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A bacteria-digesting midgut-lysozyme from Musca domestica (diptera) larvae. Purification, properties and secretory mechanism
Insect Biochem. Molec. Biol. Vol. 23, No. 4, pp. 533 541, 1993 Printed in Great Britain. All rights reserved
0965-1748/93 $6.00 + 0.00 Copyright ~ 1993 Pergamon Press Ltd
A Bacteria-digesting Midgut-lysozyme from Musca domestica (Diptera) Larvae. Purification, Properties and Secretory Mechanism FRANCISCO J. A. LEMOS,* ALBERTO F. RIBEIRO,~" WALTER R. TERRA*~: Received 31 March 1992; revised and accepted 16 October 1992
Two lysozymes were purified to homogeneity from heated acid extracts of Musca domestica larval midguts, using an S-Sepharose column, and a semi-preparative polyacrylamide gel electrophoresis (PAGE). The final yield was 60%. Lysozymes 1 and 2 display Mr 22,000, determined by ultracentrifugation or electrophoresis in non-denaturing conditions, and Mr 17,000, determined by SDS-polyacrylamide gradient gel electrophoresis. Isoelectric focusing showed the following pI values: lysozyme 1, 7.9; lysozyme 2, 8.2. Lysozyme 1 and 2 display identical kinetic properties, which include decrease in activity, displacement of the pH optimum toward acidic values and a Km increase as the ionic strength of the medium becomes higher. The lysozymes are resistant to a cathepsin D-like proteinase present in M. domestica midgut, and display a chitinase activity which is 6-fold higher than that of chicken lysozyme. Lysozyme immunolabeling revealed that lysozyme mainly occurs in secretory vesicles and at the outside surface of microvilli from M. domestica anterior midgut cells. The results showed that M. domestica lysozymes are similar to ruminant stomach lysozyme in being more active at acid pH values, when present in media with physiological ionic strengths, and in being resistant to an acid proteinase derived from the same animal as the lysozyme. Furthermore, the data support the assertion that M. domestica midgut lysozyme is secreted by exocytosis, partly remaining adsorbed to the cell glycocalyx. Lysozyme properties Lysozymepurification Digestive lysozyme Lysozymesecretion Ruminant lysozyme
INTRODUCTION Lysozyme (EC 3.2.1.17) catalyzes the hydrolysis of glycosidic bonds of the peptidoglycan present in bacterial cell walls and causes bacterial cell lysis. Lysozyme is part of the defense mechanism against bacteria and has been described in most animals (Joll~s and Joll6s, 1984), including insects (Dunn, 1986). Lysozyme is also involved in the midgut digestion of bacteria in some organisms, such as ruminants, which harbor a bacterial culture in their foreguts. Bacteria are digested in the ruminant stomach with the aid of a lysozyme (Dobson et al., 1984). This lysozyme displays distinctive properties to be active in stomach, such as a low pH optimum and resistance to pepsin digestion (Dobson et al., 1984). Musca domestica larvae kill bacteria (their major food) in the middle region of the midgut through the combined action of low pH, lysozyme and a cathepsin D-like proteinase (Espinoza-Fuentes and Terra, 1987;
Lemos and Terra, 1991a). Among the insects, the capacity of digesting bacteria in the midgut seem to be an ancestral trait of Diptera Cyclorrhapha (Lemos and Terra, 1991b), which agrees with the fact that most Diptera Cyclorrhapha larvae are saprophagous feeding largely on bacteria (Terra, 1990). Since midgut lysozyme functions in Diptera Cyclorrhapha in a way resembling to that in ruminants, it is probable that the fly lysozyme displays properties similar to that of ruminants. In this paper, the purification and characterization of the midgut lysozyme of M. domestica larvae are described and showed to be similar to the ruminant stomach lysozyme. Furthermore, evidence is presented that lysozyme is secreted by exocytosis from M. domestica midgut cells.
MATERIALS AND METHODS Animals
Larvae of M. domestica (Diptera, Cyclorrhapha, Muscidade) were reared in a mixture of fermented commercial pig food and rice hull (1:2, v/v). The larvae used in this study were actively feeding individuals at third larval instar.
*Departamento de Bioquimica, lnstituto de Quimica, Universidade de Silo Paulo, C.P. 20780, 01498 Silo Paulo, Brasil. t D e p a r t a m e n t o de Biologia, Instituto de Bioci6ncias, Universidade de Sa6 Paulo C.P. 11461, 05499 S~,o Paulo, Brasil. SAuthor for correspondence. 533
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FRANCISCO J. A. LEMOSet al.
Preparation of midgut samples Larvae were rinsed in water, blotted in filter paper, immobilized by placing them on ice and dissected in cold 150mM NaCI. Midguts with contents were stored at - 2 0 ° C until used. Purification of midgut lysozyme About 800 midguts with contents were thawed and homogenized in 0.1 M sodium acetate buffer pH 3.5 using a Potter-Elvehjen homogenizer. The homogenate was centrifuged at 20,000g for 30min at 4°C and the resulting supernatant passed through glass wool. The filtrate was adjusted with the same acetate buffer to contain material from 16.5 animals/ml (this corresponds to about 1.2 mg protein/ml) and was maintained 5 min in a boiling water bath. After cooling the suspension was centrifuged at 10,000g for rain at 4°C. The resulting supernatant was dialysed overnight against 150vol of 20 mM sodium phosphate buffer pH 5.5. A dialysate containing about 13.8 mg of protein was applied to a column (8.4 × i.2 cm i.d.) of S-Sepharose equilibrated with 20 mM sodium phosphate pH 5.5. The column was washed with the same buffer and then was eluted with 0.05~0.5 M NaC1. The active fractions were pooled and dialyzed overnight against 100 vol of water. Density-gradient ultracentrifugation Samples (0.2 ml) of preparations containing 1.5 mg of bovine hemoglobin and 50 #g of bovine liver catalase were layered on the top of 10ml glycerol gradients (10 30%, w/v) made up in 50 mM sodium acetate buffer pH 3.5 or in 50 mM citrate-sodium phosphate pH 6.2. Centrifugations and collection of fractions were performed as described previously (Terra and Ferreira, 1983). Mr values of enzymes assayed in the fractions were calculated by the method of Martin and Ames (1961), using the sedimentation rates of bovine hemoglobin (Mr 64,500) and bovine liver catalase (Mr 232,000) as reference standards. Recoveries of the activities applied to the gradients were 100%. Polyacrylamide gel electrophoresis Electrophoresis in native conditions were run in polyacrylamide gels prepared as described by Hedrick and Smith (1968), using the pH 2.3 system of Saponov (Altschul and Evans, 1967), in glass tubes of 5 mm i.d. and 100 mm length. Samples in water were made up 10% (w/v) in glycerol and 0.02% (w/v) in methyl green before being loaded onto the gel cylinders. The electrophoretic separation was achieved with a current of 5 mA/column at 4'~C. The gels were fractionated in an acrylamide gel fractionator (Autogeldivider Savant Instruments, U.S.A,) with water. Fractions of six drops (corresponding to 1.3 mm of gel) were collected with the aid of a fraction collector, and after standing 3 h at 4°C were used as a source of enzyme. Recoveries of the activities applied to the gels were 60-80%. The densitometric tracings of the gel cylinders stained with 0.2% Coomassie brilliant blue in water, methanol and acetic
acid (4:5:1, v/v/v) were performed with the aid of a densitometer from E-C Apparatus Corp. (U.S.A.). Electrophoresis in denaturing conditions were performed as follows. Samples were combined with sample buffer containing 60 mM Tris-HCl buffer pH 6.8, 2.5% (w/v) SDS, 0.36mM /~-mercaptoethanol, 0.5 mM EDTA, 10% (v/v) glycerol and 0.005% (w/v) bromophenol blue. The samples were heated for 2 rain at 95°C in a water bath before being loaded onto a 10-20% (w/v) polyacrylamide gradient gel slab containing 0.1% (w/v) SDS (Laemmli, 1970). The gels were run at constant voltage of 100 V and stained for protein using a silver stain (Blum et al., 1987). Lysozymes Mr values were calculated according to Lambin et al. (1987) using the following Mr standards (kit MW-SDS-70L purchased from Sigma): albumin, 66,000; ovalbumin, 45,000; glyceraldehyde three phosphate dehydrogenase, 36,000; carbonic anhydrase, 29,000, trypsinogen, 24,000; soybean trypsin inhibitor, 20,100; ~-lactalbumin, 14,200.
Isoelectric focusing in polyaerylamide gels Isoelectric focusing was performed as described by Terra et al. (1978), in columns of 7% polyacrylamide gels containing 1% ampholytes pH 3-10. Samples were applied after polymerization and pre-focusing (30 rain at 31 v/cm) on the top of the alkaline side of the gels. Recoveries of the activities applied to the gels were 70-90%. Protein determination and hydrolase assays Protein was determined according to Bradford (1976) using ovalbumin as a standard. Lysozyme activity was determined by measuring the decrease in turbidity at 650nm of a suspension (1 mg/ml) of Micrococcus lisodeikticus in (except otherwise specified) 50 mM citrate-sodium phosphate pH 3.5 containing 150mM NaC1, in a reaction volume of 1 ml. The reaction was stopped by the addition of 0.5ml of 0.5 M sodium carbonate and the absorbance was read. One lysozyme unit is the amount of enzyme that causes a change in the absorbance of the reaction medium of 0.01 U/min. Chitinase activity was measured using the viscometric assay of Winicur and Mitchell (1974), as modified by Lemos and Terra (1991b), with chitosan as substrate. The slopes of the plots of the inverse of specific viscosity against time in min were calculated. One chitinase unit is the amount of enzyme that results in a slope of 1000/min. In each determination of all the enzyme activities, incubations were carried out at 30°C for at least four different periods of time and the initial rates of hydrolysis were calculated. All assays were performed under conditions such that activity was proportional to protein concentration and to time. Controls without enzyme or without substrate were included. Kinetic studies The effect of pH and ionic strength of reaction media on the activity of the midgut lysozyme was determined
A MIDGUT LYSOZYME using the following buffers (Miller and Golder, 1950): glycine-HC1 (pH 2.0-3.5), sodium acetate (pH 4.0-5.5), sodium phosphate (6.0-7.5) and glycine-NaOH (pH 8.0-9.0) prepared with ionic strengths from 0.02 up to 0.2. The effect of M. lysodeikticus concentration on midgut lysozyme activity was determined in different buffers at 30°C. Km values (means and SEM) were determined by a weighted linear regression by the procedure of Wilkinson (1961), using a program written in BASIC by Oestreicher and Pinto (1983).
