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Exo-β-N-acetylglucosaminidase and chitobiase in Bombyx mori
Insect Biochem., 1977, Vol. 7, pp. 237 to 245. Pergamon Press. Printed in Great Britain
EXO-fl-N-ACETYLGLUCOSAMINIDASE AND CHITOBIASE IN B O M B Y X M O R I SHIGERU KIMURA Sericultural Experiment Station, Wada, Suginami-ku, Tokyo 166, Japan (Received 12 February 1977) Abstract--Two enzymes, one from the haemolymph and the other from the moulting fluid of the silkworm, Bombyx mori, hydrolyzing aryl N-acetyl-fl-D-glucosaminide, were investigated. As compared with the kinetic behavior of the two enzymes using p-nitrophenyl N-acetyl-fl-o-glucosaminide and N,N'-diacetylchitobiose as substrates, the haemolymph enzyme showed more affinity to the aryl glycoside than the moulting fluid enzyme, but the latter showed more affinity to the natural substrate than the former. By a double immunodiffusion test and a quantitative precipitin reaction with an anti-haemolymph enzyme serum, it was apparent that the moulting fluid enzyme was completely distinct from the haemolymph enzyme. When a study was made on the distribution of the two enzymes in various tissues at metamorphosis by assaying their activities separately with the immunoprecipitin method, the haemolymph enzyme was detected in the plasma, whereas the moulting fluid enzyme was observed in the integument and the posterior silkgland in accordance with the occurrence of a chitinase. Two enzymes were found to be in the midgut, fat body, and testis at a certain rate with respect to the developmental age. Especially during regeneration of the midgut tissues, the haemolymph enzyme disappeared followed by the appearance of the moulting fluid enzyme, which synchronized with the occurrence of a chitinase. The present data indicate that the haemolymph enzyme is an exo-fl-N-acetylglucosaminidase, whereas the moulting fluid enzyme is likely to be a chitobiase on the basis of its circumstantial association with a chitinase.
INTRODUCTION
regarded as ch]tobiase, the author investigated the kinetic behavior of the hacmolymph and moulting fluid enzymes using p-nitrophenyl glycoside and N,N'-diacetylchitobiose as substrates, and also immunologically studied them with an anti-haemolymph enzyme serum. Further, a study was made on the distribution of /~-N-acetylglucosaminidases and chitinase in various tissues on the process of the larval-pupal transformation to make clear the relation of these enzymes to the chitinase system.
CHITOBIASE ACTIVITY is often assayed using aryl
N-acetyl-fl-o-glucosaminide which has been also employed as substrate for fl-N-acetylglucosaminidase assay (BERGER and REYNOLDS, 1958; OHTAKARA, 1964; KIMURA, 1973a). All the fl-N-acetylglucosaminidase purified from various sources has been found to be capable of hydrolyzing N,N'-diacetylchitobiose to a considerable extent as well as aryl N-acetyl-fl-Dgalactosaminide (BAHLand AGRAWAL, 1969; LI and LI, 1970; TARENTINO and MALEY, 1970; MEGA et al., MATERIALS AND METHODS 1972; BERKELEY et al., 1973). There are two types of enzymes hydrolyzing an aryl Materials. Bombyx mori. strain, rearing, and collection N-acetyl-fl-t~-glucosaminide in the haemolymph and of haemolymph and moulting fluid were described premoulting fluid of the silkworm, Bombyx mori viously (KIMURA, 1976a, b). N,N'-Diacetylchitobiose was (KIMLrRA, 1974a). Recently the moulting fluid enzyme kindly given by Dr. T. Imoto, Faculty of Agriculture, Yamaguchi University, Japan. p-Nitrophenyl N-acetyl-fl-Dwas shown to be associated with a chitinase, which glucosaminide was purchased from Sigma Chem. Co., splits chitin to liberate N,N'-diacetylchitobiose U.S.A. (KIMURA, 1976a), whereas the haemolymph enzyme Enzyme preparation. Moulting fluid exo-fl-N-acetylgluwas purified to electrophoretically homogeneous and cosaminidase was obtained from the larval moulting fluid was shown to hydrolyze N,N'-diacetylchitobiose to by ammonium sulfate fractionation {K~t:RA, 1976a). Puria lesser extent than aryl N-acetyl-fl-D-glucosaminide fied haemolymph exo-fl-N-acetylglucosaminidase was pre(KIMURA, 1976b). JEUNIAUX(1961), using a chitin hyd- pared from the full-grown larval blood as described prerolysate as substrate, has detected chitobiase activity viously (KIMtmA, 1976b). In the experiment of the distribuin the haemolymph of the silkworm, B. mori at high tion of fl-N-acetylglucosaminidases and chitinase, the inconcentration. But POWNING and IRZYKIEWICZ (1964) tegument with attached trachea and muscle, midgut, fat body, silkgland, malphigian tube and testis, being collected assert that insect exo-fl-N-acetylglucosaminidase has from ten male larvae or pupae at each developmental the same function in the chitinolytic system as chito- stages, were washed, three times with cold 0.005 M phosbiase. In order to answer the question of whether or phate buffer (pH 7.2) containing 0.8°..o NaCI and a small not insect exo-B-N-acetylglucosaminidase should be amount of phenylthiourea. After weighing the tissues they 237
238
SHIGERU KIMURA
were homogenated in the same buffer with 3 or 4 times volume of their wet weight, and then centrifuged at 10,000 g for 20 min. The supernatants obtained were used for the measurement of total fl-N-acetylglucosaminidase and chitinase activities and for the immunoprecipitin reaction test. In the case of blood, 0.05 ml of blood drawn from an animal at each stage were mixed with 0.45 ml of the above mentioned buffer, and centrifuged to remove blood cells. The supernatants were used for enzyme assay. Blood volume per animal was estimated, based on the data reported by HOR|E et al. (1971). Enzyme assay. Each exo-fl-N-acetylglucosaminidase activities was assayed by colorimetric measurement of p-nitrophenol liberated from p-nitrophenyl glycoside substrate according to the method of KIMURA (1974a). One unit of enzyme was defined as the amount of enzyme which liberated 1/2mole of p-nitrophenol from the substrate per min at pH 5.0 and 35:C. When N,N'-diacetylchitobiose was used as substrate, the N-acetylglucosamine liberated was determined by the method of REISSIG et al. (1955L Chitinase activity was assayed by incubating the enzyme preparation with colloidal chitin and recording the appearance of N-acetylglucosamine, as previously reported (KIMURA, 1973b, 1976a). Amounts of N-acetylglucosamine liberated were determined by the above method. One unit of enzyme was defined as the quantity of enzyme required to catalyze the formation of 1 iLmole N-acetylglucosamine per rain at 35 C. Antiserum preparation. Antiserum IASI against the purified haemolymph exo-fl-N-acetylglucosaminidase was prepared as follows: a male rabbit was injected subcutaneously with Freund's incomplete adjuvant at a final concentration of 0.8 mg of the purified enzyme protein. Four weeks later it was intravenously injected with 1.4 mg of the protein, and I week later was provided with 1.2 mg of the protein after checking the titre of the antibody. Blood was drawn from the carotid artery 6 weeks after first injecting the protein and serum obtained by normal methods. Xl 0-3 2 .D
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Fig. 1. Effects of time on the hydrolysis of N,N'-diacetylchitobiose by exo-fl-N-acetylglucosaminidases from the haemolymph and moulting fluid. Either haemolymph exofl-N-acetylglucosaminidase (enzyme activity 7.65 unitsl (O-O) or moulting fluid exo-fl-N-acetylglucosaminidase (enzyme activity 8.38 units) (O-O) was incubated with N,N'-diacetylchitobiose (2 mg) and 0.08 M acetate buffer (pH 5.0) at 35°C. The N-acetylglucosamine (NAcGm) liberated was determined as described in Methods.
