homogenates (70 mg of protein) previously dialyzed against 1 mM

Tris/HCl buffer, pH 7.3, was applied to a carboxymethyl-Sephadex column (1.5 x 30 cm) equilibrated with the same buffer. Elution was carried out with stepwise increasing concentrations of buffered so- dium chloride. Fractions of 12.5 ml were collected and promptly dialyzed against distilled water to remove salt before biological assays. Protein, bactericidal (not shown), permeability-increasing (PI), and phospholipase AS (PLA) activities were determined as described under “Experimental Procedures.” Protein recovery was approxi- mately 85%.

0 io too 150 260

Elution Volume (ml)

FIG. 2. Sephadex G-50 chromatography of Fraction G ob- tained by carboxymethyl-Sephadex chromatography (Fig. 1). Pooled Fractions G from several carboxymethyl-Sephadex chromat- ographic separations, containing 8.9 mg of protein, were concentrated as described under “Experimental Procedures” and applied in 2.0 ml of column buffer to a Sephadex G-50 (superfine) column (1.6 X 85 cm) equilibrated in 1.0 M NaCl containing 2.5 mM Tris-HCl buffer, pH 7.5. Elution with column buffer was carried out at a constant flow rate of 13 ml/h maintained by a peristaltic pump. Fractions of 2.0 ml were collected. Protein, bactericidal (not shown), permeability-in- creasing, and phospholipase AZ activities were determined as de- scribed under “Experimental Procedures.” Protein recovery was >95%. The arrows indicate the elution volumes of blue dextran 2000 ( VO) and chymotrypsinogen A standards. Inset, SDS-polyacrylamide gel electrophoresis of pooled Sephadex G-50 void volume fractions containing bactericidal and permeability-increasing activities. Elec- trophoresis was performed as described under “Experimental Proce- dures.” Fifteen micrograms of protein was applied. Protein migration was toward bottom of gel (anode).

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Bactericidal Protein and Phospholipase AP from Rabbit Leukocytes 11003

TABLE I

Purification of bactericidal/permeability-increasing protein from rabbit polymorphonuclear leukocytes

Rabbit polymorphonuclear leukocytes (2.1 x lo”‘), pooled from several collections, were used as starting material for purification. Protein, permeability-increasing, and bactericidal activities were determined as described under “Experimental Procedures.” The values shown represent the mean of at least three independent assays carried out in duplicate.

Purification step

Homogenate 0.16 N H2S04 extract Carboxymethyl-Sephadex

chromatography Sephadex G-50 chromatogra-

pb

Protein

mt? %

4200 100 680 16

34.2 0.8

10.0 0.2

Permeability-increasing activ- Specific activity ity Purification Ractericidal activity”

units

6.3 x IO’” 1; unLls/mg ~fdrc

1.5 1 125-250’

6.6 x lo” 105 9.7 6.5 20-30 3.5 x 10” 56 103’ 69 2.5-3.5

1.1 x lo” 17 110” 73 2.5-3.5

Sephadex G-100 chromatog- raphy

4.3 0.1 7.9 x 10’ 13 183 122 1.5-2.5

n Micrograms of protein required to kill more than 90% of 5 x 10’ B. coli S15. ’ Data obtained from earlier publications (1, 6). ’ In eight different preparations, the mean specific activity is 110 f 8.8 (SE.) units/mg of protein. ” In five different preparations, the mean specific activity is 110 t 6.2 (SE.) units/mg of protein.

nongranule constituents contaminate Fraction G from whole homogenates or substantially contribute to its activities.

Vo 43K 14K Vt

Gel Filtration-The bactericidal and permeability-increas- ing activities can be completely separated from the phospho- lipase A2 activity by Sephadex G-50 chromatography of Frac- tion G (Fig. 2). All eluted bactericidal and permeability-in- creasing activities appear in the void volume. The phospho- lipase AZ activity is retarded and is eluted as a discrete peak that coincides with a small portion of the recovered protein. Despite the fact that the protein in the void volume represents only 33 + 2.2% (mean + SE. of eight chromatographic sepa- rations) of the applied protein (recovery 96 +- 4.0%), its specific permeability-increasing and bactericidal activities are the same as those of Fraction G (Table I). Available evidence does not permit us to distinguish between 1) partial dissocia- tion of an active complex into inactive components to account for the proportionate loss of total activity and protein in the active fraction and 2) the removal of an activator. In the latter case the constant specific activity would have to be fortuitous.

