Purification and characterization of membrane-bound chitin synthase.

The membrane-bound chitin synthase, a key enzyme of chitin biosynthesis, was purified, for the first time to homogeneity as a zymogen form. Digitonin could solubilize the enzyme from microsomal fraction of the filamentous fungus Absidia glauca, with 60-70% of the enzyme activity. The solubilized form of the enzyme was effectively purified by a sequence of chelating Sepharose, concanavalin A-Sepharose, and Mono Q column. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme gave a single band with a molecular weight of 30,000. IgG prepared against this 30-kDa species on SDS-polyacrylamide gel electrophoresis immunoprecipitated chitin synthase. The purified enzyme existed as a zymogen, was converted into active form by treatment with trypsin, and the limited digestion with trypsin produced a little smaller polypeptide (28.5 kDa) of which the amino-terminal sequence was identical to the zymogen. The purified enzyme was the glycoprotein and showed a requirement for Mg2+. N-Acetylglucosamine stimulated the enzyme activity approximately 5-fold and polyoxin D, an analogue of substrate, and UDP, a byproduct of enzyme reaction, strongly inhibited the enzyme activity.

Absidia glauca is a typical representative of Zygomycetes, a class of relatively primitive filamentous fungi characterized by hyphae with few septa, and which is sometimes used as a model organism for studying sexual development (20). The genus Absidia has often been reported as pathogenic to various animals, including man, and as contaminant of food (21). Recently, attention was also focused on the high content of chitin and its derivative chitosan in Absidia (22).
We have found a high activity of chitin synthase in growing mycelia of A. glauca. So far, no chitin synthases have been purified from fungi or any other organism. We report here, for the first time, the purification and characterization of membrane-bound chitin synthase as a zymogen form, which plays an important role in chitin biosynthesis and the regulation of fungal growth.

Microorganisms and Culture Conditions
A . glauca (National Food Research Institute type collection No. 1180) was maintained on potato dextrose agar. Spores were washed with water from potato dextrose agar slant grown a t 25 "C for 10 days. The spores were inoculated (final concentration: 6 X 104/ml) into liquid YEPG medium (0.3% yeast extract, 1% peptone, and 2% glucose, pH 4.5). Cultures were grown aerobically in twenty 500-ml Erlenmeyer flasks each containing 100 ml of YEPG medium a t 25 "C on a reciprocating shaker for 20 h. The growing mycelia (at the late logarithmic stage) were collected by aspiration on filter paper in a Biichner funnel and washed with 1 liter of cold buffer A (50 mM KH2P04, 10 mM magnesium acetate, pH 6.5).

Chitin Synthase Assay
Chitin synthase activity was assayed by measuring the incorporation of GlcNAc into chitin, essentially as described by Ruiz-Herrera and . Enzyme preparation required previous activation with protease. Before the assay of chitin synthase activity, the enzyme was activated by proteolysis with 100 ccglml trypsin (Sigma P-8253) for 10 min at 30 "C, and the reaction was terminated by adding 150 Gg/ml soybean trypsin inhibitor (Sigma T-9003). Standard reaction mixture contained: 20 mM GlcNAc, 0.2 mM ATP, 1 mM UDP-GlcNAc including 10 nCi UDP-[U-'4C]Gl~NAc, 50 mM KH,P04, 10 mM magnesium acetate, pH 6.5, and activated enzyme in a total volume of 50 PI. Incubation was at 30 "C for 10 min, and the reaction was terminated by adding 1 ml of ice-cold 10% (w/v) trichloroacetic acid. The precipitate was collected on a glass filter (Whatman GF/C), washed twice with 1 ml of trichloroacetic acid, and finally with 95% ethanol, then the filters were dried and the radioactivity was counted. One unit of the enzyme is the amount catalyzed the incorporation of 1 nmol of GlcNAc/min into chitin.