lmmunoanalysis. Micro-double diffusion tests were carried out as outlined by CROWLE (1973). The quantitative precipitin reaction was performed as follows: various doses of AS were incubated with either haemolymph enzyme or moulting fluid enzyme, and the total incubation volume was made up to 0.4ml with 0.1 M phosphate buffer IpH 7.4) (final concentration 0.025 M). Controls were included in which AS was replaced by distilled water. After incubation for 3 hr at 35~C, solutions were centrifuged in a bench centrifuge (3500 rev/min for 20 min) and the supernatants were assayed for the unprecipitated enzyme activity using the p-nitrophenyl glycoside. When examined the form (haemolymph or moulting fluid enzyme) and activity of the fl-N-acetylglucosaminidase in the tissues or plasma, excess amounts of undiluted AS were added to the enzyme in the incubation system to precipitate all the enzyme reacting with the anti-haemolymph enzyme serum, based on the data obtained in Fig. 5. This system was routinely composed of 0.2 ml of enzyme preparation. 0.2 ml of undiluted AS and 0.2ml of 0.1 M phosphate buffer. RESULTS
Characterization of the enzymes with fl-N-acetylglucosaminidase acticity in the haemolyrnph and moulting fluid Time courses of N,N'-diacetylchitobiose hydrolysis by the h a e m o l y m p h and moulting fluid enzymes are shown in Fig. l. A curve linear until l hr was obtained in the haemolymph enzyme with a low rate of hydrolysis. O n the other hand, the moulting fluid enzyme, of which activity against p-nitrophenyl glycoside was adjusted to be almost equal to that of the h a e m o l y m p h enzyme, hydrolyzed the substrate faster and its reaction velocity reached a plateau at 30 rain although the substrate was in excess. Thus, the incubation time used for subsequent experiment are 10 rain for moulting fluid enzyme and 60 rain for haemolymph enzyme. The apparent Michaelis constant (Kin) values of each enzyme for N,N'-diacetylchitobiose and p-nitrophenyl N-acetyl-fi-D-glucosaminide were measured at various concentrations ranging from 0 . 2 5 r a M to 10 m M and the results given in Fig. 2 and Table l. The Km value for h a e m o l y m p h enzymes with N,N'diacetylchitobiose was found to be 5.6-fold higher than that obtained with synthetic substrate. The values obtained with the moulting fluid enzyme for both substrates were, on the other hand, almost the same. With this enzyme excess substrate inhibition was noted using the p-nitrophenyl substrate, but not detected with N,N'-diacetylchitobiose. This phenomenon was similar to the previous results obtained for phenyl N-acetyl-fl-D-glucosaminide and -galactosaminide (KIMURA, 1974a) and the results of RE~S and BYRDE (1973} in the fungus, Sclerotinia fructigena. The l~,,,x values of each enzyme for the two substrates and the value of the h a e m o l y m p h enzyme for the synthetic substrate was about 460 times as great as that obtained for the natural substrate. When the ~ , ~ values of the moulting fluid enzyme were calculated to compare them with the values of the haemolymph
239
Fig. 3. Agar-immunodiffusion test of the haemolymph and moulting fluid enzymes with anti-haemolymph enzyme serum. The centre well contained 8 pl of the non-diluted antiserum raised against the purified haemolymph exo-fl-N-acetylglucosaminidase. The outer wells contained 8 pl of either the purified haemolymph enzyme (enzyme activity 0.76 units/10/tl, h) or the partially purified moulting fluid enzyme (enzyme activity 0.50 units/10/al, m).
Exo-fl-N-acetylglucosaminidase and chitobiase in B. mori
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Fig. 2. Lineweaver Burk plot of haemolymph and moulting fluid exo-fl-N-acetylglucosaminidases Either haemolymph enzyme (enzyme activity 7.65 units, O-O) or moulting fluid enzyme (enzyme activity 5.01 units, H ) was incubated with various concentration of these substrates, N,N'-diacetylchitobiose (left) and p-nitrophenyl N-acetyl-fl-D-glucosaminide (right) and 0.04 M acetate buffer (pH 5.0) at 35°C. Each reaction product was determined as described in Methods. enzyme, although the former were not homogeneous, they were estimated to be 43.5/~mole/min/mg for the synthetic substrate and 0.5/~mole/min/mg for the natural substrate. The ratio in the Vmax values obtained with the synthetic substrate to that found with the natural substrate was calculated to be 87.0. The results for agar-double immunodiffusion tests with an anti-haemolymph enzyme serum are shown in Fig. 3. Moulting fluid enzymes did not give the precipitin line. The immunotitration of the haemolymph and moulting fluid enzymes with the antibody produced against the haemolymph enzyme is shown in Fig. 4. The haemolymph enzyme (4/~g protein, 1.13 units enzyme activity for synthetic substrate) was coupled with its antibody, and this enzyme activity in the unprecipitated part of the incubation system completely disappeared by adding the undiluted antiserum with one-eighth concentration relative to the amount of the enzyme. This result was also supported by the data on the level of the antibody measured with the agar immunodiffusion test, in which the visible precipitin line was observed at the one-eighth concentration of the antiserum under the immunoviewer. On the other hand, the moulting fluid exo-fl-
N-acetylglucosaminidase activity, although it was about two-thirds of the haemolymph enzyme activity used, was not absorbed by adding the antiserum at any dose. Figure 5 shows the results of the measurement of the titre of the antibody raised against the haemolymph enzyme. With the immunoprecipitin method used here, the haemolymph enzyme activities were less than about 22 units, the antiserum completely precipitated the enzyme, and also it was possible to cross-react the enzyme even though its activities were not less than about 0.11 units. Based on the data obtained, I measured separately the haemolymph and moulting fluid enzyme activities in the plasma and tissues, adjusting the enzyme activities in the enzyme preparation from 0.1 to 20 units by dilution or concentration. Distribution of exo-fl-N-acet),l#lucosaminidases various tissues at metamorphosis
Blood. fl-N-acetylglucosaminidase activity in blood was found to be at the highest level during feeding (22.6 units) of those obtained in the developmental stages (Fig. 6). As soon as the larva began spinning,
Table l. Km and Vmaxvalues of enzymes with exo-/~-N-acetylglucosaminidase activity in haemolymph and moulting fluid of the silkworm Enzyme source Haemolymph Moulting fluid*
Substrate p-Nitrophenyl N-acetyl#-D-glucosaminide N,N'-diacetylchitobiose p-Nitrophenyl N-acetylfl-D-glucosaminide N,N'-diacetylchit obiose
* Not purified to homogeneity.