25 31 38 65

Fraction Number

Apparent dissociation might be caused by proteolytic activ- ity in carboxymethyl-Sephadex Fraction G (Fig. 1). We have found no evidence of such activity using a range of artificial substrates (including tosyl-arginine methylester, tosyl-lysine methylester, andp-nitrophenylacetate) or biosynthetically la- beled leukocyte or microbial protein. Moreover, during incu- bation for 2 h at 37°C of biosynthetically labeled [Yllysine and [‘%]arginine Fraction G, no acid-soluble radioactivity is released. We have also found no support for the presence of

an activator in Fraction G because recombination of the protein in the void volume with protein that entered the column (either the total pool or subfractions) does not increase the total bactericidal or permeability-increasing activity.

FIG. 3. Sephadex G-100 chromatography of pooled Sepha- dex G-50 void volume fractions. The pooled fractions (10 mg of protein) were applied in 2.5 ml of column buffer to a Sephadex G-100 (medium) column (1.5 x 84 cm) equilibrated in 2.5 mrvr Tris-HCl- buffered 1.0 M NaCl (pH 7.5). Elution and fraction collection were carried out as described in the legend to Fig. 2. Protein recovery was >95%. The arrows indicate elution volumes of blue dextran 2000 ( V,,), ovalbumin (43K), ribonuclease A (14K), and l -DNP-lysine ( V,).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the void volume fraction (Fig. 2, inset) shows two compo- nents with apparent molecular weights of approximately 50,000 and 12,000. The nonentry into the column of the low molecular weight component and the nearly constant specific activity of the fractions across the peak suggest that these two components form a complex. This apparent complex mostly dissociates upon rechromatography of the void volume frac- tion (Fig. 2) on Sephadex G-100 in 1.0 M NaCl (Fig. 3). A single protein peak is obtained with apparent molecular weight of 50,000 f 1,000 (mean & S.E.; ~6). Evidence will be presented in the following sections that this chromatographic step yields the bulk of the 50,000-dalton protein free of the 12,000-dalton component.

fate-polyacrylamide gel electrophoresis of individual fractions of the Sephadex G-100 column eluate (Fig. 3) shows that Fractions 25 through 31 contain essentially one protein com- ponent with apparent molecular weight of 50,000 (Fig. 4, Lane a). Later fractions (Fig. 4, Lanes h through f), however, contain increasing amounts of a faster moving component (apparent molecular weight, 12,000). What accounts for the elution of the 12,000.dalton protein (Fig. 3) considerably ear- lier than the ribonuclease protein standard (M, = 13,700) is unclear. It possibly reflects a weak association of the 50,000- and 12,000.dalton proteins which is partially broken during gel filtration in 1.0 M NaCl.

The pooled Fractions 25 through 31 also migrate as a single band during disc gel electrophoresis in p-alanine buffer at pH 4.3 and in 0.9 M acetic acid with or without 6 M urea, pH 2.7 (not shown).

Estimation of Isoelectric Point-Isoelectric focusing of the 50,000-dalton protein, obtained by Sephadex G-100 chroma- tography, in a commercial gel containing an ampholine mix- ture that at equilibrium produces a gradient from pH 2.5 to 10 (see “Experimental Procedures”) yields a single discrete pro- tein band at pH >9.5 (not shown).

Polyacrylamide Gel Electrophoresis-Sodium dodecyl sul- Permeability-increasing and Bactericidal Activities of Pu-

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11004 Bactericidal Protein and Phospholipase A2 from Rabbit Leukocytes

FIG. 4. SDS-polyacrylamide slab gel electrophoresis of Sephadex G-100 protein fractions. Ten micrograms of protein was applied in Lanes a to g; 10 pg of each standard (std) protein in Lane h. Electrophoresis was performed as described under “Experimental Procedures.” Protein migration was toward bottom of gel (anode). Protein fractions applied: (a) pooled Sephadex G-100 Fractions 25- 31 (Fig. 3); (b) pooled Fractions 32-33; (c) pooled Fractions 34-35; (d) Fraction 36; (e) Fraction 37; (f) Fraction 38; (g) purified human leukocyte bactericidal/permeability-increasing protein (estimated M, = 60,000 (4)), and (h) protein standards, from top to bottom, bovine serum albumin (68,000), ovalbumin (43,000), chymotrypsinogen A (25,000), and ribonuclease A (13,700).

TABLE II Specific permeability-increasing and bactericidal activities of

Sephadex G-100 fractions

Permeability-increasing and bactericidal activities were measured as described under “Experimental Procedures.” All values represent the mean of at least duplicate determinations.