Chitinase Assay
Chitinase activity was measured by two methods. In method o n e 4-methylumbelliferyl-N-acetyl-fi-~-glucosaminide (MUF-NAGI,' Sigma M-2133), 4-methylumbelliferyl-fi-~-diacetylchitobioside (MUF-NAG2, Sigma M-9763), and 4-methylumbelliferyl-fi-~triacetylchitotrioside Sigma "5639) were used as substrates essentially according to Havukkala: and glycolchitin was used for method two by the procedure as described by Koga et al. (25). In the case of MUF-NAG1, -2, and -3, the enzyme reaction was performed by including 10 p~ substrates with 200 pl of enzyme preparations (50 mM KH,P04, pH 6.5) in 96-well microplates kept in darkness a t 30 "C. After incubation for 10 min, the plate was exposed t o UV light at a wavelength of 366 nm, and the fluorescence intensity was checked visually, with MUF solutions and Streptomyces griseus chitinase (Sigma C-6137) as standards. In the case of glycolchitin, 1 ml of reaction mixture (50 mM KH2P04, p H 6.5), containing 0.05% glycol chitin and 200 p1 of enzyme preparation, was incubated for 12 h at 30 "C, and the amount of reducing sugars was measured.

Purification of Chitin Synthase
All the operations were performed at 0-5 "C, unless otherwise indicated.
Step I: Preparation of Microsomal Fraction-The washed mycelia (combined from twenty 500-ml flasks, wet weight 62 g) were suspended in 180 ml of buffer A, 180 ml of glass beads (0.5 mm in diameter) were added, mycelia broken with a Braun MSK cell homogenizer for 45 s with CO, cooling, and the homogenate was centrifuged (1,000 X g, 5 min). The supernatant solution (crude extract, 190 ml) was centrifuged (100,000 X g, 45 rnin), and the resultant pellet was used as the microsomal fraction.
Step 2: Solubilization of Chitin Synthase-Microsomal fraction was suspended in 180 ml of buffer A containing 0.5% digitonin (w/v), and the homogenate was incubated at 30 "C for 15 rnin with shaking and centrifuged (105,000 X g, 60 min). The supernatant (158 ml) containing the solubilized enzyme was concentrated to 10 ml by ultrafiltration through an Amicon YM-100 filter.
Step 3: Sepharose CL-GB Chromatography-A Sepharose CL-GB column (2.6 X 95 cm) was equilibrated with buffer B (buffer A containing 10% glycerol and 0.1% digitonin). The concentrated solubilized enzyme was applied on and eluted with the same buffer (400 ml) at the flow rate of 60 ml/h, and 10-ml fractions were collected. T h e active fractions (90 ml) were pooled and concentrated to 10 ml.
Step 4: Sephacryl S-300 Chromatography-The concentrated fraction was applied to a Sepharose S-300 column (1.6 X 95 cm) equilibrated with buffer B. The column was eluted with the same buffer (200 ml) at a flow rate of 60 ml/h, and 4-ml fractions were collected. T h e active fractions (36 ml) were pooled.
Step 5 Copper-chelate Chromatography-Chelating Sepharose was packed in a column (1.0 X 16 cm), the upper half of the column was saturated with copper ions by passage of a solution of CuS04 (1 mg/ ml), and then the column was equilibrated with buffer B. The pooled fraction from the above step was applied and washed with the same buffer (60 ml), then elution was carried out in three steps as follows. As the order of values for the stability constant of copper ion is glycine < histamine < histidine (26), the first elution was with 60 ml of buffer B containing 20 mM glycine, the second with 10 mM histamine, and the third with 10 mM histidine a t a flow rate of 30 ml/h. One-ml fractions were collected, and the active fractions (8 ml) were pooled.
Step 6: ConA-Sepharose Chromatography-The pooled active fractions from step 5 was applied to the ConA-Sepharose column (1 X 7 cm) equilibrated with buffer B. Non-bound components were washed out with 20 ml of buffer B, eluted with 20 ml of buffer B containing 0.1 M methyl a-D-mannopyranoside a t a flow rate of 4 ml/h, and 1ml fractions were collected. I. Havukkala, manuscript in preparation.
Step 7: Mono Q Chromatography-The buffer of the collected fractions at step 6 was changed to buffer C (15 mM histidine, 20% glycerol, and 0.1% digitonin, pH 6.3) by passing through AmpureTMSA (Amersham), a prepacked minicolumn of gel filtration for desalting.
Then the sample was applied to a Mono Q HR 5/5 column equilibrated with buffer C. The column was washed once with 5 ml of buffer C and then eluted with a linear gradient of NaCl (20 ml, 0-0.25 M) in the same buffer at the flow rate of 1 ml/min a t room temperature in a Pharmacia fast protein liquid chromatography system. 0.5-ml fractions were collected, immediately placed on ice, and 1 ml of cold buffer D (50 mM KH2P04, 20% glycerol, and 0.1% digitonin, pH 6.5) added. The active fractions (1.5 ml) were pooled.