Preparation of antibodies and double immunodiffusion A 1-ml sample containing 50/~g of purified lysozyme 1 in 0.1 M NaC1 was combined with 1 ml of Freund's complete adjuvant, and the resulting emulsion was injected subcutaneously into the rabbit. After 4 weeks, a similar injection was administered, but with Freund's incomplete adjuvant. Two weeks after the last injection, the rabbit was bled. The blood was left standing 1 h at 37°C and overnight at 4°C before being centrifuged at 3,000 g, 10 min at 4°C. The clear supernatant was added to a suitable solution to become 50% saturated in ammonium sulfate, pH 6.8. After 18 h at room temperature (25°C), the resulting suspension was centrifuged at 50,000g, 15 min, 4°C, the pellet was suspended in 50% saturated ammonium sulfate, pH 6.8, and the suspension was pelleted at 5000g, 15 min, 4°C. The last step was repeated once again, and the final pellet was solubilized in 0.1 M NaC1 and dialyzed against 1000 vol of 0.1 M NaC1 overnight with one change of solvent. The sample was centrifuged at 10,000 g for 10min at 4°C and the supernatant kept frozen at -20°C until used as antiserum. Double immunodiffusion was performed according to the method of Ouchterlony (1968) using 1% agarose in 0.1 M sodium phosphate buffer, pH 7.0, containing 0.15 M NaC1 and 0.1% sodium azide. The samples were left diffusing for 48 h at room temperature in a humid chamber containing a piece of cotton moistened with 5 % (w/v) phenol to inhibit the growth of fungi. The slabs were washed in 0.15 M NaC1 for 48 h at 4°C, dried at 45°C, stained for 5 min with 0.09% (w/v) Coomassie blue in ethanol : acetic acid :water (25 : 8 : 85). The antiserum produced against lysozyme 1, when tested by double immunodiffusion against purified lysozyme 1, was reactive up until a 50-fold dilution. Electron microscopy and immunocytochem&try For work with electron microscope, larvae were dissected in their own hemolymph and anterior midgut (fore-midgut) pieces were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 for 2 h at 4°C. After rinsing with 0.2 M sucrose in the same cacodylate buffer, the midgut pieces were post-fixed in 1% osmium tetroxide in the same cacodylate buffer for 1 h at 4°C and washed in 0.1 M NaC1. En-block staining was performed in uranyl acetate for 16-18h. After dehydration in graded ethanol at room temperature, the materials were
535
embedded in Polylite 8001 polyester resin (Resana S/A, Brazil; Coiro et al., 1972). Ultra-thin sections were cut using a LKB Nova ultramicrotome, stained with lead acetate and examined in a Zeiss EM 9 S2 electron microscope. For immunolocalization of lysozyme in tissue sections, fore-midgut pieces were fixed in 4% formaldehyde with 0.3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 3 h at 4°C. After rinsing with phosphate buffer, the material was dehydrated in graded ethanol, at room temperature, and embedded in L. R. White acrylic resin (Ladd Research Industries Inc., U.S.A.). Ultra-thin sections were cut on the ultramicrotome and collected on 200 mesh colloidon-coated nickel grids. The grids were then floated on drops of saturated sodium metaperiodate for 10min, rinsed in 0.02 M Tris-HC1 buffer pH 7.4 containing 0.83% (w/v) NaC1 (TBS) and placed on drops of 10% fetal calf serum (FCS) in TBS plus 0.025% Tween 20 and 0.025% Triton X-100, for 15 min, to block nonspecific reactions. The sections were then incubated with the primary antibody diluted 1: 500 in TBS containing 5% FCS, 0.025% Tween 20 and 0.025% Triton X-100, overnight at 4°C. As controls, sections of the same blocks were incubated with non-immune serum in the same conditions. Non-immune serum was treated as described above for immune serum. The grids were washed in TBS with detergents and floated on drops of 10% CFS in the same solution for 10min at room temperature. The material was then incubated with protein A conjugated to 10 nm Gold (E. Y. Labs Inc., U.S.A.) diluted 1:40 in the solution above, with 5% FCS, for 1 h at room temperature. Finally the grids were washed in TBS followed by distilled water, stained with uranyl acetate and lead citrate and examined in the electron microscope. RESULTS
Purification of the M. domestica midgut lysozymes Lysozyme activity in M. domestica larval midgut homogenates is stable at pH 3.5 (Espinoza-Fuentes and Terra, 1987), and it was found that a large amount of midgut proteins is rendered insoluble at this pH. Due to that, midgut homogenates were prepared at pH 3.5, and after centrifuging the resulting supernatants were used as a source of lysozyme. This acid extract has a lysozyme specific activity c 6-fold higher than the specific activities of midgut homogenates prepared in water. Two lysozymes from the M. domestica midgut acid extracts are resolved by electrophoresis [Fig. I(A)], although they display identical Mr values, as determined by density-gradient ultracentrifugation at pH 3.5 [Mr 22,000 ___1000, Fig. I(B)] and pH 6.2 (not shown), or by electrophoresis at different polyacrylamide gel concentrations [Mr 22,000 + 500, Fig. I(C)]. These Mr values coincide with that one determined previously using M. domestica midgut homogenates prepared in neutral media (Terra et al., 1988). These results suggest that the lysozymes differ only in net charge. Isoelectric focusing
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FIGURE 1. Physical properties of lysozymes in the acid extract of midgut homogenates from M. domestica larvae. (A) Electrophoretic separation at pH 2.3 in 6%, polyacrylamide gel cylinder. (B) Sedimentation profile in glycerol gradient at pH 3.5. Fractions were collected from the bottom of the tube. M r marker: H, bovine hemoglobin (Mr 64,500). (C) Determination of the M Fof lysozymes (L) by electrophoresis in cylinders of different concentrations of polyacrylamide gel by the method of Hedrick and Smith (1968). M r markers: C, chicken lysozyme (Mr 14,500), M, myoglobin (M, 17,800), T, bovine trypsin (M r 22,500). m in ordinate is the slope of a plot (100 × log Rm) against concentration of the gel. (D) Isoelectric focusing. of the acid extract confirms this hypothesis, showing the existence of m a j o r lysozymes with pI values of 7.90 _ 0.05 and 8.20 _+ 0.03 [Fig. I(D)]. The m i n o r lysozyme activity which displays pI 5.5 [Fig. I(D)] is very variable, being absent from some preparations. Heating the acid extract at 95°C for 5 min results in an increase in the lysozyme specific activity (Table 1), as could be anticipated by the finding that this enzyme is stable in that temperature (Terra et al., 1988). Lysozyme activity is retained in a S-Sepharose column, from which it is eluted with a NaC1 gradient (Fig. 2). The yield of this purification was 80% and the specific activity of the purified lysozyme was 8 9 3 0 U / m g protein (Table 1). Electrophoresis o f the lysozyme preparation resulted in the resolution of two lysozyme activities, corresponding to the m a j o r proteins in the gel, and of a m i n o r a m o u n t o f contaminants (5% o f the proteins in the gel) (Fig. 3). Further purification of the lysozymes were accomplished
eluting gel fractions corresponding to lysozyme 1 and 2 from electrophoretic runs similar to that shown in Fig. 3. The specific activities (U/mg) o f the lysozymes purified by electrophoresis were: lysozyme 1, 6860 + 140, lysozyme 2, 7850 + 140. Since the recovery from polyacrylamide gels is a b o u t 70%, the final yield of the purification is then a b o u t 60%. S D S - P A G E o f purified lysozyme 1 and 2 resulted in a single protein band for each preparation, with an identical Mr value of 17,000+ 1000 (Fig. 4). Isoelectric focusing of the purified lysozymes showed that lysozymes 1 and 2 have pI values of 7.9 and 8.2, respectively (Fig. 5). The m i n o r lysozyme with pI 5.5 was lost during the purification steps. Midgut extracts challenged against anti-lysozyme 1 serum result in a single precipitation line in double diffusion test [Fig. 6(A)]. This demonstrates that the serum is mono-specific. Antiserum produced against
TABLE 1. Purification of lysozymes from M. domestica larval midgut*
Fraction Acid extract Heated extract S-Sepharose eluate
Volume (ml)
Total protein (mg)
Total activity (Units)
Specific activity (Units/rag)
Purification factor
Yield (%)
49 56 22
57.5 13.8 2.8
31,200 24,200 25,000
542 1754 8930
1 3.2 16.5
100 78 80
*Acid extract is the supernatant obtained after centrifuging M. domestica midguts homogenized in 0.1 M sodium acetate buffer pH 3.5. This extract displays a lysozyme specific activity about 6-fold higher than the specific activities of midgut homogenates prepared in water.