Permeability-increas- ina activitv” Bactericidal activity”

25-31 (a)’ 183 1.5-2.5 32-33 (b) 171 N.T.” 34-35 (c) 131 N.T. 36 (d) 115 3.0-4.0 37 (e) 90 3.0-4.0 38 (0 N.T. 6-10

n Units of activity per milligram of protein. * Micrograms of protein required to produce >90% killing of 5 X

10’ E. coliS15. - ’ Fraction numbers refer to Fig. 3; the letters within parentheses

refer to Fig. 4. ” N.T., not tested.

rified Fraction-Table II shows that the specific permeabil- ity-increasing and bactericidal activities are maximal in the fractions containing almost exclusively the 50,000-dalton pro- tein (see Fig. 4, Lanes a through f). As the proportion of the 12,000-dalton component increases and that of the 50,000- dalton protein decreases, specific activity falls. These results are consistent with the conclusions that the 50,000-dalton protein carries both the bactericidal and the permeability- increasing activities and that the 12,000-dalton molecule does not contribute to either of these activities.

Removal of the 12,000-dalton protein would then also ac- count for the approximately 60% higher specific permeability- increasing and bactericidal activities of the pooled Fractions 25 through 31 (Fig. 3) compared to the Sephadex G-50 void volume fraction (Fig. 2; Tables I and II).

Amino Acid Analysis of Purified Bactericidal/Permeabil- ity-increasing Protein-Preparative sodium dodecyl sulfate- slab gel electrophoresis was employed to obtain the 50,000- dalton protein free of any residual 12,000-dalton protein (see “Experimental Procedures”). Table III shows the amino acid

composition of the isolated 50,000-dalton protein. Table III also shows that the bactericidal/permeability-increasing pro- tein from human polymorphonuclear leukocytes, recently pu- rified to near homogeneity (4), and the rabbit protein have a similar amino acid composition. The main exception is the arginine content of the two proteins.

The amino acid compositions (not shown) of the pooled Fractions 25 through 31 (see Fig. 3), containing almost exclu- sively the 50,000-dalton protein (Fig. 4, Lane a) and of Frac- tion 38 (see Fig. 3), containing mostly 12,000-dalton protein (Fig. 4, Lane f) are so similar that the possibility must be considered that the two proteins are related. Further study of such a relationship must await collection of adequate amounts of the 12,000-dalton protein for more complete analysis. Prog- ress in this regard has been slow because of extensive losses during preparation and purification of this low molecular weight protein.

Comparison of the Biological Activities of the Purified Bactericidal/Permeability-increasing Proteins from Rabbit and Human Polymorphonuclear Leukocytes-The dose-de- pendent effects of the purified rabbit and human proteins on viability and on permeability to actinomycin D of E. coli are closely similar (Fig. 5). Both proteins initiate net phospholipid degradation in E. coli S15 (Fig. 5), but not in a phospholipase A-deficient mutant (E. coli S17), indicating activation of bacterial phospholipase activity in the parent strain. Hydrol- ysis triggered by the rabbit protein is about two times greater than that produced by the human protein, showing that in this respect the two proteins are not equivalent. Harvesting of E. coli during stationary phase does not alter the effects of the two proteins on bacterial viability and phospholipids.

The two proteins show practically the same antimicrobial potency and specificity (Table IV). The bactericidal activity of either protein is evident only toward gram-negative bacte- ria. All gram-negative bacterial strains tested that are suscep- tible to the bactericidal action of the proteins also show an almost immediate increase in envelope permeability to acti- nomycin D. Of both proteins higher concentrations are re-

TABLE III Comparison of amino acid compositions ofpurified bactericidal/

permeability-increasingproteins from rabbit and human polymorphonuclear leukocytes

Lyophibzed protein samples acid hydrolyzed (6 N HCl), 20 h at 100°C. The results shown are the mean of two closely similar deter- minations.

Amino acid Rabbit” Human” % of total

Aspartic acid 8.4 8.5 Threonine 4.3 4.2 Serine 8.2 7.8 Glutamic acid 8.5 9.1 Proline 6.5 7.9 Glycine 9.2 5.9 Alanine 7.1 5.5 Half-cystine N.D.’ N.D. Valine 7.1 7.9 Methionine 1.6 3.3

Isoleucine 4.7 5.5

Leucine 13.7 10.6

Tyrosine 2.1 3.3 Phenylalanine 3.7 5.8 Histidine 2.8 3.6 Lysine 6.2 7.9 Arginine 6.0 3.3

n 50,000-dalton protein obtained by preparative SDS-polyacryl- amide gel electrophoresis as described under “Experimental Proce- dures.”

