Enhanced Bactericidal Action of Lysozyme to Escherichia coli by Inserting a Hydrophobic Pentapeptide into Its C Terminus*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY by The Amerian Soiety for Biohemistry and Moleular Biology, In. Vol. 269, No. 7, Issue of February 18, pp ,1994 Printed in U. S. A. Enhaned Bateriidal Ation of Lysozyme to Esherihia oli by Inserting a Hydrophobi Pentapeptide into Its C Terminus* (Reeived for publiation, July 6, 1993, and in revised form, Otober 25, 1993) Hisham Radwan Ibrahim, Mamoru Yamada, Kazunobu Matsushita, Kunihiko Kobayashi, and Akio KatoS From the Department of Biologial Chemistry, Faulty of Agriulture, Yamaguhi University, Yamnguhi 753, Japan The mehanism of the enhaned bateriidal ation to Esherihia oli of the lysozyme having a hydrophobi pentapeptide (Phe-Phe-Val-Ala-Pro) at its C terminus was investigated. The modified lysozyme, hydrophobi pentapeptide-fused lysozyme (HLz), was sereted in the ulture medium from yeast harboring the expression plasmid, in whih a syntheti DNA fragment enoding a hydrophobi pentapeptide was introdued to the 3 -end of the oding region of the lysozyme DNA. Although CD analysis showed that HLZ was onsiderably different from wild-type lysozyme (WLz) in the seondary and tertiary strutures, it retained 76% of the lyti ativity of WLz. When E. oli ells were exposed to the WLz or HLz, the survival ells were signifiantly redued only in the ase of HLz. Periplasmi proteins from the HLz-treated ells were released to an extent similar to that from the WLztreated ells, indiating that HLz has nearly the same ation as WLz with respet to the disruption of the outer membrane and peptidoglyan. Experiments with E. oli phospholipid liposomes revealed that HLz dissipated the valinomyin-indued transmembrane eletrohemial potential, but WLz did not. These results suggest that the enhaned bateriidal ation of HLz to E. oli is due to disruption of the eletrohemial potential of the inner membrane in ooperation with the inherent funtion of the lysozyme to the outer membrane and peptidoglyan. The bateriidal ation of lysozyme is intensive to Grampositive bateria but muh weaker to Gram-negative bateria. The differenes are thought to be due to the unique ell envelopes of the latter, whih onsist of the outer membrane (l), inner membrane, and peptidoglyan layer (2,3). Lysozyme atalyzes the hydrolysis of the /3-(1,4)-glyosidi linkage between N-aetylgluosamine and murami aid of the peptidoglyan in the baterial ell wall (4), interats with the lipopolysaharide (LPS) layer in the outer membrane (5), and subsequently distorts the normal paking between phosphate groups of phospholipids and LPS by its polyationi * This work was supported by Grant-in-aid for Sientifi Researh from the Ministry of Eduation, Siene and Culture of Japan. The osts of publiation of this artile were defrayed in part by the payment of page harges. This artile must therefore be hereby marked advertisement in aordane with 18 U.S.C. Setion 1734 solely to indiate this fat. To whom orrespondene should be addressed Dept. of Biologial Chemistry, Yamaguhi University, Yamaguhi 753, Japan. Tel.: (ext. 4); Fax: The abbreviations used are: LPS, lipopolysaharide; WLz, wildtype lysozyme; HLz, hydrophobi pentapeptide-fused lysozyme; dis- C3-(5), 3,3 -diisopropylthiodiarboyanine. properties (6). The distortion results in the perturbation of the outer membrane struture and the stimulation of the suseptibility to lysozyme of the peptidoglyan layer (7). Despite suh ations of lysozyme on the outer membrane, it does not adversely affet on the viability of the Gram-negative bateria, while the ations lead Gram-positive bateria to be lethal. We have reently found that the ovalent attahment of palmiti aid residues to the lysyl residues of lysozyme (8) or the fusion of a hydrophobi pentapeptide (Phe-Phe-Val- Ala-Pro), whih an form onformation with the same length as a palmiti aid residue, to its C terminus greatly enhaned the bateriidal ation to Esherihia oli (9). These findings suggest that suh hydrophobi domains may promote the penetration of the ationi moleule into the inner membrane and the subsequent loss of the eletrohemial membrane potential. In this paper, we intended