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Fig. 4. The quantitative precipitin reaction of the haemolymph and moulting fluid enzymes for the anti-haemolymph enzyme serum. A constant amount of either the purified haemolymph enzyme or moulting fluid enzyme was incubated with various amount of antibody as shown on the abscissa. The amounts of each enzymes added initially was 1.13 units for haemolymph enzyme, and 0.68 units for moulting fluid enzyme. Exo-fl-N-acetylglucosaminidase activity in the haemolymph and moulting fluid enzymes was assayed as described in the Methods section. 0--0 Haemolymph enzyme, 0 - 0 Moulting fluid enzyme. the enzyme activity gradually decreased to values of 6.6 units, being proportional to the blood volume per animal. Plasma enzyme at all stages was completely precipitated by an anti-haemolymph enzyme serum. No activities of the enzyme uncoupled with the antibody and chitinase were detectable throughout the experimental period. Integument. As presented in Fig. 7, the total fl-Nacetylglucosaminidase activity in the integument increased sharply from the commencement of spinning, and 24 hr later, that is the beginning of apolysis, its O--O
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value reached the maximum {32.5 units). Just after pupation the enzyme recovered the same low level as that obtained before wandering stage (1.5 units). The enzyme activities at all stages were not absorbed by addition of the anti-haemolymph enzyme serum at the immunoprecipitin test. Chitinase was almost undetected until the beginning of apolysis, and thereafter its activity rapidly increased to the maximum level (45 milliunits) at the spinning-stopped larval stage, which was estimated to be twenty-fold those obtained before wandering stage and after pupation. The fluctuation of the chitinase activity showed a slight difference from that of the fl-N-acetylglucosaminidase. The fates of two enzyme activities were similar to the previous results found in the integument during the larval moulting cycle (KIMURA, 1973a, 1974b) and the results reported for chitinase at apolysis and metamorphosis by JEUNIAUX (1963). Midgut. fl-N-acetylglucosaminidase and chitinase activities in the midgut tissue showed more characteristic changes at metamorphosis than those obtained ~,
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Fig. 6. The change of fl-N-acetylglucosaminidase activity in the plasma at metamorphosis. Stage, V-5; the 5 day-old larva, M; the mature larva (wandering stage) $1; the larva at the beginning of spinning, $2; the larva during spinning, Sp; the larva which had stopped spinning, P,,; the white pupa just after pupation, P~; the 1 day-old pupa. fl-NAcGmase; fl-N-acetylglucosaminidase.
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I 0 M S1 S2SpPoP~ Stage Fig. 8. The changes of fl-N-acetylglucosaminidaseand chitinase activities in the midgut at metamorphosis. O O fl-N-acetylglucosaminidase activity. H chitinase activity, O - - - O ratio of unabsorbed enzyme activity (UEA) to total enzyme activity (TEA). Symbols are shown in footnotes of Fig. 6.
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in the other tissues (Fig. 8). The total B-N-acetylglucosaminidase activity gradually increased from the final larval ecdysis, and its value reached the maximum at the wandering stage (3.25 units), but thereafter decreased rapidly until pupation. However, in contrast to the changes of the activity, the enzyme with the fl-N-acetylglucosaminidase activity which was not absorbed with the antiserum appeared from the beginning of spinning, and the ratio of the unabsorbed enzyme activity to the total enzyme activity gradually increased until pupation with its value of 0.8. In parallel to the increasing of the unabsorbed enzyme activity, chitinase activity was found to increase in the same pattern as that of that enzyme although it was almost undetected until the larva stopped spinning. The maximal value of the enzyme activity was about one-eighth of that obtained at the maximal level in the integument.
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Fat body. Total fl-N-acetylglucosaminidase activity in the tissue, which was the major occupant in the body cavity from the wandering stage to the pupal stage, remained constant with the value of about 1 unit throughout the experimnetal period (Fig. 9). Almost half of the enzyme in the tissue at each stage was found to cross-react with the antiserum. When chitinase activity was assayed, the undetermined substances in the tissue were found to disturb the colour formation in the Morgan-Elson test (REISSIG et al., 1955). Thus, all the samples were fractionated with sat. (NH,)2SO4 at a final concentration of 0.75, dialyzed against the homogenizing buffer, and then used for the enzyme assay. Chitinase activity did not increase until the larva stopped spinning, but it reached the value of 1.3 milliunits just before histolysis in the tissue occurred. Silkgland. No enzyme activities in the posterior silkgland at the developmental stages were detected until histolysis of the gland took place (Fig. 10). The fl-N-acetylglucosaminidase in the tissues appeared as soon as the larva stopped spinning and was at the maximum level (0.14 units), although it was very low as compared with that obtained in the other tissues. The activity was never absorbed with the antiserum in the immunoprecipitin test. Chitinase activity showed the same changes as that found in the E-Nacetylglucosaminidase activity. Malphigian tubes. Exo-fl-N-acetylglucosaminidase and chitinase activities were not detected during the experimental period. Testis. Although this organ was the smallest of all the tissues of which fl-N-acetylglucosaminidase activity was assayed, the activity was detected at an early developmental age (0.07 units), and was found to increase gradually according to the increasing of the wet weight (Fig. 11). The enzymes in the organ were in the two forms, as in the fat body, and the ratios of unabsorbed enzyme activity to total enzyme activity ranged from 0.6 to 0.7. Chitinase activity was
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244
SHIGERU
KIMURA
cuticle of the cockroach. The differences in the enzyme pattern between the insects used may be considerable. Thus, further studies on properties of these ."Z '~0~0 enzymes from various insects will be necessary. 6_> E In the experiments of the distribution of these gg enzymes with 3-N-acetylglucosaminidase activity in various tissues and plasma on the process of the lar~g o cval-pupal transformation, all the tissues and plasma 2----7 ~ except Malphigian tubes were found to contain one g 0 ~ g_,E or two forms of the enzyme with variation in their , , a • O a 0 0 a activities. In the integument when apolysis occurs, it V-5 M S~ $2 Sp Po P~ is well-known that the chitinolytic system develops Stoge most rapidly (JEUNIAUX, 1963, KIMURA, 1973a, 1974a). Fig. 11. The changes of fl-N-acetylglucosaminidase and In these experiments, the facts that all the fl-N-acechitinase activities in the testis at metamorphosis. © © tylglucosaminidase prepared from the integuments at fl-N-acetylglucosaminidase activity, O - - - - e chitinase ac- all the developmental ages never completely crosstivity. Symbols are shown in footnotes of Fig. 6. reacted with an anti-haemolymph fl-N-acetylglucosaminidase serum, and the increase of its activity very low, maximal activity = 0.37 milliunits. This ac- synchronized with the appearance of a chitinase, inditivity is likely to be attributable to the trachea which cate that this enzyme is the chitobiose related to chisurrounded with the testis and underwent apolysis at tinase. In reality, the accumulation of the large amounts of N-acetylglucosamine (2.4pmole/integumetamorphosis. ment) and traces of chitobiose (KIMURA, 1974a) suggest to us the existence of a fl-N-acetylglucosaminiDISCUSSION dase with a higher affinity to chitobiose than the haeThe chitinolytic system of insect moulting fluid, molymph enzyme. It