’ Data obtained from an earlier publication (4). ’ N.D. = not detected.

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Bactericidal Protein and Phospholipase AZ from Rabbit Leukocytes 11005

Sur viva/

0 2’5 5:o pig Protein

PL Degradafion

Permeability fo Act. D

0 2.5 5.0 pry Protein

z EF ? 0 25 5.0

pg Protein

FIG. 5. Effects of increasing concentrations of rabbit and human bactericidal/permeability-increasing proteins on E. coli. The effects were compared of the rabbit (circles) and the human (triangles) purified bactericidal leukocyte proteins on viability, en- velope permeability to actinomycin D (Act. D), and phospholipids (PL) of E. coli S15 (wild type: closed symbols) and phospholipids of E. coli S17 (mutant: open symbols). All assays were conducted for 30 min at 37°C with 5 x IO7 E. coli in the standard incubation mixture. Effects on envelope permeability were determined by measuring bacterial [‘%]leucine incorporation in the presence (broken lines) and absence (solid lines) of actinomycin D as described under “Ex- perimental Procedures.” Results are expressed as per cent of E. coli incubated alone (>2000 cpm; 1 to 2 nmol). Effects on viability are also expressed as per cent of E. coli incubated alone. Bacterial phospholipid degradation is shown as the accumulation of 14C-labeled monoacylphosphorylglycerol compounds plus free fatty acids (see “Experimental Procedures”) and is expressed as per cent of total E. coli lipid radioactivity. All results shown represent the mean of three or more closely similar experiments.

quired to produce the bactericidal and permeability-increasing effects in smooth strains of E. coli and S. typhimurium than in the corresponding rough strains with incomplete lipopoly- saccharides. Manyfold higher concentrations of both proteins have no effect on viability of any of five gram-positive bacterial species either at pH 7.0 or 5.5.

Other common features (not shown) of the proteins isolated from rabbit and human leukocytes include: 1) optimal bacte- ricidal and permeability-increasing activities at neutral pH; 2) inactivation by the divalent cations Ca2+ and Mg”’ (l-4); and 3) moderate heat stability. (Bactericidal and permeability- increasing activities of both proteins dissolved in distilled water are unaffected by heating at 60°C for 10 min, but are destroyed by heating at 80°C.)

Effect of Removal of Phospholipase AZ on Bactericidal and Permeability-increasing Activities of the Purified Rabbit Protein-In Fig. 5 (preceding section) we showed that at bactericidal concentrations the purified proteins from rabbit and human leukocytes cause net phospholipid degradation in E. coli by activating bacterial phospholipase(s) A. We have recently established (3), by using the phospholipase-deficient E. coli mutant S17, that the leukocyte phospholipase AZ participates in an attack on bacterial phospholipids when E. coli is exposed to bactericidal concentrations of Fraction G (Fig. l), which is rich in phospholipase A,. The onset of net phospholipid degradation is coincident with the almost in- stantaneous increase in permeability to actinomycin D and

with the rapid killing phase (2). It is conceivable, therefore, that an attack on envelope phospholipids plays some role in these other two effects of the protein fraction. This appears not to be the case, however., Table V shows that recombination of the peak phospholipase AZ fraction obtained by Sephadex G-50 chromatography with the 50,000-dalton protein does not enhance the bactericidal and permeability-increasing effects on the phospholipase A-deficient E. coli S17, even though this recombination does produce net hydrolysis of the E. coli phospholipids (see below).

Purification of Rabbit Polymorphonuclear Leukocyte Phospholipase AS

Amino acid analysis (Table VI) of an oxidized sample of the phospholipase AZ-rich fraction obtained by Sephadex G-50 chromatography (Fig. 2) revealed an extraordinarily high percentage of lysine, alanine, and proline residues and no detectable cysteic acid. Since all phospholipases AZ thus far obtained in pure form (16, 17) contain a high number of intramolecular S-S bridges, this composition suggested that the enzyme was only a minor component of the fraction.