to eluidate the mehanism of the bateriidal ation to E. oli ells of the lysozyme having the hydrophobi pentapeptide at its C terminus. EXPERIMENTAL PROCEDURES Materials-Synthesized oligodeoxyribonuleotides were supplied by Takara Shuzo, Japan. Authenti lysozyme was purified from fresh hen egg white and rerystallized five times as previously reported (10). Miroous lysodeiktius ells and LPS from E. oli Olll:B4 were purhased from Sigma. Phospholipids were isolated from E. oli aording to the method of Viitanen et al. (11). Unless otherwise stated, all other hemials were of the highest grade ommerially available. Baterial Strains and Phmids-E. oli K-12 TG1 (A(la-pro), supe, thi, hsd D5/F tra 036, proa+b+, lap, lazaml5) used as a host ell and M13 mp19 bateriophage were supplied by Amersham Japan and Takara Shuzo, respetively. E. oli K-12 IF (la1 la0 laz lay) was used primarily as a representative miroorganism for Gram-negative bateria. pyg- (12), an E. oli-yeast shuttle vetor, was provided by Dr. K. Matsubara, Osaka University. Saharomyes erevisiae AH22 (a, Leu2, His4, Cir+), an expression strain, 5059 pkk-1 plasmid (13), whih ontains the full length of the prelysozyme DNA in the same orientation as laz in puc18, and pygkk-1 (13) that was onstruted by inserting the blunt-ended, full-length DNA into the SalI site of pyg- were provided by Dr. I. Kumagai, University of Tokyo. Constrution of Expression Phmid of Fusion Lysozyme (HLz)-A PstI site was introdued into the 3 -end of the oding region of the prelysozyme DNA by the oligonuleotide-direted mutagenesis using a primer, 5 -AGAGGCTGCAGGCTGTGA-3, as reported previously (9). The resultant plasmid was digested with PstI and then ligated with two synthesized 28-mer oligonuleotide A (5 GGCTGTT- TTTTGTCGCTCCCTGACTGCA-3 ) and B (5 GTCAGGGAGC- GACAAAAAACAGCCTGCA-3 ), whih ontain the odons of Arg- 128 and Leu-129 of the lysozyme, the hydrophobi sequene (Phe- Phe-Val-Ala-Pro), and the stop odon. The orretly inserted sequene was identified by hybridization of bateriophage plaques using 32P-labeled fragment A as a DNA probe. The reombination site of the onstruted plasmid (phlz-5) was further onfirmed bydna sequening using a sequening kit from Takara Shuzo and a syntheti primer (5 -GGCAGGACCCCAGGCTCCAGG-3 ) omplementary to

2 50 Bateriidal Enhaned Ation the oding sequene from Gly-67 to Arg-73 of the lysozyme. The small fragment MluI-SalI (14) inluding the inserted sequene of phlz-5 was ligated with the large fragment MluI-SalI of pygkk- 1 to onstrut the expression plasmid of HLz (pyghl-5). Expression of HLz-pYGKK-1 and pyghl-5 were introdued into S. ereuisiae AH22 by the lithium aetate proedure (15). Leu+ transformants were sreened on modified Burkholder minimum medium plates supplemented with histidine (20 pg/ml) at 30 "C. The transformants were grown in the 5-ml yeast medium, and then lones with the highest ativity of lysozyme were sreened. For large-sale ultivation, olonies of the overexpression lones were diretly inoulated and ultured in the yeast minimum medium at 30 "C for 5 days. Purifiation of HLz-Several liters of the ulture medium were entrifuged at 00 X g for 15 min to remove ells at 4 "C and diluted with deionized water at least twie. The solution was applied to a CM-Toyopearl 650 olumn (7.5 X 1.7 m) equilibrated with 50 mm Tris-HC1 buffer, ph 7.5, and, after washing the olumn, protein was then eluted with the same buffer ontaining 0.5 M NaCl. Frations ontaining the lysozyme ativity were pooled, diluted 10 times with the same buffer, and rehromatographed on a CM-Toyopearl 650 olumn (12 X 1.5 m). Lysozyme was eluted with a linear gradient of M NaCl in the same buffer. The ative peak was olleted, dialyzed in a Spetra/por dialysis bag against deionized water, and used in the following experiments. The purity of HLz was examined by SDS-polyarylamide gel eletrophoresis. Cirular Dihroism-CD spetra of 55 pg/ml wild-type lysozyme (WLz) and HLz in 50 mm Tris-HC1 buffer, ph 7.4, were reorded at either the far-uv (from 200 to 250 nm) or near-uv (from 250 to 300 nm) regions at room temperature in 1.0-m uvettes using a Jaso Model spetropolarimeter. The auray of the data was improved by averaging eight sans integrated with a