should be noted that on the which is secreted from epidermal cells just before apo- degradation of the posterior silkgland at metamorlysis, contains an exo-fl-N-acetylglucosaminidase phosis, the moulting fluid enzyme appeared most classified as chitobiase (KIMURA, 1976a). In insect hae- active in accordance with the increase of chitinase molymph an enzyme hydrolyzing chitin hydrolysate activity, because the chitinous substances are not deis abundant (JEUNIAUX, 1961}. It also hydrolyzes tectable in the gland. For the reason mentioned p-nitrophenyl N-acetyl-fl-D-glucosaminide (KIMURA, above, it is very interesting also that during the pro1974a, 1976b). In the experiments reported here exo- cess of regeneration of the midgut tissue, the haemofl-N-acetylglucosaminidases in the haemolymph and lymph enzyme disappeared followed by the appearmoulting fluid of the silkworm, Bombyx mori, were ance of the moulting fluid enzyme, which synchronfound to have different Km and Pmax values for ized with the occurrence of chitinase. Because the lamella structure which shows the existence of chitin p-nitrophenyl N-acetyl-fl-D-glucosaminide and N,N'diacetylchitobiose (Table 1) and to be immunologi- has been observed on the cuticular intima of the cally distinct (Figs. 3 and 4). As compared with the anterior and hind guts, and anterior silkgland by elecKm values of the other exo-f-N-acetylglucosamini- tromicroscopic level (H. AKA[, personal communicadase for N,N'-diacetylchitobiose, the value (6.7 mM) tion), these enzymes in these tissues seem to play a of the moulting fluid enzyme almost resembles that r61e in digesting the chitinous substances at metamor(5 mM) obtained with an enzyme for Aspergillus sp. phosis, as they are already known to do in the integu(OHTAKARA, 1972), but differs from the values ment. The function and the site of synthesis of the haemo(0.52 mM and 0.31 mM) for the enzymes found in the fungus, Sclerotiniafructiyena (REYESand BYRDE, 1973) lymph enzyme are not yet clear. But the enzyme is and the bacterum, Bacillus subtillis B (BERKELEY et likely to have other functions different from those of al., 1973). Furthermore, this value of the moulting the moulting fluid enzyme. This opinion is based on fluid enzyme was higher than those reported for the data of the enzyme observed with a very high enzymes in the gut, blood, and cuticle of the cock- level in the blood and midgut, until the wandering roach, Periplaneta americana, which ranged from stage, at which the disintegration of the tissues had 0.31 mM to 0.37 mM (PowNING and IRZYKIEWICZ, not taken place yet. SIAKOTOS (1960) reported that 1964). On the other hand, no enzymes from other a marked change in the plasma glycoprotein occurred sources are known to have Km values higher than during the developmental periods of the cockroach, that of the haemolymph enzyme (20mM). But this Periplaneta americana, and discussed the transport of may be because few purified exo-f-N-acetylglucosa- carbohydrate for storage or utilization. Further. minidases have the kinetics of hydrolysis of N,N'-dia- L~PKE et al. (1965) suggested that on the study the cetylchitobiose have been examined in detail. POWN- carbohydrate bound protein of the cockroach, the ING and [RZYKIEWICZ (1964) unfortunately did not carbohydrate moieties of the plasma glycoprotein distinguish between these enzymes in the blood and were composed of mannose, glucose, galactose, araXIO 2
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8
Exo-fl-N-acetylglucosaminidase and chitobiase in B. mori
245
KIMURA S. (1973b) The control of chitinase activity by ecdysterone in larvae of Bombyx mori. J. Insect Physiol. 19, 115-123. KIMtraA S. (1974a) The fl-N-acetylglucosaminidase of Bombyx mori L. Comp. Biochem. Physiol. 4913, 345-351. KIMURA S. (1974b) On the metabolic fate of the carbohydrates during larval-larval transformation in the silkworm, Bombyx mori L with special reference to the lytic and synthetic system of polysaccharides. Jap. J. appl. Ent. Zool. 18, 183-188. KIMURA S. (1976a) The chitinase system in the cuticle of the silkworm, Bombyx mori. Insect Biochem. 6, 479-482. KIMURAS. (1976b) Insect haemolymph exo-fl-N-acetylglucosaminidase from Bombyx mori. Purification and properties. Biochim. biophys. Acta 446, 399406. LI Y-T. and LI S-C. (1970) Studies on the glycosidases of Jack bean meal. III Crystallization and properties of fl-N-acetylhexosaminidase. J. b i o l . Chem. 245, 5153-5160. Acknowledgements--My sincere thanks are due to Dr. R. C. W. BERKELEY,Department of Bacteriology, Univer- LtPKE H., GRAINGER M. M., and SIAKOTOSA. N. (1965) Polysaccharide and glycoprotein formation in the cocksity of Bristol, U.K. for critical reading of the manuscript. roach. I Identity and titer of bound monosaccharides. Thanks are also due to Dr. H. INOUE,in our experiment J. biol. Chem. 240, 594-600. Station for immunological techniques. MEGA T., IKENAKAT., and MATSUSHIMAY. (1972) Studies on N-acetyl-fl-o-glucosaminidase of Aspergillus oryzae. II Substrate specificity of the enzyme. J. Biochem. 71, REFERENCES 107-114. OHTAKARA A. (1964) Studies on chitinolytic enzymes of BAHL O. P. and AGRAWALK. M. L. (1969) Glycosidases Black-koji mold. Part VI Isolation and some properties of Aspergillus niger 1. Purification and characterization of N-acetylglucosaminidase. Agric. Biol. Chem. 28, of ct- and fl-galactosidases and fl-N-acetylglucosamini74~751. dase. J. biol. Chem. 244, 2970-2978. BERGER L. R. and REYNOLDSD. M. (1958) The chitinase OHXAKARAA. (1972) fl-N-acetylglucosaminidase and chitobiase of enzyme product from Aspergillus sp. Bull. Hirsystem of a strain of Streptomyces griseus. Biochim. bigoshima Woman's Unit,. 7, 53-64. phys. Acta 29, 522-534. BERKELEYR. C. W., BREWERS. J., Ogrlz J. M., and GILLES- POWNING R. F. and IRZVKIEWtCZH. (1964) fl-N-acetylglucosaminidase in the cockroach (Periplaneta americana) PIE J. B. (1973) An exo-fl-N-acetylglucosaminidase from and in the pull-ball (Lycoperdon perlatum). Comp. BigBacillus subtilis B; Characterization. Biochim. biophys. Acta 309, 157-168. chem. Physiol. 12, 405-415. CROWLE A. J. (1973) lmmunodiffusion (2nd ed.) Academic REISSIG J. L., STROMINGERJ. L., and LELOm L. F. (1955) A modified colorimetric method for the estimation of press, New York and London. HORIE Y., INOKUCHIT., WATANABEK., and YANAGAWA N-acetylamino sugar. J. biol. Chem. 217, 959-966. H. (1971) On the haemolymph volume of the silkworm, REYES F. and BVROE R. J. W. (1973) Partial purification Bombyx mori. J. sericult. Sci. Japan 40, 33(~334. and properties of a fl-N-acetylglucosaminidase from the JEUNIAUX C. (1961) Activit6 chitinolytique de l'hemofungus Sclerotinia fructigena. Biochem. J. 131, 381-388. lymphe de Bombyx mori L au cours des metamorphoses. SIAKOTOSA. M. (1960) The conjugated plasma protein of Arch. int. Physiol. Biochem. 69, 750 751. the American cockroach. II Changes during the molting JEUNIAUXC. (1963) Chitine and ehitinolyse. Masson, Paris. and clotting processes. J. 9en. Physiol. 43, 1015-1030. KIMt~A S. (1973a) Chitinolytic enzymes in the larval devel- TARENTINOA. L. and MALEY F. (1970) Multiple forms of opment of the silkworm, Bombyx mori L (Lepidoptera: a highly purified fl-N-acetylhexosaminidase from hen Bombycidael. Appl. Ent. Zool. 8, 234-236. oviduct. Arch. Biochem. Biophys. 147, 446-456. binose, and xylose. On the studies of the Bombyx plasma glycoprotein, of which fluctuation at metamorphosis was seen in the same pattern as reported in the cockroach by SIAKOTOS(1960), the terminal and non-reducing sugar in the plasma glycoprotein was found to be ct-mannose by examination of the glycosidase digestion of the glycopeptide prepared from the mature larval blood (unpublished data). Thus, it can be said that the haemolymph enzyme did not contribute directly on the breakdown of the plasma glycoprotein. The significance of the co-existence of the haemolymph and moulting fluid enzymes in the fat body and testis throughout the experimental period cannot be explained from the data in this paper.