Preparation of Anti-chitin Synthase Antibodies
Approximately 200 pg of partially purified chitin synthase (fractions a t step 6) was subjected to SDS-PAGE, and after electrophoresis the band of polyacrylamide containing the 30-kDa polypeptide was cut from the gel. The acrylamide was then broken into small fragments and then emulsified in an equal volume of Freud's complete adjuvant and injected intradermally at many sites into a New Zealand White rabbit. The rabbit was given booster shots containing 50 pg of enzyme prepared mentioned above and bled every week following the booster shots. Higher titer antiserum was obtained 3 weeks after the forth injection, and IgG was purified by chromatography on Protein A-Sepharose.

Analytical Methods
Native polyacrylamide gel electrophoresis (PAGE) was performed in a 5% polyacrylamide gel (1 mm thick for silver staining and 2 mm thick for the chitin synthase activity, X 10 cm long, 15 mA for 2 h a t 4 "C) by a modification of the procedure of Davis (27), with gel and buffer containing 0.1% digitonin. The amount of the enzyme applied to each lane was 1 pg for silver staining and 5 pg for enzyme activity. After electrophoresis, protein was visualized by silver staining using 2D-Silver Staining I1 (Daiichi Pure Chemicals, Tokyo), and the chitin synthase activity was detected as follows. The gel was cut into 2-mm sections, and the sliced gel pieces were incubated in 100 p1 of standard assay mixture containing 10 pg of trypsin a t 30 "C for 24 h. And then gel slices were dissolved by immersion for 3 h at 50 "C in hydrogen peroxide (1 ml, 30% w/v), filtered on a Whatman GF/C glass filter, washed, dried as described under "Chitin Synthase Assay," and counted for radioactivity.
SDS-PAGE was carried out essentially according to Laemmli (28). Samples were precipitated by adding 10% (w/v, final concentration) trichloroacetic acid, and the pellets were washed twice with 1 ml of cold acetone and dried under nitrogen, then dissolved in sample buffer and kept at room temperature overnight. Electrophoresis was performed in a 5-20% linear gradient polyacrylamide gel (1 mm thick, 6.5 cm long, 15 mA for 80 min). Proteins were visualized by silver staining. The molecular weight was estimated using molecular weight marker proteins (rabbit muscle phosphorylase b, 97,400; bovine serum albumin, 66,267; rabbit muscle aldolase, 42,400; bovine erythrocyte carbonic anhydrase, 30,000; soybean trypsin inhibitor, 20,400; and egg white lysozyme, 14,400 (from Daiichi Pure Chemicals)).
The amino-terminal sequence of the proteins was determined after SDS-PAGE and electrotransfer to polyvinylidene difluoride membrane (PVDF, Immobilon, from Millipore) using transblot apparatus (Sartoblot 11-S, from Sartorius) according to Towbin et al. (31). The transferred membrane was washed with 25 mM NaCl, 20 mM boric acid, pH 8.0, and proteins were stained with 0.1% Coomassie Blue R-250. After the membrane was destained with 60% methanol and washed with distilled water, the bands corresponding to the proteins sequenced were excised and the amino-terminal sequence was determined with an Applied Biosystems gas-phase amino acid sequenator (model 477A).
Colloidal gold staining and the glycoprotein staining procedure were performed after SDS-PAGE and electrotransfer to PVDF membrane of each fraction from the chitin synthase purification step. The total protein was visualized with the colloidal gold stain and the glycoproteins were detected with the enzyme-linked glycan detection kit (G. P. Sensor, from Honen Corporation research (32)). Transferrin was used as a control glycoprotein and creatinase as a non-glycosylated control protein.
Protein was measured by a sodium deoxycholate-trichloroacetic acid protein precipitation technique as described by Peterson (33).

Limited Tryptic Digestion of Chitin Synthase
Chitin synthase in buffer B was incubated at 30 "C with L-1-tosylamino-2-phenylethylchloromethyl ketone-treated trypsin (Sigma T-8642) at 1:500 (w/w) ratio of trypsin/chitin synthase. The digest was quenched with phenylmethylsulfonyl fluoride to 1 mM after various times. The digests were then prepared for and subjected to SDS-PAGE as described above.