model DP-501 data proessor. CD spetra were represented in terms of mean residue elliptiity (degrees m2 dm01 -'). Assay of Lysozyme Ativity-Lysis of M. lysodeiktius ells was estimated aording to the turbidometri method (16) based on the derease in turbidity of a 1.9-ml ell suspension (170 pg/ml) in 0.1 M sodium aetate buffer, ph , or 0.1 M sodium phosphate buffer, ph , after the addition of p1 of lysozyme solution (10 pg/ ml). The derease in the absorbane at 25 "C was monitored using a Hitahi U-2000 reording spetrophotometer. One unit of lysozyme ativity is defined as the amount of enzyme that dereases of the absorbane at 450 nm/min at 25 "C. Lyti ativity was,represented as a perentage of the ativity of WLz at ph 5.0. LPS Binding Assay-Lyti ativity of WLz and HLz against M. lysodeiktius ells was monitored in the presene of LPS as desribed previously (5) exept that the various onentrations of LPS were mixed with a single onentration of the protein (final onentration, 1 pg/ml). The mixtures were inubated at 37 "C for 15 min before adding the M. lysodeiktius ell suspension (final onentration, 170 pglml, AIM) of 0.8). AntibaterMl Assay-The suspension of E. oli ells (10' ells/ml) from the mid-logarithmi phase ulture was mixed with lysozyme (final onentration, 50 pg/ml) in 50 mm phosphate buffer, ph 7.0. The mixture was inubated at 37 "C for various lengths of time. At a given time, -pl portions or dilutions were plated onto two separate nutrient agar plates (Maonkey). All plates were inubated at 35 "C overnight (-14 h), and then the viable ell numbers were ounted. Perent survival was represented to the olony number from a ontrol solution without lysozyme. The experiment was done in tripliate. Release of p-galotosidase and Alkaline Phosphatase from E. oli Cells-E. oli ells (log ells/ml) were exposed to WLz or HLz (5 pg/ ml) for various lengths of time in 50 mm potassium phosphate buffer, ph 7.0, and the ativities of released /3-galatosidase(17) and alkaline phosphatase (18) in the resultant supernatants after entrifugation (00 rpm/min at 4 "C) were measured. Control was done in the absene of lysozyme. The results were represented as a perentage of the total ativity obtained from ell extrats prepared by soni osillation. Preparation of Liposomes-A -pl aliquot (50 mg of dry weight/ ml of argon-saturated 2 mm /3-meraptoethanol) from lipid suspension was added to 900 pl of 50 mm potassium phosphate buffer, ph 7.5, in a miroentrifuge tube. The mixture was soniated for 2 s at maximum output followed by a 3-5 pause, and the yle was repeated until the turbid suspension had been larified. The solution was entrifuged at 356,000 X g for 1 h at 4 "C. The obtained liposome pellet was suspended in p1 of the same buffer, and soniation followed by entrifugation was repeated. Finally, the soniated liposome suspension was entrifuged for 3 min at 10,000 rpm to disard any multila- of Lysozyme mellar liposomes. The liposome preparation was kept in an ie water bath under argon and used within a few hours in the following experiments. Measurements of Membrane Potential (A$) of E. oli Phospholipid Liposomes-A$ (interior negative) was determined by measuring the fluoresene quenhing of 3,3'-diisopropylthiodiarboyanine (dis- C3-(5)) using a Hitahi s fluoresene spetrophotometer (19). Experiments were performed at 25 "C under the ondition of exitation at 622 nm and emission at 670 nm. The reation mixture ontained (in final onentrations) 50 mm sodium phosphate, ph 7.5, 1 p~ dis-cs-(5), and 70 pg/ml phospholipid liposomes in a total volume of 1.0 ml. K+-ontaining liposomes were energized (inside negative) by a valinomyin-mediated potassium diffusion. Lysozyme was then added at the indiated onentrations. The data were ompared with that of the ontrol experiment of the nigeriin-mediated eletroneutral exhange of K+ for H+. Fluoresene quenhing was reported as a perentage hange (AFIF), where F is the maximum fluoresene intensity after addition of nigeriin, and AF is the fluoresene signal in the presene of lysozyme as a funtion of time. Protein Assay-Protein ontent was determined by the Lowry method modified by Miller (20) using bovine serum albumin as a standard. RESULTS AND DISCUSSION HLz sereted in