EXO-fl-N-ACETYLGLUCOSAMINIDASE AND CHITOBIASE IN B O M B Y X M O R I SHIGERU KIMURA Sericultural Experiment Station, Wada, Suginami-ku, Tokyo 166, Japan (Received 12 February 1977) Abstract--Two enzymes, one from the haemolymph and the other from the moulting fluid of the silkworm, Bombyx mori, hydrolyzing aryl N-acetyl-fl-D-glucosaminide, were investigated. As compared with the kinetic behavior of the two enzymes using p-nitrophenyl N-acetyl-fl-o-glucosaminide and N,N'-diacetylchitobiose as substrates, the haemolymph enzyme showed more affinity to the aryl glycoside than the moulting fluid enzyme, but the latter showed more affinity to the natural substrate than the former. By a double immunodiffusion test and a quantitative precipitin reaction with an anti-haemolymph enzyme serum, it was apparent that the moulting fluid enzyme was completely distinct from the haemolymph enzyme. When a study was made on the distribution of the two enzymes in various tissues at metamorphosis by assaying their activities separately with the immunoprecipitin method, the haemolymph enzyme was detected in the plasma, whereas the moulting fluid enzyme was observed in the integument and the posterior silkgland in accordance with the occurrence of a chitinase. Two enzymes were found to be in the midgut, fat body, and testis at a certain rate with respect to the developmental age. Especially during regeneration of the midgut tissues, the haemolymph enzyme disappeared followed by the appearance of the moulting fluid enzyme, which synchronized with the occurrence of a chitinase. The present data indicate that the haemolymph enzyme is an exo-fl-N-acetylglucosaminidase, whereas the moulting fluid enzyme is likely to be a chitobiase on the basis of its circumstantial association with a chitinase.
INTRODUCTION
regarded as ch]tobiase, the author investigated the kinetic behavior of the hacmolymph and moulting fluid enzymes using p-nitrophenyl glycoside and N,N'-diacetylchitobiose as substrates, and also immunologically studied them with an anti-haemolymph enzyme serum. Further, a study was made on the distribution of /~-N-acetylglucosaminidases and chitinase in various tissues on the process of the larval-pupal transformation to make clear the relation of these enzymes to the chitinase system.
CHITOBIASE ACTIVITY is often assayed using aryl
N-acetyl-fl-o-glucosaminide which has been also employed as substrate for fl-N-acetylglucosaminidase assay (BERGER and REYNOLDS, 1958; OHTAKARA, 1964; KIMURA, 1973a). All the fl-N-acetylglucosaminidase purified from various sources has been found to be capable of hydrolyzing N,N'-diacetylchitobiose to a considerable extent as well as aryl N-acetyl-fl-Dgalactosaminide (BAHLand AGRAWAL, 1969; LI and LI, 1970; TARENTINO and MALEY, 1970; MEGA et al., MATERIALS AND METHODS 1972; BERKELEY et al., 1973). There are two types of enzymes hydrolyzing an aryl Materials. Bombyx mori. strain, rearing, and collection N-acetyl-fl-t~-glucosaminide in the haemolymph and of haemolymph and moulting fluid were described premoulting fluid of the silkworm, Bombyx mori viously (KIMURA, 1976a, b). N,N'-Diacetylchitobiose was (KIMLrRA, 1974a). Recently the moulting fluid enzyme kindly given by Dr. T. Imoto, Faculty of Agriculture, Yamaguchi University, Japan. p-Nitrophenyl N-acetyl-fl-Dwas shown to be associated with a chitinase, which glucosaminide was purchased from Sigma Chem. Co., splits chitin to liberate N,N'-diacetylchitobiose U.S.A. (KIMURA, 1976a), whereas the haemolymph enzyme Enzyme preparation. Moulting fluid exo-fl-N-acetylgluwas purified to electrophoretically homogeneous and cosaminidase was obtained from the larval moulting fluid was shown to hydrolyze N,N'-diacetylchitobiose to by ammonium sulfate fractionation {K~t:RA, 1976a). Puria lesser extent than aryl N-acetyl-fl-D-glucosaminide fied haemolymph exo-fl-N-acetylglucosaminidase was pre(KIMURA, 1976b). JEUNIAUX(1961), using a chitin hyd- pared from the full-grown larval blood as described prerolysate as substrate, has detected chitobiase activity viously (KIMtmA, 1976b). In the experiment of the distribuin the haemolymph of the silkworm, B. mori at high tion of fl-N-acetylglucosaminidases and chitinase, the inconcentration. But POWNING and IRZYKIEWICZ (1964) tegument with attached trachea and muscle, midgut, fat body, silkgland, malphigian tube and testis, being collected assert that insect exo-fl-N-acetylglucosaminidase has from ten male larvae or pupae at each developmental the same function in the chitinolytic system as chito- stages, were washed, three times with cold 0.005 M phosbiase. In order to answer the question of whether or phate buffer (pH 7.2) containing 0.8°..o NaCI and a small not insect exo-B-N-acetylglucosaminidase should be amount of phenylthiourea. After weighing the tissues they 237
238
SHIGERU KIMURA
were homogenated in the same buffer with 3 or 4 times volume of their wet weight, and then centrifuged at 10,000 g for 20 min. The supernatants obtained were used for the measurement of total fl-N-acetylglucosaminidase and chitinase activities and for the immunoprecipitin reaction test. In the case of blood, 0.05 ml of blood drawn from an animal at each stage were mixed with 0.45 ml of the above mentioned buffer, and centrifuged to remove blood cells. The supernatants were used for enzyme assay. Blood volume per animal was estimated, based on the data reported by HOR|E et al. (1971). Enzyme assay. Each exo-fl-N-acetylglucosaminidase activities was assayed by colorimetric measurement of p-nitrophenol liberated from p-nitrophenyl glycoside substrate according to the method of KIMURA (1974a). One unit of enzyme was defined as the amount of enzyme which liberated 1/2mole of p-nitrophenol from the substrate per min at pH 5.0 and 35:C. When N,N'-diacetylchitobiose was used as substrate, the N-acetylglucosamine liberated was determined by the method of REISSIG et al. (1955L Chitinase activity was assayed by incubating the enzyme preparation with colloidal chitin and recording the appearance of N-acetylglucosamine, as previously reported (KIMURA, 1973b, 1976a). Amounts of N-acetylglucosamine liberated were determined by the above method. One unit of enzyme was defined as the quantity of enzyme required to catalyze the formation of 1 iLmole N-acetylglucosamine per rain at 35 C. Antiserum preparation. Antiserum IASI against the purified haemolymph exo-fl-N-acetylglucosaminidase was prepared as follows: a male rabbit was injected subcutaneously with Freund's incomplete adjuvant at a final concentration of 0.8 mg of the purified enzyme protein. Four weeks later it was intravenously injected with 1.4 mg of the protein, and I week later was provided with 1.2 mg of the protein after checking the titre of the antibody. Blood was drawn from the carotid artery 6 weeks after first injecting the protein and serum obtained by normal methods. Xl 0-3 2 .D
/
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Fig. 1. Effects of time on the hydrolysis of N,N'-diacetylchitobiose by exo-fl-N-acetylglucosaminidases from the haemolymph and moulting fluid. Either haemolymph exofl-N-acetylglucosaminidase (enzyme activity 7.65 unitsl (O-O) or moulting fluid exo-fl-N-acetylglucosaminidase (enzyme activity 8.38 units) (O-O) was incubated with N,N'-diacetylchitobiose (2 mg) and 0.08 M acetate buffer (pH 5.0) at 35°C. The N-acetylglucosamine (NAcGm) liberated was determined as described in Methods.