A MIDGUT LYSOZYME
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FIGURE 2. Chromatography on S-Sepharose of M. domesticamidgut homogenates after treatment with acid and heat. The column (8.4 x 1.2 cm i.d.) was equilibrated with 20 mM sodium acetate pH 5.5. About 14 mg of protein in the same buffer was applied to the column with a flow rate of 17ml/h. The column was washed with 20mM sodium acetate pH 5.5 and then eluted with 0.05--0.5M NaC1. Fractions of 2 ml were collected at a flow rate of 45 ml/h. Nacl (. . . . ); protein (O); lysozyme activity (O). lysozyme 1 is reactive with purified lysozyme 2 [Fig. 6(B)], suggesting that the lysozymes are immunologically identical.
Kinetic properties of M. domestica midgut lysozymes The kinetic properties o f lysozymes 1 and 2 seem to be similar in relation to the effect o f pH, ionic strength and the concentration o f substrate (Fig. 7). Due to that, we performed the last experiments with only one o f the lysozymes, assuming that the results would be identical whichever lysozyme be used. 2
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FIGURE 4. Electrophoresis in SDS 10-20% polyacrylamide gradient gel slab. Lane A, Mr markers, top to bottom: 66, 45, 36, 29, 24, 20.1 and 14.2 K. Lane B, purified lysozyme 1 (0.19#g). Lane C, purified ]ysozyme 2 (0.60pg). Lane D, soluble fraction of anterior midgut homogenate (11/~g of proteins). Other details in Materials and Methods. The M. domestica midgut lysozyme activity decreases [Fig. 7(A),(B)], its p H o p t i m u m is displaced toward acidic p H values [Fig. 7(A),(B)] and its Km value increases (Fig. 8) as the ionic strength o f its assay medium becomes higher. Similar results were also found for other enzymes (Maurel and D o u z o u , 1976, D o b s o n et al., 1984). Fig. 7(C) shows a decrease o f lysozyme activity caused by excess substrate. Similar results were found with goose lysozyme (Locquet et al., 1968). Lysozyme 2 maintained in the presence o f M. domestica midgut homogenates containing 200 m U o f cathepsin D for 180min in 1 0 r a M acetate buffer p H 3.0 at 30°C retains full activity. This confirms previous observations made with crude preparations o f lysozyme, according to which this enzyme is stable in the acid M. domestica middle region o f the midgut that contains a cathepsin D (Espinoza-Fuentes and Terra, 1987). Nevertheless, lysozyme 2 incubated with 85 m U o f bovine pepsin loses 40% o f its activity after 120 min in 10 m M acetate buffer p H 3.0 at 30°C.
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FIGURE 5. Isoelectric focusing of purified lysozyme 1 (O) and 2 (O). Profiles obtained from other preparations are similar to those shown.
FRANCISCO J. A. LEMOS et al.
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DISCUSSION
A
B
Properties of the M. domestica midgut lysozyme
FIGURE 6. Ouchterlony double diffusion test. A, the center well contains serum against lysozyme1; wells 1~ contain different amounts (690, 340, 170, 90, 40, 20 #g of protein, respectively)of the soluble fraction of anterior midgut homogenate. B, the center well contains serum against lysozyme I; wells 1, 3 and 5 contain 0.17/~g of lysozyme 1; wells 2, 4 and 6 contain 0.55/~g of lysozyme 2. Figure 9 shows that 37 units of M. domestica lysozyme displays 1.7 m U of chitinase, whereas 80 units of chicken lysozyme presents 0 . 6 m U of chitinase. Thus, the M. domestica midgut lysozyme shows a chitinase activity about 6-fold higher than that of chicken lysozyme.
Fore-midgut immunolocalization of M. domestica lysozyme There are in fore-midgut cells morphological features which are usually associated with secretory activity, such as many elements of Golgi, plenty of rough endoplasmic reticulum and conspicuous secretory vesicles [Fig. 10(A)]. Figure 10(B) shows that lysozyme immunolabeling is mainly observed in secretory vesicles and at the outside surface of microvilli. It should be noted, however, that only part of the secretory vesicles is labeled. Control preparations showed no immunolabeling above background (results not shown), confirming that immunoreactivity of vesicles content and microvillar surface was due specifically to the presence of lysozyme.
M. domestica larvae display two lysozymes which have an identical Mr of 22,000 + 1000 when measured by density-gradient ultracentrifugation (pH 3.5) and by electrophoresis (pH 2.3) in native conditions performed in polyacrylamide gel of different concentrations. When electrophoresis in SDS-polyacrylamide gradient gel was employed, the two enzymes display Mr 17,000_+ 1000. The last mentioned value is probably the best estimate for the lysozyme Mr value. Thus, M. domestica midgut lysozyme has a Mr value which is similar to that of most lysozymes, including those from insect hemolymph (Jolles et al., 1979; Hultmark et al., 1980; Zachary and Hoffman, 1984; Schneider, 1985). The M. domestica midgut lysozymes differ in pI values, which are: lysozyme 1, 7.9; lysozyme 2, 8.2. The kinetic properties of lysozymes 1 and 2 seem to be similar in relation to the effect of pH, ionic strength and the concentration of substrate. The purified lysozymes are also immunologically identical. Thus, it is probable that lysozymes 1 and 2 have polypeptide chains differing in a few amino acid residues. These results agree with the findings in another Diptera Cyclorrhapha, Drosophila melanogaster. This insect displays several lysozyme genes which are expressed in the midgut (Kylsten et al., 1992). The pl values of M. domestica midgut lysozymes are lower than those of most animals (Imoto et al., 1972), although they are similar to that of ruminant lysozymes (Pahud and Widner, 1982). The lower pI values displayed by these enzymes are supposed to result from a few amino acid substitutions in the surface of the lysozyme molecule (Dobson et al., 1984). M. domestica midgut lysozyme shows a chitinase activity about 6-fold higher than that of chicken
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FIGURE 7. Kinetic properties of purified lysozyme 1 and 2. (A) Activity of lysozyme 1 in function of pH and ionic strength. The ionic strengths used were: 0.02 (0); 0.04 (/X); 0.1 (O); 0,2 (riD- (B) Activity of lysozyme2 in function of pH and ionic strength. Ionic strengths as before. (C) Effectof substrate concentration on the activities(pH, 4; ionic strength, 0.04) of lysozyme 1 (Q) and 2 (O). [S], concentration of M. lysodeikticusin mg/ml. The straight line was adjusted to the points using a weighted linear regression procedure. Each data point in this figure is the result of four assays in the same sample.
A MIDGUT LYSOZYME
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Recently, it was shown that the Lys D lysozyme gene, which is expressed in D. melanogaster midguts, becomes depressed after bacterial injections into the fly hemocelle (Kylsten et al., 1992). This confirms that lysozyme is not part of the defense mechanism against bacteria in Diptera Cyclorrhapha, and lends support to the proposal that lysozyme is a true digestive enzyme in these insects.
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FIGURE 8. Effectof ionicstrengthon Kmvaluesof lysozyme2 at pH 4.0. Kmvaluesdeterminedusinga weightedlinearregressionprocedure (n = 5). lysozyme (see Results), although only 20% of the M. domestica midgut chitinase activity can be accounted for by the midgut lysozyme (Lemos and Terra, 1991b). A high chitinase activity seems to be usual among insect lysozymes. The lysozyme isolated from Ceratitis capitata eggs is 350-fold more active on chitin than the chicken lysozyme (Fernandez-Sousa et al., 1977). Midgut lysozymes 1 and 2 become less active and their pH-activity profile is displaced towards the acid side as the ionic strength of the medium increases. Although this was found with other lysozymes (Maurel and Douzou, 1976), the magnitude of the changes observed are only comparable with ruminant lysozymes. Thus, the pH optimum of the cow lysozyme varies from 6.5 to 5.0 as the ionic strength is increased from 0.02 to 0.1 (Dobson et al., 1984), whereas that of M. domestica midgut lysozyme, from 6.5 to 4.5. At an ionic strength of 0.2, the pH optimum of the midgut lysozyme is as low as 3.8. These results may be explained by the polyanionic structure of the bacterial cell walls which, due to a negative electrostatic field, influences the physico-chemical properties of the microenvironment where the catalytic reaction proceeds, decreasing the local pH. At high ionic strength, the electrostatic potential is decreased and the local pH approaches that of the bulk solution (Maurel and Douzou, 1976). The linear dependence of the logarithm of the lysozyme Km with the logarithm of the ionic strength (Fig. 8) is thought to result from the effect of ionic strength on the interaction of the negatively charged bacterial cell walls with the positively charged lysozyme molecules (Maurel and Douzou, 1976). Lysozyme as a digestive enzyme in insects and vertebrates
The ability of M. domestica to use lysozyme as a digestive enzyme seems to have evolved in parallel to that of vertebrates. Thus, lysozyme is absent from most tissues and is present in large amounts in the acidic stomachs of ruminants (Prieur, 1986) and middle midguts of M. domestica (Lemos and Terra, 1991b). M. domestica midgut lysozyme is resistant to the midgut cathepsin D-like proteinase, whereas ruminant lysozyme is resistant to pepsin (Dobson et al., 1984). Both enzymes are more active at acid pH values when present in media with physiological ionic strengths.