the ulture medium was olleted and purified by two-step ation exhange hromatography. The seretion amount of HLz was less than half of WLz. Although a onsiderable amount of HLz was retained in the periplasmi fration of yeast, the protein sereted in the ulture medium was used in this experiment. Conformation of HLz-The onformational hanges were investigated aording to CD analysis. CD spetra of WLz and HLz are shown in Fig. 1. CD spetra at and at nm reflet the seondary and tertiary strutures of protein, respetively. As shown in Fig. la, CD spetra at ph 7.4 indiated a signifiant hange in the seondary struture of HLz ompared with that of WLz, although HLz still retained a onsiderable amount of the original seondary struture. The hange may be due to the partial unfolding of the adjaent a-helix (residues 5-15) aused by the hydrophobi interation of the pentapeptide inserted at the C terminus, as predited from the rystal struture of lysozyme, whereas the CD spetrum at nm of HLz at ph 3 showed a similar negative elliptiity urve to that of WLz (data not shown). It is possible that the inserted hydrophobi pentapeptide forms whih extends toward the C terminus at low ph values without altering the adjaent a-helix. On the other hand, the CD spetrum at nm (Fig. 1B) shows signifiant hanges in the tertiary struture of HLz at neutral ph value. The similar hange in the tertiary struture A Wave Length (nm) FIG. 1. CD spetra of WLz and HLz at the far-uv (A) and near-uv (B) regions. The CD spetra were reorded for 55 pg/ml WLz (solid lines) or HLz (dashed lines) in 50 mm Tris-HC1 buffer, ph 7.4, at 25 "C. t

3 Bateriidal Enhaned Ation of Lysozyme 5061 of HLz was observed at ph 3 (data not shown). These results suggest that the tertiary struture of HLz is slightly more unstable than that of WLz. Lyti Ativity of HLz-Fig. 2 shows the lyti ativities of WLz and HLz for M. lysodeiktius ells at different ph values. The lyti ativity of HLz as well as WLz was dependent on the ph value of the reation mixture. WLz revealed the maximal ativity at ph 5.5, while the maximal ativity of HLz was observed at ph 5.0 and was 76% of that of WLz. The shift of the optimal ph value to the aidi side and the redution of the lyti ativity at the neutral ph value may be due to the onformational hanges as deteted by CD analysis. These results indiate that the hydrophobi extension has no adverse effet on the atalyti funtion of lysozyme at aidi ph value. Antibaterial Ation to E. oli of HLz-Fig. 3 shows the bateriidal ations of WLz and HLz for E. oli ells. WLz showed no detetable bateriidal ation, but HLz did show signifiant bateriidal ation, espeially at ph 5.0. The ation of HLz was enhaned up to the end of the inubation time tested (30 min). The results indiate that the fusion of the hydrophobi pentapeptide to lysozyme has rendered the enzyme ative in killing E. oli ells. Considering the fat that both WLz and HLz were ative in hydrolyzing the baterial ell walls, the differene in sensitivity of E. oli to WLz and HLz would be responsible for the amphiphili nature of the latter, whih may ontribute to the interation with the omplementary amphiphili surfae of the bateria. The differene in the antibaterial ativity between the neutral and aidi ph values may be attributed to that of the lyti ativity of HLz (see Fig. 2). Another possibility is that the hydrophobi pentapeptide is exposed at an aidi ph value and, hene, failitates the binding and insertion of the HLz moleule into the baterial membrane to produe the subsequent killing effet. It is interesting to note that HLz has a strong bateriidal ation, although the lyti ativity is redued to 76% of the wild type. Apparently the hydrophobi tail is losely involved in the killing effet against E. oli. Interation between LPS from E. oli and HLz-The interation of HLz with LPS, the most abundant omponents in the outer membrane of Gram-negative bateria, was investigated by monitoring the redution of the lyti ativity of lysozyme for M. lysodeiktius after inubation with various onentrations of LPS (5). The inubation with LPS redued the ativities of WLz and HLz in a dose-dependent manner. The ativity of WLz at ph 7.0 (Fig. 4A) was linearly dereased with the inrease in the LPS