The tight membrane association of the rabbit phospholipase Aa (1, 13) prompted us to employ hydrophobic interaction (phenyl-Sepharose) chromatography for further purification (Fig. 6). Nearly all the protein applied was recovered during elution with 1.0 M NaCl, unaccompanied by any detectable phospholipase A2 activity. More than two thirds of the applied

TABLE IV

Susceptibility of different microorganisms to permeability- increasing and bactericidal effects ofpurified rabbit and human

polymorphonuclear fractions

Incubations and assays were carried out as described under “Ex- perimental Procedures.” The number of microorganisms added per incubation mixture was 5 x 10” for each species. The designations 1 to 4+ indicate the amount either of purified rabbit (Fig. 3) or human (4) protein necessary to produce >90% effect on microbial viability and permeability, measured as detailed under “Experimental Proce- dures.“”

Rabbit protein Human protein

Bacteria Permea- hilitv

Gram-negative Escherichia coli

S15 (r) 517 (r) C600 (r) HO129 (s) ML 308 225 (s)

Salmonella typhimurium G30 (r) RlO-395 (r) MS-395 (s)

Gram-positive Staphylococcus aureus

Qui W46 Cl Ferrari (coag. +)

Streptococcus faecalis 655 Bacillus subtilis Micrococcus lysodeikticus Listeria monocytogenes

+++ +++ +++ IG. +++ +++

+

- -

I.oss of Permea viahilitv billtv

+++ +++ +++

++ ++

+++ +++

+

0 0 0 0 0 0 0 0

+++ +++ +++ N’.;. +++ +++ + - - - - - - - -

Loss of viability

+++ +++ +++

++ ++

+++ +++

+

0

0 0 0 0 0 0 0

u +++ = <1 pg; ++ = l-5 pg; + = 5-20 pg; 0 = no detectable effect with ~20 pg; N.T. = not tested; - indicates that microorganism is natively susceptible to actinomycin D. All experiments, except those with B. subtilis and M. lysodeikticus, were carried out at least two times in duplicate. Colony morphology is indicated in parentheses; r = rough, s = smooth.

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11006 Bactericidal Protein and Phospholipase AZ from Rabbit Leukocytes

TABLE V

Added rabbit polymorphonuclear leukocyte phospholipase A2 does not potentiate the activities of the rahhit leukocyte bactericidal/

permeability-increasing protein E. coli S17 (5 x 10’ bacteria) were incubated with increasing

concentrations of bactericidal/permeability-increasing protein (pooled Sephadex G-100 Fractions 25-31), in the presence and ab- sence of phospholipase AL, in the standard incubation mixture for 30 min at 37°C. Bactericidal and permeability-increasing activities were determined as described under “Experimental Procedures.” Values are expressed as per cent of values obtained with I?. coli incubated alone in the absence of actinomycin D.

Amount of bat- Phospholl- [lIcll eucine inrorporation tericidal/perme- Bacterial via- ablllt,v-increasing pase AL” -Actmomy- +Actino- hility

motein added added rin I) mvcin 11

0 80 - c

kc 0.0 - 100 99 100 0.85 - 94 81 79

+ 92 79 81 1.7 - 90 34 58

+ 80 30 45 3.3 - 73 22 11

+ 72 22 11 6.5 - 69 20 3.7

+ 65 18 2.8

“Where indicated, 400 units of phospholipase A, activity from Sephadex G-50 peak phospholipase A, fraction (see Fig. 2) was added.

TABLE VI

Amino acid compos&on ofphospholipase AZ-rich /Faction hefore nnd afterphenyl-Sepharose chromatography

Lyophilized protein samples oxidized with performic acid, then acid hydrolized (6 N HCl), 20 h at 100°C.

Aspartic acid 1.9 Threonine 2.4 Serine 4.1 Glutamic acid 1.8 I’roline 15.4 Glycine 4.3 Alanine 24.8 Half-cystine N.D. Valine 2.8 Methionine to.5 Isoleucine 0.8 Leucine 1.3 Tyrosine N.1). I’henylalanine N.1). Histidine 2.0 1,ysine 38.4 Arrinine N.D.

” See Fig. 2. ” See Fig. 7. ’ NJ). = not detected.

‘G Of totn1

1.9 2.4 3.9 3.4

13.8 4.9

20.4 N.11.

2.8 <0.5

1.0 1.3

N.1). N.1).

2.2 41.6

N.11.

10.8 5. 9 7.7 9.6 5.3

10.3 8.1 6.4

N.11. N.11.

4.4 8.1

N.1). N.11.

1.6 5.9

11.9

phospholipase A2 activity was recovered in a sharp peak by subsequent elution with 50% ethylene glycol in 1.0 M ammo- nium acetate (pH 4.0). The solvent was removed by lyophili- zation. SDS-polyacrylamide gel electrophoresis of the protein resuspended in 1.5%> SDS yields a single band with an appar- ent M,. of approximately 14,000 (Fig. 7).