The secretory mechanism lysozyme
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domestica
midgut
Lysozyme is not usually found in the luminal contents of the insect midgut (see Lemos and Terra, 1991b, for references). Furthermore, insect midgut secretory mechanisms are poorly known (Chapman, 1985). Hence, the mechanism by which M. domestica lysozyme is secreted into midgut lumen deserves attention. Lysozyme is most active in the lumen of the middle region of the M. domestica midgut. In midgut cells, lysozyme occurs mainly in the fore-midgut, which led to the hypothesis that this region is responsible for lysozyme secretion (Espinoza-Fuentes and Terra, 1987). Subcellular fractionation of fore-midgut cells showed that lysozyme is recovered in major amounts in fraction P,, characterized by the occurrence of large vesicles displaying microvilli, and in fraction P3, which displays mitochondria, unidentified vesicles and secretory vesicles. Based on the fact that freezing and thawing sets lysozyme free on both fractions Pl and P3, a secretory mechanism for lysozyme was proposed. According to this mechanism, lysozyme occurs in contents of secretory vesicles visible in the electron micrographs of fraction P3 and, after exocytosis, becomes partly adsorbed in the glycocalyx (explaining its presence in fraction P0 (Espinoza-Fuentes et al., 1987). This hypothesis was supported by the finding that fraction P3 displays large structures (apparent buoyant density 1.21 g/cm3), from which lysozyme is set free on freezing and thawing (Espinoza-Fuentes et al., 1987). Exocytosis of lysozyme and its partial adsorption to the microvillar glycocalyx of fore-midgut cells were further evidenced in the present paper. Immunolabeling has shown that lysozyme occurs 2.6
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FIGURE 9. Activityof M. domestica lysozyme,37 Units (Q) and chickenlysozyme,80 Units (O) upon chitosan.Viscometricassay.(A) Assayin 25 mM sodiumacetatebufferpH 3.5 containing0.1 M NaC1. (B) Assay in 25 mM sodium acetate buffer pH 5.5 containing0.1 M NaC1. Resultsobtainedwith other preparations were similarto those shown. The source of M. domestica lysozymewas the S-Sepharose eluate.
540
FRANCISCO J. A. LEMOS et al.
FIGURE 10. Apex of a cell from the M. domestica fore-midgut. (A) Morphological features associated with secretion. Note the elements of Golgi (G), rough endoplasmic reticulum (RER), secretory vesicles (SV) and many mitochondria (Mi). (B) Immunolocalization of lysozyme. Note labeling in part of the secretory vesicles (arrowheads) and in microvilli (Mv). Bar = 1 #m. in the c o n t e n t s o f secretory vesicles w h i c h are s i m i l a r to those seen a s s o c i a t e d to e l e m e n t s o f G o l g i . F u r t h e r m o r e , l y s o z y m e was also f o u n d at the o u t s i d e surface o f the cell microvilli. It is r e m a r k a b l e , h o w e v e r , t h a t l y s o z y m e i m m u n o l a b e l i n g o c c u r s o n l y in p a r t o f the secretory vesicles. T h i s suggests t h a t secretory vesicles in M. d o m e s t i c a f o r e - m i d g u t cells differ in c o n t e n t . T h e data, h o w e v e r , are n o t sufficient to c o n c l u d e if the differences are q u a n t i t a t i v e o r q u a l i t a t i v e . REFERENCES Altschul A. and Evans W. J. (1967) Zone electrophoresis with polyacrylamide gel. Meth. Enzym. 11, 179-194.
Blum H., Beier H. and Gross H. J. (1987) Improved silver staining of plant proteins, RNA, and DNA in polyacrylamide gels. Electrophoresis 8, 93-99. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. Chapman R, F. (1985) Structure of the digestive system. In Comprehensive Insect Physiology Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol, 4, pp. 165-211. Pergamon Press, New York. Coiro J. R. R., Weigi D. R., Kisielius J., Menezes H. and Bilotta J. A. T. (1972) A new embedding medium (Polylite 80001) for biological material. Cienc. Cult., Sg*o Paulo 24, 660462. Dobson D. E., Prager E. M. and Wilson A. C. (1984) Stomach lysozymes of ruminants. I. Distribution and catalytic properties. J. biol. Chem. 259, 11,607-11,616.
A MIDGUT LYSOZYME Dunn P. E. (1986) Biochemical aspects of insect immunology. A. rev. Ent. 31, 321 339. Espinoza-Fuentes F. P. and Terra W. R. (1987) Physiological adaptations for digesting bacteria. Water fluxes and distribution of digestive enzymes in Musca domestica larval midgut. Insect Biochem. 17, 809-817. Fernandez-Sousa J. M., Gavilanes J. G., Municio A. M., PerezAranda A. and Rodrigues R. (1977) Lysozyme from the insect Ceratitis capitata eggs. Eur. J. Bioehem. 72, 25-33. Hedrick J. L. and Smith A. J. (1968) Size and charge isomer separation and estimation of molecular weights of proteins by disc-gel electrophoresis. Archs Biochem. Biophys. 126, 155-164. Hultmark D., Steiner H., Rasmuson T. and Boman H. G. (1980) Insect immunity. Purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora ceeropia. Eur. J. Biochem. 106, 7-16. Imoto T., Johnson L. N., North A. C. T., Phillips D. C. and Rupley J. A. (1972) Vertebrate lysozymes. In The Enzymes (Edited by Boyer P. D.), 3rd Edn, Vol. 7, pp. 665 868. Academic Press, New York. Joll6s P. and Joll6s J. (1984) What's new in lysozyme research? Molec. cell. Bioehem. 63, 165 189. Joll6s J., Schoentgen F., Croizer G., Croizer L. and Joll6s P. (1979) Insect lysozymes from three species of Lepidoptera: their structural relatedness to the c (chicken) type lysozyme. J. Molec. Evol. 14, 267-271. Kylsten P., Kimbrell D. A., Daffre S., Samakovlis C. and Hultmark D. (1992) The lysozyme locus in Drosophila melanogaster: different genes are expressed in midgut and salivary glands. Mol. gen. Genet. 232, 335-343. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227, 680~585. Lambin P., Rocbu D. and Fine J. M. (1976) A new method for determination of molecular weights of proteins by electrophoresis across a sodium dodecyl sulphate (SDS) polyacrylamide gradient gel. Analyt. Bioehem. 74, 567-575. Locquet J. P., Saint-Blancard J. and Joll6s P. (1968) Apparent affinity constants of lysozymes from different origins for Mieroeoceus lysodeikticus cells. Bioehim. biophys. Aeta 51, 150-153. Lemos F. J. A. and Terra W. R. (1991a) Properties and intracellular distribution of a cathepsin D-like proteinase active at the acid region of Musca domestiea midgut. Insect Biochem. 21, 457-465. Lemos F. J. A. and Terra W. R. (1991b) Digestion of bacteria and the role of midgut lysozyme in some insect larvae. Comp. Bioehem. Physiol. 100B, 265 268. Martin R. G. and Ames B. N. (1961) A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J. biol. Chem. 236, 1372-1379.
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Maurel P. and Douzou P. (1976) Catalytic implications of electrostatic potentials: the lytic activity of lysozyme as a model. J. Molec. Biol. 102, 253-264. Miller G. L. and Golder R. H. (1950) Buffers of pH 2 to 12 for use in electrophoresis. Archs Biochem. 29, 420-423. Oestreicher E. G. and Pinto G. F. (1983) Pocket computer program for fitting the Michaelis Menten equation. Comput. biol. Med. 13, 309-315. Ouchterlony O. (1968) Handbook of lmmunodiffusion and Immunoelectrophoresis. Ann Arbor Science Publishers, Ann Arbor. Pahud J. J. and Widmer F. (1982) Calf rennet lysozyme. Bioehem. J. 201, 661-664. Prieur D. J. (1986) Tissue specific deficiency of lysozyme in ruminants. Comp. Biochem. Physiol. 85B, 349 353. Schneider P. M. (1985) Purification and properties of three lysozymes from hemolymph of the cricket, Gryllus bimaculatus (De Geer). Insect Biochem. 15, 463-470. Terra W. R. (1990) Evolution of digestive systems of insects. A. rev. Ent. 35, 181-200. Terra W. R. and Ferreira C. (1983) Further evidence that enzymes involved in the final stages of digestion of Rhynchosciara americana do not enter the endoperitrophic space. Insect Biochem. 13, 143-150. Terra W. R., Espinoza-Fuentes F. P. and Ferreira C. (1988) Midgut amylase, lysozyme, aminopeptidase, and trehalase from larvae and adults of Musca domestica. Archs Insect Bioehem. Physiol. 9, 283 297. Terra W. R., Ferreira C. and De Bianchi A. G. (1978) Physical properties and Tris inhibition of an insect trehalase and a thermodynamic approach to the nature of its active site. Biochim. biophys. Aeta 524, 131-141. Wilkinson G. N. (1961) Statistical estimations in enzyme kinetics. Biochem. J. 80, 324~332. Winicur S. and Mitchell H. K. (1974) Chitinase activity during Drosophila development. J. Insect Physiol. 20, 1795 1805. Zachary D. and Hoffmann D. (1984) Lysozyme is stored in the granules of certain hemocyte types in Locusta. J. Insect Physiol. 30, 405-411.
Acknowledgements--We are much indebted to Dr C. Ferreira, for helpful discussions; to B. P. Jord~o, M.Sc. for SDS PAGE and Ouchterlony tests; to Dr A. Sesso, for electron microscope facilities and for his help in the immunocytochemical analyses; and to Miss L. Y. Nakabayashi, Mr M. V. Cruz and Mr W. Caldeira for technical assistance. This work was supported by the Brazilian Research Agencies FAPESP, CNPq and FINEP. F. J. A. Lemos is a graduate fellow of CNPq and A. F. Ribeiro and W. R. Terra are staff members of their respective departments and research fellows of CNPq.