lmmunoanalysis. Micro-double diffusion tests were carried out as outlined by CROWLE (1973). The quantitative precipitin reaction was performed as follows: various doses of AS were incubated with either haemolymph enzyme or moulting fluid enzyme, and the total incubation volume was made up to 0.4ml with 0.1 M phosphate buffer IpH 7.4) (final concentration 0.025 M). Controls were included in which AS was replaced by distilled water. After incubation for 3 hr at 35~C, solutions were centrifuged in a bench centrifuge (3500 rev/min for 20 min) and the supernatants were assayed for the unprecipitated enzyme activity using the p-nitrophenyl glycoside. When examined the form (haemolymph or moulting fluid enzyme) and activity of the fl-N-acetylglucosaminidase in the tissues or plasma, excess amounts of undiluted AS were added to the enzyme in the incubation system to precipitate all the enzyme reacting with the anti-haemolymph enzyme serum, based on the data obtained in Fig. 5. This system was routinely composed of 0.2 ml of enzyme preparation. 0.2 ml of undiluted AS and 0.2ml of 0.1 M phosphate buffer. RESULTS
Characterization of the enzymes with fl-N-acetylglucosaminidase acticity in the haemolyrnph and moulting fluid Time courses of N,N'-diacetylchitobiose hydrolysis by the h a e m o l y m p h and moulting fluid enzymes are shown in Fig. l. A curve linear until l hr was obtained in the haemolymph enzyme with a low rate of hydrolysis. O n the other hand, the moulting fluid enzyme, of which activity against p-nitrophenyl glycoside was adjusted to be almost equal to that of the h a e m o l y m p h enzyme, hydrolyzed the substrate faster and its reaction velocity reached a plateau at 30 rain although the substrate was in excess. Thus, the incubation time used for subsequent experiment are 10 rain for moulting fluid enzyme and 60 rain for haemolymph enzyme. The apparent Michaelis constant (Kin) values of each enzyme for N,N'-diacetylchitobiose and p-nitrophenyl N-acetyl-fi-D-glucosaminide were measured at various concentrations ranging from 0 . 2 5 r a M to 10 m M and the results given in Fig. 2 and Table l. The Km value for h a e m o l y m p h enzymes with N,N'diacetylchitobiose was found to be 5.6-fold higher than that obtained with synthetic substrate. The values obtained with the moulting fluid enzyme for both substrates were, on the other hand, almost the same. With this enzyme excess substrate inhibition was noted using the p-nitrophenyl substrate, but not detected with N,N'-diacetylchitobiose. This phenomenon was similar to the previous results obtained for phenyl N-acetyl-fl-D-glucosaminide and -galactosaminide (KIMURA, 1974a) and the results of RE~S and BYRDE (1973} in the fungus, Sclerotinia fructigena. The l~,,,x values of each enzyme for the two substrates and the value of the h a e m o l y m p h enzyme for the synthetic substrate was about 460 times as great as that obtained for the natural substrate. When the ~ , ~ values of the moulting fluid enzyme were calculated to compare them with the values of the haemolymph
239
Fig. 3. Agar-immunodiffusion test of the haemolymph and moulting fluid enzymes with anti-haemolymph enzyme serum. The centre well contained 8 pl of the non-diluted antiserum raised against the purified haemolymph exo-fl-N-acetylglucosaminidase. The outer wells contained 8 pl of either the purified haemolymph enzyme (enzyme activity 0.76 units/10/tl, h) or the partially purified moulting fluid enzyme (enzyme activity 0.50 units/10/al, m).
Exo-fl-N-acetylglucosaminidase and chitobiase in B. mori
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Fig. 2. Lineweaver Burk plot of haemolymph and moulting fluid exo-fl-N-acetylglucosaminidases Either haemolymph enzyme (enzyme activity 7.65 units, O-O) or moulting fluid enzyme (enzyme activity 5.01 units, H ) was incubated with various concentration of these substrates, N,N'-diacetylchitobiose (left) and p-nitrophenyl N-acetyl-fl-D-glucosaminide (right) and 0.04 M acetate buffer (pH 5.0) at 35°C. Each reaction product was determined as described in Methods. enzyme, although the former were not homogeneous, they were estimated to be 43.5/~mole/min/mg for the synthetic substrate and 0.5/~mole/min/mg for the natural substrate. The ratio in the Vmax values obtained with the synthetic substrate to that found with the natural substrate was calculated to be 87.0. The results for agar-double immunodiffusion tests with an anti-haemolymph enzyme serum are shown in Fig. 3. Moulting fluid enzymes did not give the precipitin line. The immunotitration of the haemolymph and moulting fluid enzymes with the antibody produced against the haemolymph enzyme is shown in Fig. 4. The haemolymph enzyme (4/~g protein, 1.13 units enzyme activity for synthetic substrate) was coupled with its antibody, and this enzyme activity in the unprecipitated part of the incubation system completely disappeared by adding the undiluted antiserum with one-eighth concentration relative to the amount of the enzyme. This result was also supported by the data on the level of the antibody measured with the agar immunodiffusion test, in which the visible precipitin line was observed at the one-eighth concentration of the antiserum under the immunoviewer. On the other hand, the moulting fluid exo-fl-
N-acetylglucosaminidase activity, although it was about two-thirds of the haemolymph enzyme activity used, was not absorbed by adding the antiserum at any dose. Figure 5 shows the results of the measurement of the titre of the antibody raised against the haemolymph enzyme. With the immunoprecipitin method used here, the haemolymph enzyme activities were less than about 22 units, the antiserum completely precipitated the enzyme, and also it was possible to cross-react the enzyme even though its activities were not less than about 0.11 units. Based on the data obtained, I measured separately the haemolymph and moulting fluid enzyme activities in the plasma and tissues, adjusting the enzyme activities in the enzyme preparation from 0.1 to 20 units by dilution or concentration. Distribution of exo-fl-N-acet),l#lucosaminidases various tissues at metamorphosis
Blood. fl-N-acetylglucosaminidase activity in blood was found to be at the highest level during feeding (22.6 units) of those obtained in the developmental stages (Fig. 6). As soon as the larva began spinning,
Table l. Km and Vmaxvalues of enzymes with exo-/~-N-acetylglucosaminidase activity in haemolymph and moulting fluid of the silkworm Enzyme source Haemolymph Moulting fluid*
Substrate p-Nitrophenyl N-acetyl#-D-glucosaminide N,N'-diacetylchitobiose p-Nitrophenyl N-acetylfl-D-glucosaminide N,N'-diacetylchit obiose
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Fig. 4. The quantitative precipitin reaction of the haemolymph and moulting fluid enzymes for the anti-haemolymph enzyme serum. A constant amount of either the purified haemolymph enzyme or moulting fluid enzyme was incubated with various amount of antibody as shown on the abscissa. The amounts of each enzymes added initially was 1.13 units for haemolymph enzyme, and 0.68 units for moulting fluid enzyme. Exo-fl-N-acetylglucosaminidase activity in the haemolymph and moulting fluid enzymes was assayed as described in the Methods section. 0--0 Haemolymph enzyme, 0 - 0 Moulting fluid enzyme. the enzyme activity gradually decreased to values of 6.6 units, being proportional to the blood volume per animal. Plasma enzyme at all stages was completely precipitated by an anti-haemolymph enzyme serum. No activities of the enzyme uncoupled with the antibody and chitinase were detectable throughout the experimental period. Integument. As presented in Fig. 7, the total fl-Nacetylglucosaminidase activity in the integument increased sharply from the commencement of spinning, and 24 hr later, that is the beginning of apolysis, its O--O
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Fig. 5. The precipitation of purified haemolymph fl-N-acetylglucosaminidase by antiserum. A constant amount of the undiluted antiserum was incubated with various amounts of the enzyme as shown on the abscissa. The enzyme level corresponding to enzyme concentration 1, was 2.7l units/0.1 ml. fl-N-acetylglucosaminidase activity was assayed as described in the Method section.