RESULTS
Solubilization of Chitin Synthase-Most of the enzyme activity (95%) was present in the microsomal fraction. Efforts t o solubilize the membrane-bound chitin synthase by a chelating agent (EDTA), or chaotropic agents (NaSCN and NaClOJ, or increased ionic strength were unsuccessful. As these three methods could release proteins which were connected to the membrane by divalent cations, hydrophobic bonds and electrostatic interaction, respectively, chitin synthase was concluded to be not an extrinsic membrane protein but an intrinsic membrane protein. Accordingly, nearly 40 different detergents, including new ones both ionic (CHAPS, CHAPSO, Zwittergent, etc.) and non-ionic (MEGA series, Brij series, n-octyl P-D-thioglucoside, etc.) were tested. Only digitonin and n-heptyl P-D-thioglucoside proved to be effective. While digitonin could solubilize chitin synthase with 60-70% of the enzyme activity of the microsomal fractions, nheptyl /3-D-thioglucoside could solubilize only 30% of the activity. The most effective procedure for solubilizing the enzyme was described under "Experimental Procedures." Purification of Chitin Synthase-The solubilized crude extract was applied to gel filtration chromatography, and Sepharose CL-GB was used for the first gel filtration and Sephacryl S-300 for the second. The second gel filtration could not increase the specific activity remarkably (Table I), but this step was useful to prolong the lifetimes of the ConA and Mono Q columns used a t later steps. Sephacryl S-300 eluate was applied to a copper-chelate-Sepharose column. Chitin synthase adsorbed to copper-chelate-Sepharose and was eluted specifically by 10 mM histidine (Fig. l), but was not adsorbed t o Zn2+-, Co2+-, Ni2+-, and Cd2+-chelating Sepharose (data not shown).
Although the specific activity decreased (Table I), this step was effective in removing higher molecular weight proteins (Fig. 5 ) . The loss of activity may be due to the presence of copurified chitinase because three times as high as the chitinase activity from S. griseus (Sigma C-6137) was also found in the microsomal fractions of A. glauca and 10% of the chitinase was solubilized with digitonin performed in this study. As chitinase degrades chitin, a product of chitin synthase, this may have an effect during the whole purification sequence and so cause the apparent decline of specific activity.
However, relatively little chitinase activity (1% of the microsomal activity) was detected even at the ConA-Sepharose step?
The active fractions from a copper-chelate-Sepharose were applied a ConA-Sepharose column. It has been reported that ConA could bind to glycoprotein which contains a-D-mannopyranose or a-D-glucopyranose (34), and the solubilized membrane-bound enzyme has some affinity to ConA-Sepharose (35). Nearly 60% of chitin synthase activity was bound to ConA-Sepharose and was eluted by 0.1 M methyl-a-Dmannopyranoside (Fig. 2).
The ConA-Sepharose eluate was further subjected to Mono Q ion-exchange chromatography, and one peak of activity was observed (Fig. 3). As a high concentration of NaCl quickly caused the loss of chitin synthase activity, it is necessary to dilute each fraction immediately with buffer D. Other slower methods for reducing the concentration of NaCl, such as dialysis or desalting on gel filtration column, caused almost total loss of the enzyme activity.
Purification of membrane-bound chitin synthase from A. glauca is summarized in Table I, and the enzyme was purified about 458-fold with a yield of 0.12%. The purified enzyme was found to be homogeneous on native PAGE, and the position of the protein band was coincident with the enzyme activity (Fig. 4). SDS-PAGE of purified enzyme gave a single band, corresponding to a molecular weight of 30,000 (Fig. 4). The protein in the final enzyme purifications was shown to be chitin synthase by immunochemical means. IgG from the antiserum immunoprecipitated the native protein and depleted enzyme activity in the presence of Protein A-Sepharose (36).
The purified enzyme could be detected on a PVDF membrane by using the glycoprotein detection kit, and the position of the glycoprotein band was coincident with the protein band visualized with the colloidal gold stain (Fig. 5 ) . This evidence demonstrates that the membrane-bound chitin synthase from A. glauca is a glycoprotein having a carbohydrate residue attached. The specificity of the kit allowed the clear distinction of the glycoprotein (transferrin) from the non-glycosylated protein (creatinase) (Fig. 5 ) .
The amino-terminal sequence of the purified chitin synthase is shown in Fig. 7. Comparison of the chitin synthase amino-terminal sequence revealed homology with ConA from Sword bean (37), Jack bean (38), and lectin a chain from Mucana (39). Among lectin these sequences include amino acid residues (i.e. Glu8 and Asp") which contribute to metal binding to achieve sugar binding activity (40).
The result of the gel filtration revealed that the molecular weight under non-denaturing conditions (with 0.1% digitonin) was about 520,000, and the molecular weight estimated from the sedimentation coefficient was calculated as 540,000. This suggested that chitin synthase aggregates to large complexes S. Machida and M. Saito, unpublished results.