0965-1748/93 $6.00 + 0.00 Copyright ~ 1993 Pergamon Press Ltd
A Bacteria-digesting Midgut-lysozyme from Musca domestica (Diptera) Larvae. Purification, Properties and Secretory Mechanism FRANCISCO J. A. LEMOS,* ALBERTO F. RIBEIRO,~" WALTER R. TERRA*~: Received 31 March 1992; revised and accepted 16 October 1992
Two lysozymes were purified to homogeneity from heated acid extracts of Musca domestica larval midguts, using an S-Sepharose column, and a semi-preparative polyacrylamide gel electrophoresis (PAGE). The final yield was 60%. Lysozymes 1 and 2 display Mr 22,000, determined by ultracentrifugation or electrophoresis in non-denaturing conditions, and Mr 17,000, determined by SDS-polyacrylamide gradient gel electrophoresis. Isoelectric focusing showed the following pI values: lysozyme 1, 7.9; lysozyme 2, 8.2. Lysozyme 1 and 2 display identical kinetic properties, which include decrease in activity, displacement of the pH optimum toward acidic values and a Km increase as the ionic strength of the medium becomes higher. The lysozymes are resistant to a cathepsin D-like proteinase present in M. domestica midgut, and display a chitinase activity which is 6-fold higher than that of chicken lysozyme. Lysozyme immunolabeling revealed that lysozyme mainly occurs in secretory vesicles and at the outside surface of microvilli from M. domestica anterior midgut cells. The results showed that M. domestica lysozymes are similar to ruminant stomach lysozyme in being more active at acid pH values, when present in media with physiological ionic strengths, and in being resistant to an acid proteinase derived from the same animal as the lysozyme. Furthermore, the data support the assertion that M. domestica midgut lysozyme is secreted by exocytosis, partly remaining adsorbed to the cell glycocalyx. Lysozyme properties Lysozymepurification Digestive lysozyme Lysozymesecretion Ruminant lysozyme
INTRODUCTION Lysozyme (EC 3.2.1.17) catalyzes the hydrolysis of glycosidic bonds of the peptidoglycan present in bacterial cell walls and causes bacterial cell lysis. Lysozyme is part of the defense mechanism against bacteria and has been described in most animals (Joll~s and Joll6s, 1984), including insects (Dunn, 1986). Lysozyme is also involved in the midgut digestion of bacteria in some organisms, such as ruminants, which harbor a bacterial culture in their foreguts. Bacteria are digested in the ruminant stomach with the aid of a lysozyme (Dobson et al., 1984). This lysozyme displays distinctive properties to be active in stomach, such as a low pH optimum and resistance to pepsin digestion (Dobson et al., 1984). Musca domestica larvae kill bacteria (their major food) in the middle region of the midgut through the combined action of low pH, lysozyme and a cathepsin D-like proteinase (Espinoza-Fuentes and Terra, 1987;
Lemos and Terra, 1991a). Among the insects, the capacity of digesting bacteria in the midgut seem to be an ancestral trait of Diptera Cyclorrhapha (Lemos and Terra, 1991b), which agrees with the fact that most Diptera Cyclorrhapha larvae are saprophagous feeding largely on bacteria (Terra, 1990). Since midgut lysozyme functions in Diptera Cyclorrhapha in a way resembling to that in ruminants, it is probable that the fly lysozyme displays properties similar to that of ruminants. In this paper, the purification and characterization of the midgut lysozyme of M. domestica larvae are described and showed to be similar to the ruminant stomach lysozyme. Furthermore, evidence is presented that lysozyme is secreted by exocytosis from M. domestica midgut cells.
MATERIALS AND METHODS Animals
Larvae of M. domestica (Diptera, Cyclorrhapha, Muscidade) were reared in a mixture of fermented commercial pig food and rice hull (1:2, v/v). The larvae used in this study were actively feeding individuals at third larval instar.
*Departamento de Bioquimica, lnstituto de Quimica, Universidade de Silo Paulo, C.P. 20780, 01498 Silo Paulo, Brasil. t D e p a r t a m e n t o de Biologia, Instituto de Bioci6ncias, Universidade de Sa6 Paulo C.P. 11461, 05499 S~,o Paulo, Brasil. SAuthor for correspondence. 533
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FRANCISCO J. A. LEMOSet al.
Preparation of midgut samples Larvae were rinsed in water, blotted in filter paper, immobilized by placing them on ice and dissected in cold 150mM NaCI. Midguts with contents were stored at - 2 0 ° C until used. Purification of midgut lysozyme About 800 midguts with contents were thawed and homogenized in 0.1 M sodium acetate buffer pH 3.5 using a Potter-Elvehjen homogenizer. The homogenate was centrifuged at 20,000g for 30min at 4°C and the resulting supernatant passed through glass wool. The filtrate was adjusted with the same acetate buffer to contain material from 16.5 animals/ml (this corresponds to about 1.2 mg protein/ml) and was maintained 5 min in a boiling water bath. After cooling the suspension was centrifuged at 10,000g for rain at 4°C. The resulting supernatant was dialysed overnight against 150vol of 20 mM sodium phosphate buffer pH 5.5. A dialysate containing about 13.8 mg of protein was applied to a column (8.4 × i.2 cm i.d.) of S-Sepharose equilibrated with 20 mM sodium phosphate pH 5.5. The column was washed with the same buffer and then was eluted with 0.05~0.5 M NaC1. The active fractions were pooled and dialyzed overnight against 100 vol of water. Density-gradient ultracentrifugation Samples (0.2 ml) of preparations containing 1.5 mg of bovine hemoglobin and 50 #g of bovine liver catalase were layered on the top of 10ml glycerol gradients (10 30%, w/v) made up in 50 mM sodium acetate buffer pH 3.5 or in 50 mM citrate-sodium phosphate pH 6.2. Centrifugations and collection of fractions were performed as described previously (Terra and Ferreira, 1983). Mr values of enzymes assayed in the fractions were calculated by the method of Martin and Ames (1961), using the sedimentation rates of bovine hemoglobin (Mr 64,500) and bovine liver catalase (Mr 232,000) as reference standards. Recoveries of the activities applied to the gradients were 100%. Polyacrylamide gel electrophoresis Electrophoresis in native conditions were run in polyacrylamide gels prepared as described by Hedrick and Smith (1968), using the pH 2.3 system of Saponov (Altschul and Evans, 1967), in glass tubes of 5 mm i.d. and 100 mm length. Samples in water were made up 10% (w/v) in glycerol and 0.02% (w/v) in methyl green before being loaded onto the gel cylinders. The electrophoretic separation was achieved with a current of 5 mA/column at 4'~C. The gels were fractionated in an acrylamide gel fractionator (Autogeldivider Savant Instruments, U.S.A,) with water. Fractions of six drops (corresponding to 1.3 mm of gel) were collected with the aid of a fraction collector, and after standing 3 h at 4°C were used as a source of enzyme. Recoveries of the activities applied to the gels were 60-80%. The densitometric tracings of the gel cylinders stained with 0.2% Coomassie brilliant blue in water, methanol and acetic
acid (4:5:1, v/v/v) were performed with the aid of a densitometer from E-C Apparatus Corp. (U.S.A.). Electrophoresis in denaturing conditions were performed as follows. Samples were combined with sample buffer containing 60 mM Tris-HCl buffer pH 6.8, 2.5% (w/v) SDS, 0.36mM /~-mercaptoethanol, 0.5 mM EDTA, 10% (v/v) glycerol and 0.005% (w/v) bromophenol blue. The samples were heated for 2 rain at 95°C in a water bath before being loaded onto a 10-20% (w/v) polyacrylamide gradient gel slab containing 0.1% (w/v) SDS (Laemmli, 1970). The gels were run at constant voltage of 100 V and stained for protein using a silver stain (Blum et al., 1987). Lysozymes Mr values were calculated according to Lambin et al. (1987) using the following Mr standards (kit MW-SDS-70L purchased from Sigma): albumin, 66,000; ovalbumin, 45,000; glyceraldehyde three phosphate dehydrogenase, 36,000; carbonic anhydrase, 29,000, trypsinogen, 24,000; soybean trypsin inhibitor, 20,100; ~-lactalbumin, 14,200.
Isoelectric focusing in polyaerylamide gels Isoelectric focusing was performed as described by Terra et al. (1978), in columns of 7% polyacrylamide gels containing 1% ampholytes pH 3-10. Samples were applied after polymerization and pre-focusing (30 rain at 31 v/cm) on the top of the alkaline side of the gels. Recoveries of the activities applied to the gels were 70-90%. Protein determination and hydrolase assays Protein was determined according to Bradford (1976) using ovalbumin as a standard. Lysozyme activity was determined by measuring the decrease in turbidity at 650nm of a suspension (1 mg/ml) of Micrococcus lisodeikticus in (except otherwise specified) 50 mM citrate-sodium phosphate pH 3.5 containing 150mM NaC1, in a reaction volume of 1 ml. The reaction was stopped by the addition of 0.5ml of 0.5 M sodium carbonate and the absorbance was read. One lysozyme unit is the amount of enzyme that causes a change in the absorbance of the reaction medium of 0.01 U/min. Chitinase activity was measured using the viscometric assay of Winicur and Mitchell (1974), as modified by Lemos and Terra (1991b), with chitosan as substrate. The slopes of the plots of the inverse of specific viscosity against time in min were calculated. One chitinase unit is the amount of enzyme that results in a slope of 1000/min. In each determination of all the enzyme activities, incubations were carried out at 30°C for at least four different periods of time and the initial rates of hydrolysis were calculated. All assays were performed under conditions such that activity was proportional to protein concentration and to time. Controls without enzyme or without substrate were included. Kinetic studies The effect of pH and ionic strength of reaction media on the activity of the midgut lysozyme was determined
A MIDGUT LYSOZYME using the following buffers (Miller and Golder, 1950): glycine-HC1 (pH 2.0-3.5), sodium acetate (pH 4.0-5.5), sodium phosphate (6.0-7.5) and glycine-NaOH (pH 8.0-9.0) prepared with ionic strengths from 0.02 up to 0.2. The effect of M. lysodeikticus concentration on midgut lysozyme activity was determined in different buffers at 30°C. Km values (means and SEM) were determined by a weighted linear regression by the procedure of Wilkinson (1961), using a program written in BASIC by Oestreicher and Pinto (1983).