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value reached the maximum {32.5 units). Just after pupation the enzyme recovered the same low level as that obtained before wandering stage (1.5 units). The enzyme activities at all stages were not absorbed by addition of the anti-haemolymph enzyme serum at the immunoprecipitin test. Chitinase was almost undetected until the beginning of apolysis, and thereafter its activity rapidly increased to the maximum level (45 milliunits) at the spinning-stopped larval stage, which was estimated to be twenty-fold those obtained before wandering stage and after pupation. The fluctuation of the chitinase activity showed a slight difference from that of the fl-N-acetylglucosaminidase. The fates of two enzyme activities were similar to the previous results found in the integument during the larval moulting cycle (KIMURA, 1973a, 1974b) and the results reported for chitinase at apolysis and metamorphosis by JEUNIAUX (1963). Midgut. fl-N-acetylglucosaminidase and chitinase activities in the midgut tissue showed more characteristic changes at metamorphosis than those obtained ~,
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Fig. 6. The change of fl-N-acetylglucosaminidase activity in the plasma at metamorphosis. Stage, V-5; the 5 day-old larva, M; the mature larva (wandering stage) $1; the larva at the beginning of spinning, $2; the larva during spinning, Sp; the larva which had stopped spinning, P,,; the white pupa just after pupation, P~; the 1 day-old pupa. fl-NAcGmase; fl-N-acetylglucosaminidase.
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Exo-fl-N-acetylglucosaminidaseand chitobiase in B. mori 5
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V-5
in the other tissues (Fig. 8). The total B-N-acetylglucosaminidase activity gradually increased from the final larval ecdysis, and its value reached the maximum at the wandering stage (3.25 units), but thereafter decreased rapidly until pupation. However, in contrast to the changes of the activity, the enzyme with the fl-N-acetylglucosaminidase activity which was not absorbed with the antiserum appeared from the beginning of spinning, and the ratio of the unabsorbed enzyme activity to the total enzyme activity gradually increased until pupation with its value of 0.8. In parallel to the increasing of the unabsorbed enzyme activity, chitinase activity was found to increase in the same pattern as that of that enzyme although it was almost undetected until the larva stopped spinning. The maximal value of the enzyme activity was about one-eighth of that obtained at the maximal level in the integument.
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243
Fat body. Total fl-N-acetylglucosaminidase activity in the tissue, which was the major occupant in the body cavity from the wandering stage to the pupal stage, remained constant with the value of about 1 unit throughout the experimnetal period (Fig. 9). Almost half of the enzyme in the tissue at each stage was found to cross-react with the antiserum. When chitinase activity was assayed, the undetermined substances in the tissue were found to disturb the colour formation in the Morgan-Elson test (REISSIG et al., 1955). Thus, all the samples were fractionated with sat. (NH,)2SO4 at a final concentration of 0.75, dialyzed against the homogenizing buffer, and then used for the enzyme assay. Chitinase activity did not increase until the larva stopped spinning, but it reached the value of 1.3 milliunits just before histolysis in the tissue occurred. Silkgland. No enzyme activities in the posterior silkgland at the developmental stages were detected until histolysis of the gland took place (Fig. 10). The fl-N-acetylglucosaminidase in the tissues appeared as soon as the larva stopped spinning and was at the maximum level (0.14 units), although it was very low as compared with that obtained in the other tissues. The activity was never absorbed with the antiserum in the immunoprecipitin test. Chitinase activity showed the same changes as that found in the E-Nacetylglucosaminidase activity. Malphigian tubes. Exo-fl-N-acetylglucosaminidase and chitinase activities were not detected during the experimental period. Testis. Although this organ was the smallest of all the tissues of which fl-N-acetylglucosaminidase activity was assayed, the activity was detected at an early developmental age (0.07 units), and was found to increase gradually according to the increasing of the wet weight (Fig. 11). The enzymes in the organ were in the two forms, as in the fat body, and the ratios of unabsorbed enzyme activity to total enzyme activity ranged from 0.6 to 0.7. Chitinase activity was
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0 M S~ 52 Sp P0 PI Stage Fig. 9. The changes of fl-N-acetylglucosaminidaseand chitinase activities in the fat body at metamorphosis. O----O fl-N-acetylglucosaminidase activity, H chitinase activity, O - - - O ratio of unabsorbed enzyme activity (UEA) to total enzyme activity (TEA). Symbols are shown in footnotes of Fig. 6. m.
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244
SHIGERU
KIMURA
cuticle of the cockroach. The differences in the enzyme pattern between the insects used may be considerable. Thus, further studies on properties of these ."Z '~0~0 enzymes from various insects will be necessary. 6_> E In the experiments of the distribution of these gg enzymes with 3-N-acetylglucosaminidase activity in various tissues and plasma on the process of the lar~g o cval-pupal transformation, all the tissues and plasma 2----7 ~ except Malphigian tubes were found to contain one g 0 ~ g_,E or two forms of the enzyme with variation in their , , a • O a 0 0 a activities. In the integument when apolysis occurs, it V-5 M S~ $2 Sp Po P~ is well-known that the chitinolytic system develops Stoge most rapidly (JEUNIAUX, 1963, KIMURA, 1973a, 1974a). Fig. 11. The changes of fl-N-acetylglucosaminidase and In these experiments, the facts that all the fl-N-acechitinase activities in the testis at metamorphosis. © © tylglucosaminidase prepared from the integuments at fl-N-acetylglucosaminidase activity, O - - - - e chitinase ac- all the developmental ages never completely crosstivity. Symbols are shown in footnotes of Fig. 6. reacted with an anti-haemolymph fl-N-acetylglucosaminidase serum, and the increase of its activity very low, maximal activity = 0.37 milliunits. This ac- synchronized with the appearance of a chitinase, inditivity is likely to be attributable to the trachea which cate that this enzyme is the chitobiose related to chisurrounded with the testis and underwent apolysis at tinase. In reality, the accumulation of the large amounts of N-acetylglucosamine (2.4pmole/integumetamorphosis. ment) and traces of chitobiose (KIMURA, 1974a) suggest to us the existence of a fl-N-acetylglucosaminiDISCUSSION dase with a higher affinity to chitobiose than the haeThe chitinolytic system of insect moulting fluid, molymph enzyme. It should be noted that on the which is secreted from epidermal cells just before apo- degradation of the posterior silkgland at metamorlysis, contains an exo-fl-N-acetylglucosaminidase phosis, the moulting fluid enzyme appeared most classified as chitobiase (KIMURA, 1976a). In insect hae- active in accordance with the increase of chitinase molymph an enzyme hydrolyzing chitin hydrolysate activity, because the chitinous substances are not deis abundant (JEUNIAUX, 1961}. It also hydrolyzes tectable in the gland. For the reason