dose, while the derease in the ativity of HLz was more pronouned. Although the inubation with LPS at ph 5.0 had a milder effet on the ativity of WLz than that at ph 7.0, the substantial redution of the ativity was observed in HLz (Fig. 4B). These results suggest that HLz has muh higher binding ativity to LPS ompared with WLz. The enhaned interation of LPS with HLz at ph 5.0 may be due to the hydrophobi pentapeptide, beause a low ph value is known to indue exposure of the hydrophobi region of proteins (21). The dereased enzymati ativity of lysozyme in response to LPS may be largely attributed to the S 9 PH FIG. 2. Lyti ativities of WLz and HLz as a funtion of ph values. The derease in the turbidity at 450 nm of M. lysodeiktius ell suspensions in 2 ml of mm buffer was determined at different ph values. The lysis was monitored in the presene of 0.5 pg/ml WLz (0) or HLz (0) at 25 "C. The ativity is expressed as a perentage of that observed for WLz at ph % 20 0 I I I 1 I I I 1 I I ILj 1 1 \... I inubation Time (min) FIG. 3. Bateriidal ation of WLz and HLz to E. oli ells M a funtion of inubation time at 37 OC. The purified 50 pg/ml WLz (open symbols) or HLz (solid symbols) were inubated with E. oli ells (10' ells/ml) in 50 mm potassium phosphate buffer, ph 7.0 (irles), and in 50 mm sodium aetate buffer, ph 5.0 (squares). Values are the means from three independent experiments, and the bars indiate the standard deviation. Ratio of LPS to Lysozyme (dm) FIG. 4. Lyti ativities of WLz and HLz at neutral (A) and aidi (B) ph values in the presene of LPS. One pg/ml WLz (0) or HLz (0) were inubated with different onentrations of the E. oli LPS at 37 "C for 15 min in 50 mm sodium phosphate, ph 7.0 (A), or sodium aetate buffer, ph 5.0 (E). After oolingdown to 25 "C, the solution (0.1 ml) was mixed with 1.9 ml of M. lysodeiktius ell suspensions in the same buffer (final onentration, 170 pglml), and the derease in the absorbane at 450 nm (25 'C) was monitored. The ativity is expressed as a perentage of that observed in the absene of LPS.

4 5062 Enhaned Bateriidal Ation of Lysozyme formation of the bound lysozyme-lps omplex through hydrophobi interations (5). Thus, the binding of HLz to the baterial membrane permeability barrier (LPS) appeared to be enhaned by the hydrophobi pentapeptide at its C terminus, resulting in disruption of the integrity of the outer membrane. Release of Periplasmi and Cytoplasmi Proteins from E. oli Cells-In order to assess the apability of HLz to damage the outer membrane of E. oli ells, the releases of marker proteins suh as alkaline phosphatase (a periplasmi enzyme) galatosidase (a ytoplasmi enzyme) from the ells treated with HLz and WLz were ompared (Table I). No signifiant differenes in the releases of alkaline phosphatase galatosidase were observed between WLz and HLz. Further evidene for the disruption of the outer membrane integrity was obtained from the SDS-polyarylamide gel eletrophoresis analysis of the supernatants of the E. oli treated with WLz. and HLz. The major protein bands orresponding to membrane proteins and periplasmi proteins were observed in both preparations (data not shown). These results suggest that the interation of HLz with the outer membrane is almost the same as that of WLz. Therefore, the hydrophobi interation between HLz and the inner membrane may be important for the antibaterial ation, thereby failitating the moleule to penetrate into the inner membrane and kill the ells as other proteins do (22-24). Dissipation of the Membrane Potential of E. oli Phospho- lipid Liposomes by HLz-The effet of HLz on the dis-ca-(5) fluoresene quenhing indued by valinomyin in the E. oli phospholipid liposomes was investigated (Fig. 5). The addition of HLz (5 pg/ml) to the hyperpolarized liposomes by valinomyin reversed the fluoresene quenhing, but the addition of WLz at the same onentration had no effet on the signal. The reversal of the fluoresene quenhing by HLz may reflet its ability to disrupt the generated eletrohemial potential. The inrease in the onentration of HLz (10 pg/ml) resulted in a very rapid and signifiant ollapse of the membrane potential in a way similar to the addition of nigeriin. On the other hand, the inrease in the