The amino acid composition of the protein in the NaCl eluate closely resembles that of the protein applied to the phenyl-Sepharose column (Table VI). The very different amino acid composition of the protein in the ethylene glycol fraction includes a substantial proportion of half-cystine and arginine, neither of which is detected in the protein applied to the column. This indicates that the protein in the ethylene

i-------i Sample Elut~on Volume (ml)

LOW

Fro. 6. Phenyl-Sepharose chromatography of pooled Seph- adex G-50 phospholipase AZ-rich fractions. The pooled fractions (2.6 mg of protein obtained from several Sephadex G-50 chromato- graphic separations) were applied in 40 ml of column buffer to a I’henyl-Sepharose column (0.9 x 2.0 cm) equilibrated in 10 mM sodium acetate-buffered 1.0 M NaCl (pH 4.0). Elution was maintained at a slow flow rate (2 to 4 ml/h) by hydrostatic pressure. The buffer wash was collected as a single batch. Subsequent elution was carried out successfully with 10 ml of column buffer, 10 ml of distilled water, and 12 ml of 50% ethylene glycol in 1.0 M ammonium acetate, pH 4.0. Fractions of 1.0 ml were collected. Protein and phospholipase AZ activity was determined as described under “Experimental I’roce- dures.” Protein recovery was >90%,. No protein was detectable in fractions containing phospholipase AZ.

FIG:. 7. SDS-polyacrylamide gel electrophoresis of purified phospholipase A2 fraction obtained by Phenyl-Sepharose chromatography. Of this fraction, a sample containing 40,000 units of activity was applied after lyophilization and treatment as described under “Experimental Procedures.” After Coomassie blue staining, the gel was scanned, densitometrically, at 590 nm using a Schoeffel SD 3000 spectrodensitometer. The anode is at the left. The arrows indicate the migration of bovine serum albumin (68K), ovalbumin (44K), chromotrypsinogen A (25K), and ribonuclease A ( 24K).

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Bactericidal Protein and Phospholipase AZ from Rabbit Leukocytes 11007

TABLE VII

Purification of rabbit polymorphonuclear leukocyte phospholipase AP

Rabbit polymorphonuclear leukocytes (2.1 x lO”‘), pooled from several collections, were used as starting material for purification. Protein and phospholipase activities were determined as described under “Experimental Procedures.” The values shown represent the mean of at least three independent assays carried out in duplicate.

Purification step

Homogenate 0.16 N HSOz ex-

tract Carboxymethyl-

Sephadex chro- matography

Sephadex G-50 chromatogra- phy

Phenyl-Sepha- rose chroma- tography

Protan

1.6 0.04

<0.08 <0.00114.9 x 10” x 10’

I

I’urifi- cation

-fold

1 5.4

70

500

>8000

” Data obtained from earlier publication (13)

glycol fraction cannot represent more than 5% of the applied protein and that its specific phospholipase activity is at least 8000-fold greater than that in the whole homogenate (Table VII).

I f we assume purification of the phospholipase Ae to near homogeneity and a molecular weight of 14,000 (on the basis of SDS-polyacrylamide gel electrophoresis), then the amino acid analysis accounts for eight half-cystines per molecule. The phospholipases A? whose sequence has been determined con- tain at least four disulfide bridges (16). The number of half- cystines in the leukocyte phospholipase AP may well have been underestimated, either because during performic acid oxidation one or more disulfide links may have remained unoxidized or because the phospholipase A% might still be contaminated with a small amount of extraneous protein that is not detected as a separate band by SDS-polyacrylamide gel electrophoresis. At present the number and range of analyses of the purified phospholipase AZ are severely restricted by the very small amounts of protein in the ethylene glycol fraction and by the propensity of the purified protein to adhere to vessel walls.

Facilitation of the Action ofRabbit Leukocyte Phospholipase A4 on Phospholipids of E. coli by the Purified Rabbit

Bactericidal/Permeability-increasing Protein

The phospholipase Az present in Fraction G (Fig. 1) de- grades the phospholipids of a phospholipase A-deficient E. coli mutant (S17) (3). In contrast, more purified phospholipase AZ fractions, completely devoid of the bactericidal/permeabil- ity-increasing protein, are inactive toward these intact E. coli (Table VIII). Recombination of the phospholipase AZ-rich fraction (Fig. 2) with the 50,000-dalton bactericidal/permea- bility-increasing protein restores the activity of the enzyme toward intact E. coli S17 (Table VIII). Similar results are obtained by recombining the most purified form of the phos- pholipase An (Figs. 6 and 7) with the bactericidal/permeabil- ity-increasing protein (results not shown). These findings in- dicate that, although neither bactericidal nor permeability- increasing activities depend on the leukocyte phospholipase AZ, the two purified proteins act in concert in a degradative attack on the microbial envelope. The extent of degradation produced by the combination of the two purified proteins is almost the same as that produced by the cruder protein