14"
, I by digestion with trypsin. When chitin synthase was subjected to limited proteolysis with trypsin, it produced a little smaller polypeptide (28.5 kDa) as analyzed by SDS-PAGE (Fig. 6). There were no cleavage sites by trypsin at the 20-residue region near the amino terminus, and the amino-terminal sequence of the 28.5-kDa polypeptide was essentially identical with the 30-kDa polypeptide (Fig. 7).
Stability-The purified enzyme was stable only when stored at -80 "C in 20% glycerol. Although glycerol was a good

Effect of various cations on chitin synthase activity
The reaction was carried out at 30 "C for 10 min in the standard assay mixture except magnesium acetate and GlcNAc. Activity measured in the presence of magnesium acetate and GlcNAc was taken as 100%. stabilizer, chitin synthase lost 50% of its activity during storage a t 4 "C for several days. During the purification sequence (5-6 days), loss of enzyme activity occurred. Addition of dithiothreitol to protect -SH groups, or EDTA to protect the enzyme from denaturation by heavy metals, or phenylmethylsulfonyl fluoride to prevent the enzyme from proteolysis by serine protease did not increase the stability of the enzyme.

Cation
Effect of Assay Conditions on the Enzyme Activity-The purified enzyme showed an optimal activity at approximately p H 6.5. Although 40% of the highest activity was obtained at p H 5.5, almost all (95%) was lost at pH 8.0. HEPES buffer, p H 7.0, and MES buffer, pH 6.0. could maintain the same reaction rate as the standard assay buffer (KH2P04, pH 6.51, but Tris buffer, pH 7.0, obtained only 7%. The optimal temperature for activity was 30 "C, and 50% of the enzyme activity was lost at 40 "C. The enzyme lost its activity completely after incubation for 5 min a t 60 "C. Cofactor Requirements-The purified enzyme required a divalent cation for maximum activity (Table 11). M F increased the enzyme activity most, but Mn2+only slightly. As chloride and sulfate ions inhibited the enzyme a t 10 mM, it is necessary to add Mg2+ in the acetate form. Co2+, Zn2+, and Cu2+ could not stimulate the enzyme activity. On the contrary, these divalent cations caused 80-95% loss of activity a t 10 mM under the condition of excess M P (Table 111). Free GlcNAc stimulated the enzyme activity in the presence of 10 mM manganese acetate. The addition of 20 mM GlcNAc increased the enzyme activity about &fold (Table 11). The apparent K,,, for UDP-GlcNAc under 10 mM manganese acetate and 20 mM GlcNAc was 0.7 mM. Inhibition of the Enzymatic Activity-Polyoxin D is a powerful and specific competitive inhibitor of chitin synthase (41). Chitin synthase purified from A. glauca in this study was also sensitive to polyoxin D. An 80% loss of activity was caused by 10 p~ polyoxin D, and 100 p~ polyoxin D inhibited the enzyme activity completely a t 1 mM substrate concentration (Table 111). UDP is a by-product of the enzyme reaction and also caused inhibition of the enzyme activity (Table 111).
Half of the activity was lost at the concentration of 50 pM, and the activity was lost completely at 100 pM.