onentration of WLz had very little effet on the fluoresene quenhing. This experiment was further performed by employing right-side-out membrane vesiles of E. oli prepared as desribed previously (25). The same effet of HLz on the membrane potential generated by the addition of gluose was observed (data not shown). These results suggest that HLz an dissipate the membrane eletrohemial potential in E. oli liposomes as a result of the speifi or nonspeifi interation. It is probable that the perturbation effet of HLz on the membrane poten- TABLE I Releases of p-galatosidase and alkaline phosphatase from E. oli ells treated with WLz or HLz Alkaline phosphatase Control WLz HLz Control WLz HLz min % % NDb ND E. oli ells (lo8 ells/ml) were inubated with 5 pg/ml wild-type lysozyme (WLz) or fusion lysozyme (HLz) in 50 mm potassium phosphate buffer, ph 7.0, for a given time, and then eah enzyme ativity was determined in the supernatants after entrifugation. Enzyme ativities are expressed as a perentage of the total enzyme ativity in the rude ell extrats obtained by the soni treatment. Control ells were treated with the buffer in the absene of lysozyme. Values are the means of three determinations. ND, not determined. Val FIG. 5. Effet of HLz on the dis-cs-(6) fluoresene quenhing indued by valinomyin in E. oli phospholipid liposomes. The reation mixtures ontained 50 mm sodium phosphate, ph 7.5, 1 p~ dis-cs-(5), and 70 pg of 50 mm potassium phosphate (ph 7.5)- ontaining liposomes in a total volume of 1.0 ml. One pl of 10 mm valinomyin (Val) was added (solid arrow) to hyperpolarize the liposomes (interior negative). After reahing a onstant fluoresene quenhing (open arrow), 5 pg (H(5)) or 10 pg (H(I0)) of HLz or 5 pg (W(5)) or 10 pg ( W( IO)) of WLz were added, and 0.2 pl of 0.25 mm nigeriin (Nig) was added as a ontrol. Fluoresene quenhing is expressed as desribed under Experimental Proedures. tial is due to the interation of HLz with the inner membrane through the hydrophobi pentapeptide and the eletrostati interation between the positively harged groups of the lysozyme moleule and the anioni polar heads of the phospholipids of the membrane. Therefore, the ability of HLz to damage the outer membrane and subsequent penetration into the inner membrane may aount for its bateriidal ation to E. oli. Our results support the hypothesis that the killing site of Gram-negative bateria would be the inner membrane (22). It is generally aepted that the ompetene of the membrane fusion orresponds to the inomplete folding of a protein and the presene of an aessible hydrophobi streth to reside within the lipid bilayer when the protein penetrates into the membranes (26, 27). Our experimental data do not exlude the possibility that the onformational hanges (partially unfolded) indiated by the CD spetra of HLz may ontribute to the enhaned bateriidal ation to E. oli by failitating its penetration into the lipid bilayer. We have reported in the previous paper (8) that the palmitoyl lysozyme has the potent antibaterial ativity to E. oli. Based on this observation, the neessary length and onfiguration of the hydrophobi pentapeptide were estimated. The amino aid sequene of the pentapeptide was seleted to provide onformation, whih is postulated to reside within the lipid bilayer (26). The length of the pentapeptide is equal to that of palmiti aid when it forms onformation. Another favorable aspet is that the aromati group-ontaining side hain of Phe and the yli imide harater of Pro might be important features for the spontaneous insertion of protein into the membranes (26). The fat that lysozyme with the inserted hydrophobi pentapeptide an effetively inhibit the growth of E. oli would suggest that suh an approah might be useful for the design of new antimirobial proteins. REFERENCES 1. Wilkinson, R. G., Gemski, P., and Stoker, B. A. D. (1972) J. Gen. MierobioL 70, De Petris, S. (1967) J. Ultrastrut. Res. 19, Murry, R. G. E., Steed, P., and Elson, H. H. (1965) Can. J. MierobioL 11. KA7-5CIl - 4. JolDs, P., and Jolles, J. (1984) Mol. Cell. Biohem. 63, Ohno, N., and Morrison, D. C. (1989) J. Bwl. Chem. 264, hive, L. (1974) Ann. N. Y. Aad. Si. 236,

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