TABLE VIII Action of rabbit leukocyte phospholipase A2 on intact E. coli (Sl7). Facilitation b.y rabbit bactericidal/permeability-increasing protein

E. coli S17 (5 x 10’ bacteria), prelabeled during growth with [‘“Cloleic acid as described under “Experimental Procedures,” were incubated for 60 min at 37°C with the indicated phospholipase Al fraction, containing 400 arbitrary units of activity. The incubation mixtures also contained 10 pmol of Tris/HCl buffer (pH 7.0), 25 al of Hanks’ solution, and 250 pg of vitamin-free casamino acid in a total volume of 0.4 ml of physiological saline. The Sephadex G-50 phos- pholipase Ae fraction alone produces no effect on bacterial viability or permeability (determined as described under “Experimental Pro- cedures”), whereas the other fractions produce >95% bacterial killing and maximal permeability-increasing effects. Phospholipid degrada- tion, measured as the accumulation of ‘%labeled breakdown prod- ucts (under these conditions only “C-free fatty acids), is expressed as percent of total E. coli lipid radioactivity. The results shown are the means f S.E. The number in parentheses indicates the number of independent determinations.

Phospholipase A, fraction l’hospholipid degrada- tion

v

Sephadex G-50 (Fig. 2) <0.2 (5) Sephadex G-50 + purified bactericidal/perme- 23.3 f 4.2 (8)

ability-increasing protein Carboxymethyl-Sephadex Fraction G (Fig. 1) 26.4 + 1.2 (10) 0.16 N H,SO, extract 26.7 * 2.5 (4)

fractions, Fraction G, and neutralized acid extract (Table VIII), suggesting that the bactericidal/permeability-increas- ing protein is indeed the principal agent of rabbit polymor- phonuclear leukocytes facilitating the action of the phospho- lipase AZ on E. coli S17. In the accompanying paper we will show that this facilitating effect of the rabbit leukocyte bac- tericidal/permeability-increasing protein is specific for the rabbit leukocyte phospholipase Az.

DISCUSSION

The granules of polymorphonuclear leukocytes are the site of several cationic antimicrobial proteins, some catalytic, such as lysozyme and myeloperoxidase, and others without known enzymatic activity (18). Upon degranulation, i.e. the fusion of the granule with the phagocytic vacuole, these proteins can exert their effects on sequestered microorganisms. The cata- lytically active proteins, including lysozyme, myeloperoxidase, and certain proteases (la-20), have limited bactericidal po- tency by themselves, but in combination with other factors and cationic proteins potentiation of antimicrobial action is evident (21). In contrast, the cationic antibacterial proteins without known catalytic activity, that have been partially purified by Zeya and Spitznagel (22, 23), appear to be capable of considerable independent bactericidal action. These pro- teins represent a heterogeneous group of low molecular weight (4,000 to lO,OOO), most active at acid pH and with different antibacterial specificities, extending to gram-positive and gram-negative bacteria (23).

The bactericidal/permeability-increasing protein isolated and purified in this laboratory is distinct from the above group of proteins. It has an apparent molecular weight of approxi- mately 50,000 and a different amino acid composition (Table III; Refs. 22 and 23), is maximally active at neutral pH, and kills a number of gram-negative bacterial species, but displays no bactericidal activity toward any of the gram-positive mi- croorganisms that we have tested.

In the intact polymorphonuclear leukocyte the ingested microorganism is met by these and other antimicrobial sys- tems and factors (18). It is difficult to establish the relative importance of one or another component in this arsenal except when decreased host resistance to infection can be attributed

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11008 Bactericidal Protein and Phospholipase A2 from Rabbit Leukocytes

to the absence or deficiency of an apparently single compo- nent, as in the case of congenital chronic granulomatous disease (18). Indirect evidence suggests, however, that the fate of susceptible gram-negative bacteria is in large measure determined by the leukocyte bactericidal/permeability-in- creasing protein. Thus, the time course of killing of E. coli and the accompanying bacterial envelope alterations, pro- duced by intact polymorphonuclear leukocytes, disrupted leu- kocytes, and the bactericidal/permeability-increasing protein at various stages of purification, are remarkably similar (1, 6, 24). No other leukocyte fractions separated during purification of the bactericidal/permeability-increasing protein exhibit comparable biological activities toward these bacteria. This can be interpreted to mean that other ingredients of the antimicrobial apparatus of the polymorphonuclear leukocyte do not greatly contribute to the demise of E. coli and certain other gram-negative bacteria.