Preparation of antibodies and double immunodiffusion A 1-ml sample containing 50/~g of purified lysozyme 1 in 0.1 M NaC1 was combined with 1 ml of Freund's complete adjuvant, and the resulting emulsion was injected subcutaneously into the rabbit. After 4 weeks, a similar injection was administered, but with Freund's incomplete adjuvant. Two weeks after the last injection, the rabbit was bled. The blood was left standing 1 h at 37°C and overnight at 4°C before being centrifuged at 3,000 g, 10 min at 4°C. The clear supernatant was added to a suitable solution to become 50% saturated in ammonium sulfate, pH 6.8. After 18 h at room temperature (25°C), the resulting suspension was centrifuged at 50,000g, 15 min, 4°C, the pellet was suspended in 50% saturated ammonium sulfate, pH 6.8, and the suspension was pelleted at 5000g, 15 min, 4°C. The last step was repeated once again, and the final pellet was solubilized in 0.1 M NaC1 and dialyzed against 1000 vol of 0.1 M NaC1 overnight with one change of solvent. The sample was centrifuged at 10,000 g for 10min at 4°C and the supernatant kept frozen at -20°C until used as antiserum. Double immunodiffusion was performed according to the method of Ouchterlony (1968) using 1% agarose in 0.1 M sodium phosphate buffer, pH 7.0, containing 0.15 M NaC1 and 0.1% sodium azide. The samples were left diffusing for 48 h at room temperature in a humid chamber containing a piece of cotton moistened with 5 % (w/v) phenol to inhibit the growth of fungi. The slabs were washed in 0.15 M NaC1 for 48 h at 4°C, dried at 45°C, stained for 5 min with 0.09% (w/v) Coomassie blue in ethanol : acetic acid :water (25 : 8 : 85). The antiserum produced against lysozyme 1, when tested by double immunodiffusion against purified lysozyme 1, was reactive up until a 50-fold dilution. Electron microscopy and immunocytochem&try For work with electron microscope, larvae were dissected in their own hemolymph and anterior midgut (fore-midgut) pieces were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 for 2 h at 4°C. After rinsing with 0.2 M sucrose in the same cacodylate buffer, the midgut pieces were post-fixed in 1% osmium tetroxide in the same cacodylate buffer for 1 h at 4°C and washed in 0.1 M NaC1. En-block staining was performed in uranyl acetate for 16-18h. After dehydration in graded ethanol at room temperature, the materials were
535
embedded in Polylite 8001 polyester resin (Resana S/A, Brazil; Coiro et al., 1972). Ultra-thin sections were cut using a LKB Nova ultramicrotome, stained with lead acetate and examined in a Zeiss EM 9 S2 electron microscope. For immunolocalization of lysozyme in tissue sections, fore-midgut pieces were fixed in 4% formaldehyde with 0.3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 3 h at 4°C. After rinsing with phosphate buffer, the material was dehydrated in graded ethanol, at room temperature, and embedded in L. R. White acrylic resin (Ladd Research Industries Inc., U.S.A.). Ultra-thin sections were cut on the ultramicrotome and collected on 200 mesh colloidon-coated nickel grids. The grids were then floated on drops of saturated sodium metaperiodate for 10min, rinsed in 0.02 M Tris-HC1 buffer pH 7.4 containing 0.83% (w/v) NaC1 (TBS) and placed on drops of 10% fetal calf serum (FCS) in TBS plus 0.025% Tween 20 and 0.025% Triton X-100, for 15 min, to block nonspecific reactions. The sections were then incubated with the primary antibody diluted 1: 500 in TBS containing 5% FCS, 0.025% Tween 20 and 0.025% Triton X-100, overnight at 4°C. As controls, sections of the same blocks were incubated with non-immune serum in the same conditions. Non-immune serum was treated as described above for immune serum. The grids were washed in TBS with detergents and floated on drops of 10% CFS in the same solution for 10min at room temperature. The material was then incubated with protein A conjugated to 10 nm Gold (E. Y. Labs Inc., U.S.A.) diluted 1:40 in the solution above, with 5% FCS, for 1 h at room temperature. Finally the grids were washed in TBS followed by distilled water, stained with uranyl acetate and lead citrate and examined in the electron microscope. RESULTS
Purification of the M. domestica midgut lysozymes Lysozyme activity in M. domestica larval midgut homogenates is stable at pH 3.5 (Espinoza-Fuentes and Terra, 1987), and it was found that a large amount of midgut proteins is rendered insoluble at this pH. Due to that, midgut homogenates were prepared at pH 3.5, and after centrifuging the resulting supernatants were used as a source of lysozyme. This acid extract has a lysozyme specific activity c 6-fold higher than the specific activities of midgut homogenates prepared in water. Two lysozymes from the M. domestica midgut acid extracts are resolved by electrophoresis [Fig. I(A)], although they display identical Mr values, as determined by density-gradient ultracentrifugation at pH 3.5 [Mr 22,000 ___1000, Fig. I(B)] and pH 6.2 (not shown), or by electrophoresis at different polyacrylamide gel concentrations [Mr 22,000 + 500, Fig. I(C)]. These Mr values coincide with that one determined previously using M. domestica midgut homogenates prepared in neutral media (Terra et al., 1988). These results suggest that the lysozymes differ only in net charge. Isoelectric focusing
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FIGURE 1. Physical properties of lysozymes in the acid extract of midgut homogenates from M. domestica larvae. (A) Electrophoretic separation at pH 2.3 in 6%, polyacrylamide gel cylinder. (B) Sedimentation profile in glycerol gradient at pH 3.5. Fractions were collected from the bottom of the tube. M r marker: H, bovine hemoglobin (Mr 64,500). (C) Determination of the M Fof lysozymes (L) by electrophoresis in cylinders of different concentrations of polyacrylamide gel by the method of Hedrick and Smith (1968). M r markers: C, chicken lysozyme (Mr 14,500), M, myoglobin (M, 17,800), T, bovine trypsin (M r 22,500). m in ordinate is the slope of a plot (100 × log Rm) against concentration of the gel. (D) Isoelectric focusing. of the acid extract confirms this hypothesis, showing the existence of m a j o r lysozymes with pI values of 7.90 _ 0.05 and 8.20 _+ 0.03 [Fig. I(D)]. The m i n o r lysozyme activity which displays pI 5.5 [Fig. I(D)] is very variable, being absent from some preparations. Heating the acid extract at 95°C for 5 min results in an increase in the lysozyme specific activity (Table 1), as could be anticipated by the finding that this enzyme is stable in that temperature (Terra et al., 1988). Lysozyme activity is retained in a S-Sepharose column, from which it is eluted with a NaC1 gradient (Fig. 2). The yield of this purification was 80% and the specific activity of the purified lysozyme was 8 9 3 0 U / m g protein (Table 1). Electrophoresis o f the lysozyme preparation resulted in the resolution of two lysozyme activities, corresponding to the m a j o r proteins in the gel, and of a m i n o r a m o u n t o f contaminants (5% o f the proteins in the gel) (Fig. 3). Further purification of the lysozymes were accomplished
eluting gel fractions corresponding to lysozyme 1 and 2 from electrophoretic runs similar to that shown in Fig. 3. The specific activities (U/mg) o f the lysozymes purified by electrophoresis were: lysozyme 1, 6860 + 140, lysozyme 2, 7850 + 140. Since the recovery from polyacrylamide gels is a b o u t 70%, the final yield of the purification is then a b o u t 60%. S D S - P A G E o f purified lysozyme 1 and 2 resulted in a single protein band for each preparation, with an identical Mr value of 17,000+ 1000 (Fig. 4). Isoelectric focusing of the purified lysozymes showed that lysozymes 1 and 2 have pI values of 7.9 and 8.2, respectively (Fig. 5). The m i n o r lysozyme with pI 5.5 was lost during the purification steps. Midgut extracts challenged against anti-lysozyme 1 serum result in a single precipitation line in double diffusion test [Fig. 6(A)]. This demonstrates that the serum is mono-specific. Antiserum produced against
TABLE 1. Purification of lysozymes from M. domestica larval midgut*
Fraction Acid extract Heated extract S-Sepharose eluate
Volume (ml)
Total protein (mg)
Total activity (Units)
Specific activity (Units/rag)
Purification factor
Yield (%)
49 56 22
57.5 13.8 2.8
31,200 24,200 25,000
542 1754 8930
1 3.2 16.5
100 78 80
*Acid extract is the supernatant obtained after centrifuging M. domestica midguts homogenized in 0.1 M sodium acetate buffer pH 3.5. This extract displays a lysozyme specific activity about 6-fold higher than the specific activities of midgut homogenates prepared in water.
A MIDGUT LYSOZYME
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FIGURE 2. Chromatography on S-Sepharose of M. domesticamidgut homogenates after treatment with acid and heat. The column (8.4 x 1.2 cm i.d.) was equilibrated with 20 mM sodium acetate pH 5.5. About 14 mg of protein in the same buffer was applied to the column with a flow rate of 17ml/h. The column was washed with 20mM sodium acetate pH 5.5 and then eluted with 0.05--0.5M NaC1. Fractions of 2 ml were collected at a flow rate of 45 ml/h. Nacl (. . . . ); protein (O); lysozyme activity (O). lysozyme 1 is reactive with purified lysozyme 2 [Fig. 6(B)], suggesting that the lysozymes are immunologically identical.