DISCUSSION
In the present study, we have for the first time purified a membrane-bound chitin synthase, a key enzyme of chitin biosynthesis, as the zymogen form. The metal-chelate chromatography is based on the binding between metal and acid residues of protein (42) and has the advantage of not being affected by detergents. Chitin synthase from A. glauca absorbed only to copper-chelate-Sepharose and was eluted by histidine at neutral pH. This result suggested that chitin synthase from the fungus A. glauca had surface-exposed histidine and cysteine residues because copper ions interact with imidazole and thiol groups of protein (42).
ConA-Sepharose chromatography was successful for purification, and the elution profile (Fig. 2) suggested that the purified chitin synthase was a glycoprotein which had asparagine-linked oligosaccharides. This conclusion was supported by the selective staining of glycoprotein immobilized on PVDF membrane. Bulawa et al. (11) reported that chitin synthase from S. cerevisiae was not glycosylated, based on their ConA-Sepharose binding experiment, although there were 11 potential glycosylation sites in the coding sequence. In general, oligosaccharides attached to protein give some characteristic properties to the protein and play an important role in protein transport, processing, etc. (43). Therefore, an analysis of the oligosaccharide structure of the purified chitin synthase would clarify the transport system including chitosome.
The molecular weight of chitin synthase from A. glauca was estimated to be 30,000 on SDS-PAGE. The evidence that IgG prepared against this 30-kDa polypeptide immunoprecipitated the chitin synthase demonstrates that a single 30-kDa species on SDS-PAGE is chitin synthase. Lending et al. (19) isolated 16 S chitin synthase particles from cell walls of M. rouxii which also belongs to Zygomycetes. They reported four polypeptide bands (21, 23, 33, and 39 kDa) that were tightly connected with chitin synthase activity and that 21 kDa was the main component. These molecular weights are not very different from our values. On the other hand, partially purified membrane-bound chitin synthase from yeast S. cerevisiae showed a major band at 63,000 (17) and the basidiomycete C. cinereus showed a band at 67,000 (18). The molecular weights estimated from the cloned genes of S. cerevisiae (11-13), C. albicans (14), and an ascomycete N . crmsa (15) were over 100,000. Bowen et al. (16) reported that, based on a chitin synthaselike sequence, chitin synthases from the same genus or family are much alike. They found a large evolutionary separation between yeast S. cerevisiae and the other filamentous fungi.
The evolutionary separation into these two groups was also suggested by the analysis of glyceraldehyde 3-phosphate gene (44). As both A. glauca and M. rouxii belong to Zygomycetes (same order, Mucorales), a division of filamentous fungi which is quite different from Ascomycetes and Basidiomycetes. It is of great interest to see whether this evolutionary separateness can be seen in the structure of chitin synthase of A. glauca.
In this study high chitinase activity (3-fold higher than S. griseus chitinase) was also detected in the microsomal fraction of A. glauca. Investigations on microsomal chitinases of another filamentous fungus, M. mucedo (45), and yeast, C.
albicans (46), have shown that the microsomal chitinase was a membrane-bound zymogen. The balance of degradation and synthesis of chitin is thought to be controlled accurately by coordinate action of chitin synthase and chitinase (47, 48). Thus the amounts of chitooligosaccharides and GlcNAc, which are the final products of chitin degradation, are crucial for the functioning of cell wall synthesis in vivo. Addition of GlcNAc stimulated the activity of purified chitin synthase of A. glauca. There has been controversy over whether chitin synthase requires any primer such as GlcNAc or not (17,49), but the data presented in this study suggest that GlcNAc plays an essential role as a primer in synthesis. Moreover, UDP, a by-product of the enzyme reaction, inhibits the enzyme activity and is thus likely to be closely involved in the regulation of chitin synthase. Although the conversion of zymogen into active form is an important point of regulating the chitin synthesis in many fungi, the activating mechanisms in vivo remain unknown. In this report the fact that the limited tryptic digestion produced polypeptide (28.5 kDa) and the time course of appearance of the polypeptide coincided with chitin synthase activity was shown. The accurate molecular weight of the enzyme is not known because of the atypical behavior of membrane-bound proteins on SDS-PAGE (24). The results demonstrate that the polypeptide of 30 kDa is zymogenic and the 28.5-kDa polypeptide is an active form. The amino-terminal sequence data obtained for both polypeptides were identical. It seems that trypsin cleaves near the carboxyl terminus of chitin synthase and converts the enzyme into active form. Preliminary experiments indicated that another protease such as acid protease from Rhizopus sp. was more effective in activating the enzyme. Thus, in uiuo, other proteases may be involved in activation. Further study is needed to elucidate the exact mechanism of the enzyme re-action. A study of the regulatory properties and the structure of chitin synthase from A. glauca is now in progress.