mentioned p-nitrophenyl N-acetyl-fl-D-glucosaminide (KIMURA, above, it is very interesting also that during the pro1974a, 1976b). In the experiments reported here exo- cess of regeneration of the midgut tissue, the haemofl-N-acetylglucosaminidases in the haemolymph and lymph enzyme disappeared followed by the appearmoulting fluid of the silkworm, Bombyx mori, were ance of the moulting fluid enzyme, which synchronfound to have different Km and Pmax values for ized with the occurrence of chitinase. Because the lamella structure which shows the existence of chitin p-nitrophenyl N-acetyl-fl-D-glucosaminide and N,N'diacetylchitobiose (Table 1) and to be immunologi- has been observed on the cuticular intima of the cally distinct (Figs. 3 and 4). As compared with the anterior and hind guts, and anterior silkgland by elecKm values of the other exo-f-N-acetylglucosamini- tromicroscopic level (H. AKA[, personal communicadase for N,N'-diacetylchitobiose, the value (6.7 mM) tion), these enzymes in these tissues seem to play a of the moulting fluid enzyme almost resembles that r61e in digesting the chitinous substances at metamor(5 mM) obtained with an enzyme for Aspergillus sp. phosis, as they are already known to do in the integu(OHTAKARA, 1972), but differs from the values ment. The function and the site of synthesis of the haemo(0.52 mM and 0.31 mM) for the enzymes found in the fungus, Sclerotiniafructiyena (REYESand BYRDE, 1973) lymph enzyme are not yet clear. But the enzyme is and the bacterum, Bacillus subtillis B (BERKELEY et likely to have other functions different from those of al., 1973). Furthermore, this value of the moulting the moulting fluid enzyme. This opinion is based on fluid enzyme was higher than those reported for the data of the enzyme observed with a very high enzymes in the gut, blood, and cuticle of the cock- level in the blood and midgut, until the wandering roach, Periplaneta americana, which ranged from stage, at which the disintegration of the tissues had 0.31 mM to 0.37 mM (PowNING and IRZYKIEWICZ, not taken place yet. SIAKOTOS (1960) reported that 1964). On the other hand, no enzymes from other a marked change in the plasma glycoprotein occurred sources are known to have Km values higher than during the developmental periods of the cockroach, that of the haemolymph enzyme (20mM). But this Periplaneta americana, and discussed the transport of may be because few purified exo-f-N-acetylglucosa- carbohydrate for storage or utilization. Further. minidases have the kinetics of hydrolysis of N,N'-dia- L~PKE et al. (1965) suggested that on the study the cetylchitobiose have been examined in detail. POWN- carbohydrate bound protein of the cockroach, the ING and [RZYKIEWICZ (1964) unfortunately did not carbohydrate moieties of the plasma glycoprotein distinguish between these enzymes in the blood and were composed of mannose, glucose, galactose, araXIO 2
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8
Exo-fl-N-acetylglucosaminidase and chitobiase in B. mori
245
KIMURA S. (1973b) The control of chitinase activity by ecdysterone in larvae of Bombyx mori. J. Insect Physiol. 19, 115-123. KIMtraA S. (1974a) The fl-N-acetylglucosaminidase of Bombyx mori L. Comp. Biochem. Physiol. 4913, 345-351. KIMURA S. (1974b) On the metabolic fate of the carbohydrates during larval-larval transformation in the silkworm, Bombyx mori L with special reference to the lytic and synthetic system of polysaccharides. Jap. J. appl. Ent. Zool. 18, 183-188. KIMURA S. (1976a) The chitinase system in the cuticle of the silkworm, Bombyx mori. Insect Biochem. 6, 479-482. KIMURAS. (1976b) Insect haemolymph exo-fl-N-acetylglucosaminidase from Bombyx mori. Purification and properties. Biochim. biophys. Acta 446, 399406. LI Y-T. and LI S-C. (1970) Studies on the glycosidases of Jack bean meal. III Crystallization and properties of fl-N-acetylhexosaminidase. J. b i o l . Chem. 245, 5153-5160. Acknowledgements--My sincere thanks are due to Dr. R. C. W. BERKELEY,Department of Bacteriology, Univer- LtPKE H., GRAINGER M. M., and SIAKOTOSA. N. (1965) Polysaccharide and glycoprotein formation in the cocksity of Bristol, U.K. for critical reading of the manuscript. roach. I Identity and titer of bound monosaccharides. Thanks are also due to Dr. H. INOUE,in our experiment J. biol. Chem. 240, 594-600. Station for immunological techniques. MEGA T., IKENAKAT., and MATSUSHIMAY. (1972) Studies on N-acetyl-fl-o-glucosaminidase of Aspergillus oryzae. II Substrate specificity of the enzyme. J. Biochem. 71, REFERENCES 107-114. OHTAKARA A. (1964) Studies on chitinolytic enzymes of BAHL O. P. and AGRAWALK. M. L. (1969) Glycosidases Black-koji mold. Part VI Isolation and some properties of Aspergillus niger 1. Purification and characterization of N-acetylglucosaminidase. Agric. Biol. Chem. 28, of ct- and fl-galactosidases and fl-N-acetylglucosamini74~751. dase. J. biol. Chem. 244, 2970-2978. BERGER L. R. and REYNOLDSD. M. (1958) The chitinase OHXAKARAA. (1972) fl-N-acetylglucosaminidase and chitobiase of enzyme product from Aspergillus sp. Bull. Hirsystem of a strain of Streptomyces griseus. Biochim. bigoshima Woman's Unit,. 7, 53-64. phys. Acta 29, 522-534. BERKELEYR. C. W., BREWERS. J., Ogrlz J. M., and GILLES- POWNING R. F. and IRZVKIEWtCZH. (1964) fl-N-acetylglucosaminidase in the cockroach (Periplaneta americana) PIE J. B. (1973) An exo-fl-N-acetylglucosaminidase from and in the pull-ball (Lycoperdon perlatum). Comp. BigBacillus subtilis B; Characterization. Biochim. biophys. Acta 309, 157-168. chem. Physiol. 12, 405-415. CROWLE A. J. (1973) lmmunodiffusion (2nd ed.) Academic REISSIG J. L., STROMINGERJ. L., and LELOm L. F. (1955) A modified colorimetric method for the estimation of press, New York and London. HORIE Y., INOKUCHIT., WATANABEK., and YANAGAWA N-acetylamino sugar. J. biol. Chem. 217, 959-966. H. (1971) On the haemolymph volume of the silkworm, REYES F. and BVROE R. J. W. (1973) Partial purification Bombyx mori. J. sericult. Sci. Japan 40, 33(~334. and properties of a fl-N-acetylglucosaminidase from the JEUNIAUX C. (1961) Activit6 chitinolytique de l'hemofungus Sclerotinia fructigena. Biochem. J. 131, 381-388. lymphe de Bombyx mori L au cours des metamorphoses. SIAKOTOSA. M. (1960) The conjugated plasma protein of Arch. int. Physiol. Biochem. 69, 750 751. the American cockroach. II Changes during the molting JEUNIAUXC. (1963) Chitine and ehitinolyse. Masson, Paris. and clotting processes. J. 9en. Physiol. 43, 1015-1030. KIMt~A S. (1973a) Chitinolytic enzymes in the larval devel- TARENTINOA. L. and MALEY F. (1970) Multiple forms of opment of the silkworm, Bombyx mori L (Lepidoptera: a highly purified fl-N-acetylhexosaminidase from hen Bombycidael. Appl. Ent. Zool. 8, 234-236. oviduct. Arch. Biochem. Biophys. 147, 446-456. binose, and xylose. On the studies of the Bombyx plasma glycoprotein, of which fluctuation at metamorphosis was seen in the same pattern as reported in the cockroach by SIAKOTOS(1960), the terminal and non-reducing sugar in the plasma glycoprotein was found to be ct-mannose by examination of the glycosidase digestion of the glycopeptide prepared from the mature larval blood (unpublished data). Thus, it can be said that the haemolymph enzyme did not contribute directly on the breakdown of the plasma glycoprotein. The significance of the co-existence of the haemolymph and moulting fluid enzymes in the fat body and testis throughout the experimental period cannot be explained from the data in this paper.