Kinetic properties of M. domestica midgut lysozymes The kinetic properties o f lysozymes 1 and 2 seem to be similar in relation to the effect o f pH, ionic strength and the concentration o f substrate (Fig. 7). Due to that, we performed the last experiments with only one o f the lysozymes, assuming that the results would be identical whichever lysozyme be used. 2
.3
0
• lOO
20
40
FIGURE 4. Electrophoresis in SDS 10-20% polyacrylamide gradient gel slab. Lane A, Mr markers, top to bottom: 66, 45, 36, 29, 24, 20.1 and 14.2 K. Lane B, purified lysozyme 1 (0.19#g). Lane C, purified ]ysozyme 2 (0.60pg). Lane D, soluble fraction of anterior midgut homogenate (11/~g of proteins). Other details in Materials and Methods. The M. domestica midgut lysozyme activity decreases [Fig. 7(A),(B)], its p H o p t i m u m is displaced toward acidic p H values [Fig. 7(A),(B)] and its Km value increases (Fig. 8) as the ionic strength o f its assay medium becomes higher. Similar results were also found for other enzymes (Maurel and D o u z o u , 1976, D o b s o n et al., 1984). Fig. 7(C) shows a decrease o f lysozyme activity caused by excess substrate. Similar results were found with goose lysozyme (Locquet et al., 1968). Lysozyme 2 maintained in the presence o f M. domestica midgut homogenates containing 200 m U o f cathepsin D for 180min in 1 0 r a M acetate buffer p H 3.0 at 30°C retains full activity. This confirms previous observations made with crude preparations o f lysozyme, according to which this enzyme is stable in the acid M. domestica middle region o f the midgut that contains a cathepsin D (Espinoza-Fuentes and Terra, 1987). Nevertheless, lysozyme 2 incubated with 85 m U o f bovine pepsin loses 40% o f its activity after 120 min in 10 m M acetate buffer p H 3.0 at 30°C.
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Fraction No.
FIGURE 3. Electrophoresis of the pooled active fractions from the eluate of the S-Sepharose column. The runs were performed at pH 2.3 in 6% polyacrylamide gel cylinders. (A) Lysozyme assays. (B) Densitometric tracing of a cylinder stained with Coomassie blue. Profiles obtained from other preparations are similar to those shown. 1 and 2 refer to the two lysozymes.
ol. . . . .
20
J
tl,
40
Fraction No.
FIGURE 5. Isoelectric focusing of purified lysozyme 1 (O) and 2 (O). Profiles obtained from other preparations are similar to those shown.
FRANCISCO J. A. LEMOS et al.
538
DISCUSSION
A
B
Properties of the M. domestica midgut lysozyme
FIGURE 6. Ouchterlony double diffusion test. A, the center well contains serum against lysozyme1; wells 1~ contain different amounts (690, 340, 170, 90, 40, 20 #g of protein, respectively)of the soluble fraction of anterior midgut homogenate. B, the center well contains serum against lysozyme I; wells 1, 3 and 5 contain 0.17/~g of lysozyme 1; wells 2, 4 and 6 contain 0.55/~g of lysozyme 2. Figure 9 shows that 37 units of M. domestica lysozyme displays 1.7 m U of chitinase, whereas 80 units of chicken lysozyme presents 0 . 6 m U of chitinase. Thus, the M. domestica midgut lysozyme shows a chitinase activity about 6-fold higher than that of chicken lysozyme.
Fore-midgut immunolocalization of M. domestica lysozyme There are in fore-midgut cells morphological features which are usually associated with secretory activity, such as many elements of Golgi, plenty of rough endoplasmic reticulum and conspicuous secretory vesicles [Fig. 10(A)]. Figure 10(B) shows that lysozyme immunolabeling is mainly observed in secretory vesicles and at the outside surface of microvilli. It should be noted, however, that only part of the secretory vesicles is labeled. Control preparations showed no immunolabeling above background (results not shown), confirming that immunoreactivity of vesicles content and microvillar surface was due specifically to the presence of lysozyme.
M. domestica larvae display two lysozymes which have an identical Mr of 22,000 + 1000 when measured by density-gradient ultracentrifugation (pH 3.5) and by electrophoresis (pH 2.3) in native conditions performed in polyacrylamide gel of different concentrations. When electrophoresis in SDS-polyacrylamide gradient gel was employed, the two enzymes display Mr 17,000_+ 1000. The last mentioned value is probably the best estimate for the lysozyme Mr value. Thus, M. domestica midgut lysozyme has a Mr value which is similar to that of most lysozymes, including those from insect hemolymph (Jolles et al., 1979; Hultmark et al., 1980; Zachary and Hoffman, 1984; Schneider, 1985). The M. domestica midgut lysozymes differ in pI values, which are: lysozyme 1, 7.9; lysozyme 2, 8.2. The kinetic properties of lysozymes 1 and 2 seem to be similar in relation to the effect of pH, ionic strength and the concentration of substrate. The purified lysozymes are also immunologically identical. Thus, it is probable that lysozymes 1 and 2 have polypeptide chains differing in a few amino acid residues. These results agree with the findings in another Diptera Cyclorrhapha, Drosophila melanogaster. This insect displays several lysozyme genes which are expressed in the midgut (Kylsten et al., 1992). The pl values of M. domestica midgut lysozymes are lower than those of most animals (Imoto et al., 1972), although they are similar to that of ruminant lysozymes (Pahud and Widner, 1982). The lower pI values displayed by these enzymes are supposed to result from a few amino acid substitutions in the surface of the lysozyme molecule (Dobson et al., 1984). M. domestica midgut lysozyme shows a chitinase activity about 6-fold higher than that of chicken
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FIGURE 7. Kinetic properties of purified lysozyme 1 and 2. (A) Activity of lysozyme 1 in function of pH and ionic strength. The ionic strengths used were: 0.02 (0); 0.04 (/X); 0.1 (O); 0,2 (riD- (B) Activity of lysozyme2 in function of pH and ionic strength. Ionic strengths as before. (C) Effectof substrate concentration on the activities(pH, 4; ionic strength, 0.04) of lysozyme 1 (Q) and 2 (O). [S], concentration of M. lysodeikticusin mg/ml. The straight line was adjusted to the points using a weighted linear regression procedure. Each data point in this figure is the result of four assays in the same sample.
A MIDGUT LYSOZYME
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Recently, it was shown that the Lys D lysozyme gene, which is expressed in D. melanogaster midguts, becomes depressed after bacterial injections into the fly hemocelle (Kylsten et al., 1992). This confirms that lysozyme is not part of the defense mechanism against bacteria in Diptera Cyclorrhapha, and lends support to the proposal that lysozyme is a true digestive enzyme in these insects.
log I
FIGURE 8. Effectof ionicstrengthon Kmvaluesof lysozyme2 at pH 4.0. Kmvaluesdeterminedusinga weightedlinearregressionprocedure (n = 5). lysozyme (see Results), although only 20% of the M. domestica midgut chitinase activity can be accounted for by the midgut lysozyme (Lemos and Terra, 1991b). A high chitinase activity seems to be usual among insect lysozymes. The lysozyme isolated from Ceratitis capitata eggs is 350-fold more active on chitin than the chicken lysozyme (Fernandez-Sousa et al., 1977). Midgut lysozymes 1 and 2 become less active and their pH-activity profile is displaced towards the acid side as the ionic strength of the medium increases. Although this was found with other lysozymes (Maurel and Douzou, 1976), the magnitude of the changes observed are only comparable with ruminant lysozymes. Thus, the pH optimum of the cow lysozyme varies from 6.5 to 5.0 as the ionic strength is increased from 0.02 to 0.1 (Dobson et al., 1984), whereas that of M. domestica midgut lysozyme, from 6.5 to 4.5. At an ionic strength of 0.2, the pH optimum of the midgut lysozyme is as low as 3.8. These results may be explained by the polyanionic structure of the bacterial cell walls which, due to a negative electrostatic field, influences the physico-chemical properties of the microenvironment where the catalytic reaction proceeds, decreasing the local pH. At high ionic strength, the electrostatic potential is decreased and the local pH approaches that of the bulk solution (Maurel and Douzou, 1976). The linear dependence of the logarithm of the lysozyme Km with the logarithm of the ionic strength (Fig. 8) is thought to result from the effect of ionic strength on the interaction of the negatively charged bacterial cell walls with the positively charged lysozyme molecules (Maurel and Douzou, 1976). Lysozyme as a digestive enzyme in insects and vertebrates
The ability of M. domestica to use lysozyme as a digestive enzyme seems to have evolved in parallel to that of vertebrates. Thus, lysozyme is absent from most tissues and is present in large amounts in the acidic stomachs of ruminants (Prieur, 1986) and middle midguts of M. domestica (Lemos and Terra, 1991b). M. domestica midgut lysozyme is resistant to the midgut cathepsin D-like proteinase, whereas ruminant lysozyme is resistant to pepsin (Dobson et al., 1984). Both enzymes are more active at acid pH values when present in media with physiological ionic strengths.