The properties of this rabbit leukocyte bactericidal protein closely resemble those of the recently purified bactericidal/ permeability-increasing protein from human polymorphonu- clear leukocytes (4). Thus, the two proteins have molecular weights of 50,000 and 60,000, respectively (estimated by gel filtration and sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis) and a rather similar amino acid composition. Both proteins are strongly cationic (isoelectric points > pH 9.5), without a sufficiently high basic amino acid content to account for the net positive charge, therefore suggesting that acidic amino acids are in the amide form (4). Further, prelim- inary observations indicate that a monospecific antiserum generated in goat against the rabbit leukocyte protein cross- reacts with the purified human leukocyte protein. Foremost in the similarity are their almost identical biological properties with respect to potency (Fig. 5) and antibacterial specificity (Table IV).

An interesting feature of the purification of the rabbit bactericidal/permeability-increasing protein that is not shared by the human protein is the close association of the rabbit protein with phospholipase A, activity during early steps in its purification (1). The complete separation of the bacteri- cidal/permeability-increasing protein from the rabbit leuko- cyte’s phospholipase Ae described herein, without loss of either bactericidal or permeability-increasing activity toward a phospholipase A-deficient E. coli mutant, establishes that neither activity depends on net deacylation of envelope phos- pholipids.

We believe that the purification we describe for the rabbit leukocyte phospholipase A, is the most extensive, to date, for a tightly membrane-associated phospholipase A2 (1, 13). Whereas numerous soluble phospholipases Aa have been pu- rified to homogeneity in recent years (25, 26), the purification and chemical analysis of mammalian tissue phospholipases A:! have proved more difficult, particularly because the enzyme tends to be present in the cell as a minute protein component (27). Although the exceedingly small amounts of purified enzyme protein we obtained prevent precise quantitative pro- tein determination, we can estimate approximately the maxi- mal quantity of recovered enzyme protein and hence the minimal specific activity of the leukocyte phospholipase AS at the present stage of purification (Table VII). This specific activity is comparable to that of pure phospholipase Aa from other sources as determined by our assay system.’

In contrast to its activity toward intact E. coli when present in less purified bactericidal protein fractions (Table VIII; Ref. 3), the purified leukocyte phospholipase AS like other purified

’ I’. Elsbach, J. Weiss, R. C. Franson, S. Bcckerdite-Quagliata, A. Schneider, and L. Harris, unpublished observations,

phospholipases AZ (28) is unable to degrade the phospholipids of intact E. coli. The restoration of this activity upon recom- bination with the bactericidal/permeability-increasing protein suggests that the rabbit leukocyte phospholipase AZ only degrades phospholipids of intact E. coli when the enzyme is associated with the rabbit bactericidal/permeability-increas- ing protein. This cooperative effect appears to be specific for these two rabbit leukocyte proteins because it is not seen with purified phospholipases A2 from other sources or with the human leukocyte bactericidal/permeability-increasing pro- tein (29) suggesting that the rabbit leukocyte bactericidal/ permeability-increasing protein and phospholipase AZ may constitute an antimicrobial package.

Although phospholipid hydrolysis is apparently nonessen- tial for effective killing of E. coli by the purified bactericidal protein, the very early degradative attack by the rabbit leu- kocyte phospholipase AZ on the envelope phospholipids may well fulfill an important function in the overall microbial destruction by the leukocyte. Making use of the experimental approaches described herein, we can now explore the possi- bility that the membrane-disruptive effect of phospholipid degradation determines the rate and extent of bacterial diges- tion.

Achnowledgments-We are indebted to Dr. E. C. Franklin and Ms. Joan Zaretsky, Department of Medicine, New York University School of Medicine, New York, NY, who performed the amino acid analyses.

We are particularly grateful for the help and advice generously given on many occasions by Dr. Isaac Schenkein, New York Univer- sity School of Medicine.

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4.

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7.

8. 9.

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18 19

20

21 22 23

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P Elsbach, J Weiss, R C Franson, S Beckerdite-Quagliata, A Schneider and L Harrispolymorphonuclear leukocytes. Observations on their relationship.

protein and a closely associated phospholipase A2 from rabbit Separation and purification of a potent bactericidal/permeability-increasing

1979, 254:11000-11009.J. Biol. Chem. 

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