The secretory mechanism lysozyme
of M.
domestica
midgut
Lysozyme is not usually found in the luminal contents of the insect midgut (see Lemos and Terra, 1991b, for references). Furthermore, insect midgut secretory mechanisms are poorly known (Chapman, 1985). Hence, the mechanism by which M. domestica lysozyme is secreted into midgut lumen deserves attention. Lysozyme is most active in the lumen of the middle region of the M. domestica midgut. In midgut cells, lysozyme occurs mainly in the fore-midgut, which led to the hypothesis that this region is responsible for lysozyme secretion (Espinoza-Fuentes and Terra, 1987). Subcellular fractionation of fore-midgut cells showed that lysozyme is recovered in major amounts in fraction P,, characterized by the occurrence of large vesicles displaying microvilli, and in fraction P3, which displays mitochondria, unidentified vesicles and secretory vesicles. Based on the fact that freezing and thawing sets lysozyme free on both fractions Pl and P3, a secretory mechanism for lysozyme was proposed. According to this mechanism, lysozyme occurs in contents of secretory vesicles visible in the electron micrographs of fraction P3 and, after exocytosis, becomes partly adsorbed in the glycocalyx (explaining its presence in fraction P0 (Espinoza-Fuentes et al., 1987). This hypothesis was supported by the finding that fraction P3 displays large structures (apparent buoyant density 1.21 g/cm3), from which lysozyme is set free on freezing and thawing (Espinoza-Fuentes et al., 1987). Exocytosis of lysozyme and its partial adsorption to the microvillar glycocalyx of fore-midgut cells were further evidenced in the present paper. Immunolabeling has shown that lysozyme occurs 2.6
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time
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FIGURE 9. Activityof M. domestica lysozyme,37 Units (Q) and chickenlysozyme,80 Units (O) upon chitosan.Viscometricassay.(A) Assayin 25 mM sodiumacetatebufferpH 3.5 containing0.1 M NaC1. (B) Assay in 25 mM sodium acetate buffer pH 5.5 containing0.1 M NaC1. Resultsobtainedwith other preparations were similarto those shown. The source of M. domestica lysozymewas the S-Sepharose eluate.
540
FRANCISCO J. A. LEMOS et al.
FIGURE 10. Apex of a cell from the M. domestica fore-midgut. (A) Morphological features associated with secretion. Note the elements of Golgi (G), rough endoplasmic reticulum (RER), secretory vesicles (SV) and many mitochondria (Mi). (B) Immunolocalization of lysozyme. Note labeling in part of the secretory vesicles (arrowheads) and in microvilli (Mv). Bar = 1 #m. in the c o n t e n t s o f secretory vesicles w h i c h are s i m i l a r to those seen a s s o c i a t e d to e l e m e n t s o f G o l g i . F u r t h e r m o r e , l y s o z y m e was also f o u n d at the o u t s i d e surface o f the cell microvilli. It is r e m a r k a b l e , h o w e v e r , t h a t l y s o z y m e i m m u n o l a b e l i n g o c c u r s o n l y in p a r t o f the secretory vesicles. T h i s suggests t h a t secretory vesicles in M. d o m e s t i c a f o r e - m i d g u t cells differ in c o n t e n t . T h e data, h o w e v e r , are n o t sufficient to c o n c l u d e if the differences are q u a n t i t a t i v e o r q u a l i t a t i v e . REFERENCES Altschul A. and Evans W. J. (1967) Zone electrophoresis with polyacrylamide gel. Meth. Enzym. 11, 179-194.
Blum H., Beier H. and Gross H. J. (1987) Improved silver staining of plant proteins, RNA, and DNA in polyacrylamide gels. Electrophoresis 8, 93-99. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. Chapman R, F. (1985) Structure of the digestive system. In Comprehensive Insect Physiology Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol, 4, pp. 165-211. Pergamon Press, New York. Coiro J. R. R., Weigi D. R., Kisielius J., Menezes H. and Bilotta J. A. T. (1972) A new embedding medium (Polylite 80001) for biological material. Cienc. Cult., Sg*o Paulo 24, 660462. Dobson D. E., Prager E. M. and Wilson A. C. (1984) Stomach lysozymes of ruminants. I. Distribution and catalytic properties. J. biol. Chem. 259, 11,607-11,616.
A MIDGUT LYSOZYME Dunn P. E. (1986) Biochemical aspects of insect immunology. A. rev. Ent. 31, 321 339. Espinoza-Fuentes F. P. and Terra W. R. (1987) Physiological adaptations for digesting bacteria. Water fluxes and distribution of digestive enzymes in Musca domestica larval midgut. Insect Biochem. 17, 809-817. Fernandez-Sousa J. M., Gavilanes J. G., Municio A. M., PerezAranda A. and Rodrigues R. (1977) Lysozyme from the insect Ceratitis capitata eggs. Eur. J. Bioehem. 72, 25-33. Hedrick J. L. and Smith A. J. (1968) Size and charge isomer separation and estimation of molecular weights of proteins by disc-gel electrophoresis. Archs Biochem. Biophys. 126, 155-164. Hultmark D., Steiner H., Rasmuson T. and Boman H. G. (1980) Insect immunity. Purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora ceeropia. Eur. J. Biochem. 106, 7-16. Imoto T., Johnson L. N., North A. C. T., Phillips D. C. and Rupley J. A. (1972) Vertebrate lysozymes. In The Enzymes (Edited by Boyer P. D.), 3rd Edn, Vol. 7, pp. 665 868. Academic Press, New York. Joll6s P. and Joll6s J. (1984) What's new in lysozyme research? Molec. cell. Bioehem. 63, 165 189. Joll6s J., Schoentgen F., Croizer G., Croizer L. and Joll6s P. (1979) Insect lysozymes from three species of Lepidoptera: their structural relatedness to the c (chicken) type lysozyme. J. Molec. Evol. 14, 267-271. Kylsten P., Kimbrell D. A., Daffre S., Samakovlis C. and Hultmark D. (1992) The lysozyme locus in Drosophila melanogaster: different genes are expressed in midgut and salivary glands. Mol. gen. Genet. 232, 335-343. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227, 680~585. Lambin P., Rocbu D. and Fine J. M. (1976) A new method for determination of molecular weights of proteins by electrophoresis across a sodium dodecyl sulphate (SDS) polyacrylamide gradient gel. Analyt. Bioehem. 74, 567-575. Locquet J. P., Saint-Blancard J. and Joll6s P. (1968) Apparent affinity constants of lysozymes from different origins for Mieroeoceus lysodeikticus cells. Bioehim. biophys. Aeta 51, 150-153. Lemos F. J. A. and Terra W. R. (1991a) Properties and intracellular distribution of a cathepsin D-like proteinase active at the acid region of Musca domestiea midgut. Insect Biochem. 21, 457-465. Lemos F. J. A. and Terra W. R. (1991b) Digestion of bacteria and the role of midgut lysozyme in some insect larvae. Comp. Bioehem. Physiol. 100B, 265 268. Martin R. G. and Ames B. N. (1961) A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J. biol. Chem. 236, 1372-1379.
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Maurel P. and Douzou P. (1976) Catalytic implications of electrostatic potentials: the lytic activity of lysozyme as a model. J. Molec. Biol. 102, 253-264. Miller G. L. and Golder R. H. (1950) Buffers of pH 2 to 12 for use in electrophoresis. Archs Biochem. 29, 420-423. Oestreicher E. G. and Pinto G. F. (1983) Pocket computer program for fitting the Michaelis Menten equation. Comput. biol. Med. 13, 309-315. Ouchterlony O. (1968) Handbook of lmmunodiffusion and Immunoelectrophoresis. Ann Arbor Science Publishers, Ann Arbor. Pahud J. J. and Widmer F. (1982) Calf rennet lysozyme. Bioehem. J. 201, 661-664. Prieur D. J. (1986) Tissue specific deficiency of lysozyme in ruminants. Comp. Biochem. Physiol. 85B, 349 353. Schneider P. M. (1985) Purification and properties of three lysozymes from hemolymph of the cricket, Gryllus bimaculatus (De Geer). Insect Biochem. 15, 463-470. Terra W. R. (1990) Evolution of digestive systems of insects. A. rev. Ent. 35, 181-200. Terra W. R. and Ferreira C. (1983) Further evidence that enzymes involved in the final stages of digestion of Rhynchosciara americana do not enter the endoperitrophic space. Insect Biochem. 13, 143-150. Terra W. R., Espinoza-Fuentes F. P. and Ferreira C. (1988) Midgut amylase, lysozyme, aminopeptidase, and trehalase from larvae and adults of Musca domestica. Archs Insect Bioehem. Physiol. 9, 283 297. Terra W. R., Ferreira C. and De Bianchi A. G. (1978) Physical properties and Tris inhibition of an insect trehalase and a thermodynamic approach to the nature of its active site. Biochim. biophys. Aeta 524, 131-141. Wilkinson G. N. (1961) Statistical estimations in enzyme kinetics. Biochem. J. 80, 324~332. Winicur S. and Mitchell H. K. (1974) Chitinase activity during Drosophila development. J. Insect Physiol. 20, 1795 1805. Zachary D. and Hoffmann D. (1984) Lysozyme is stored in the granules of certain hemocyte types in Locusta. J. Insect Physiol. 30, 405-411.
Acknowledgements--We are much indebted to Dr C. Ferreira, for helpful discussions; to B. P. Jord~o, M.Sc. for SDS PAGE and Ouchterlony tests; to Dr A. Sesso, for electron microscope facilities and for his help in the immunocytochemical analyses; and to Miss L. Y. Nakabayashi, Mr M. V. Cruz and Mr W. Caldeira for technical assistance. This work was supported by the Brazilian Research Agencies FAPESP, CNPq and FINEP. F. J. A. Lemos is a graduate fellow of CNPq and A. F. Ribeiro and W. R. Terra are staff members of their respective departments and research fellows of CNPq.