THE JOURNAL OF BIOLOUICAL CHEMISTRY

Vol. 246, No. 12, Issue of June 25, pp. 3999-4007, 1971

Printed in U.S.A.

Pathway of Chitin Synthesis and Cellular Localization of

Chitin Synthetase in Mucor rouxii*

(Received for publication, September 10, 1970)

IAN MCMURROUGH, $ A. FLORES-CARREON, $ AND S. BARTNICKI-GARCIA

From the Department of Plant Pathology, University of California, Riverside, California 9.%502

SUMMARY

Cell-free extracts of ikfucor rouxii contain enzymes which synthesize uridine diphosphate &lcetyl-D-glucosamine from IV-acetyl-n-glucosamine. Procedures are described to pre- pare radiolabeled UDP-GlcNAc using cell-free extracts or living cells of this fungus. This sugar nucleotide serves as the precursor for chitin synthesis. Free N-acetyl-n-glucos- amine is a strong activator of chitin synthesis. Chitin synthetase activity is found in the different particulate frac- tions of the cell. The cell walls are particularly rich in chitin synthetase activity. Autoradiographic studies showed that chitin synthetase is localized preferentially in the apical region of hyphal walls. This is also the region of chitin deposition in vivo. The total chitin synthetase content of a culture was shown to increase concomitantly with cell growth and with the total hexosamine content of the cells.

This is the first of a series of communications dealing with the biosynthesis of cell wall polymers, and their precursors, by the fungus Mucor rouxii. The cell wall composition of this or- ganism has been studied in some detail (1, 2). Four different polysaccharides have been recognized in vegetative walls, namely chitosan, chitin, a heteropolysaccharide of D-glucuronic acid, u-mannose, L-fucose, and n-galactose, and a polymer of n-glucuronic acid. Chitin, the p-1,4-polymer of N-acetyl-n- glucosamine, together with chitosan, its nonacetylated analogue, constitute over 40% of the mycelial walls of M. rouxii.

Initially, we have undertaken to elucidate the course of chitin biosynthesis. Chitin biosynthesis has already been examined in other fungi, crustaceans, and insects; in all cases uridine di- phosphate N-acetylglucosamine acts as the glycosyl donor (S-10). In the present communication we described the forma- tion of UDP-GlcNAc from N-acetyl-n-glucosamine, the syn- thesis of chitin from this sugar nucleotide, and the subcellular localization of chitin synthetase.

* This work was supported by Research Grant AI-05540 from the National Institutes of Health, United States Public Health Service.

$ Present address, Arthur Guinness, Son and Company, Ltd., Dublin, Ireland.

$ Present address, Escuela National de Ciencias Biologicas, Institute Politecnico National, Mexico, D.F., Mdxico.

MATERIALS AND METHODS

Cultivation Procedures

M. routii strain IM-80 was employed (1). Stock cultures were derived from a single yeast-like colony of the organism: To obtain isolated yeast-like colonies, spores were streaked over the surface of solid Medium A (yeast extract-peptone-glucose- agar) (11) and incubated for 48 hours at 30” under a 30% CO2 + 70% Nz atmosphere. Stock cultures were grown aerobically and maintained on solid Medium A slants in screw-capped tubes at 30”. Large numbers of spores (sporangiospores) were ob- tained from 4-day aerobic cultures, grown in prescription bottles containing 50 ml of solid Medium A, by irrigating the surface mat with water. The spores were washed three times and used to inoculate liquid cultures (500 ml in cotton-plugged 2000-ml Erlenmeyer flasks) of Medium A (pH 4.5) to a final concentra- tion between lo5 and 4 x lo5 spores per ml. These cultures were incubated aerobically on a reciprocating shaker at 30” and harvested after 6 to 8 hours. Microscopic examination revealed that spore germination was predominantly synchronous; at harvest time, more than 95% of the spores had emitted a germ tube. These cells were harvested by filtration through a coarse grade sintered glass filter and were washed three times by cen- trifugation (2000 X g) in ice-cold phosphate buffer (0.5 M KH2P04-KOH containing 0.01 M MgC12; pH 6.5).

Preparation of Crude Enzyme Fractions

Cells, prepared as described above, were extremely susceptible to disruption by freezing and thawing. Accordingly, suspensions containing approximately 108 cells were centrifuged in 50-ml conical tubes for 10 min at 2000 X g. Supernatant fluid was drained from the hard-packed pellet by inverting the tube for 30 s. The compact cell mass was then rapidly frozen by im- mersing the base of the centrifuge tube in a solid COz-acetone mixture for 5 min. After thawing at 20’ the cell mass was cen- trifuged at 2000 x g for 10 min at 4”. The compacted cell mass was thus reduced to about 3 its original volume, the remainder being replaced by a turbid supernatant. The supernatant was withdrawn and the pellet (“cell residues”) was washed with 5 volumes of phosphate buffer. Washings were combined to yield about 20 ml of crude extract. After one freeze-thaw cycle, 99.98% of the cells were killed and 45 to 65% of the total cell protein was released into the supernatant.

Cell fractions were prepared by centrifuging the crude extract first at 10,000 X g for 10 min to yield a solid pellet and a turbid supernatant. The latter was centrifuged at 100,000 x g for 20

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min to yield another pellet and a clear supernatant. The clear supernatant was withdrawn and dialyzed for 10 hours against phosphate buffer at 4’ and the nondialyzable material (dialyzed supernatant I) was stored at -20”. Both pellets were in- dividually washed twice in phosphate buffer, finally resuspended in 2.0 ml of phosphate buffer, and stored at -20”. For con- venience, the designations “mitochondrial” and “microsomal” will be assigned to the 10,000 X g and 100,000 X g pellets, re- spectively.

Dialyzed Supernatant II was obtained by dialyzing super- natant I against 0.05 M Tris-HCl buffer (pH 7.5) containing 0.01 M MgClt for 8 hours at 4”. Dialyzed Supernatant II was not amenable to storage at -20”; considerable precipitation of protein was observed on subsequent thawing.

For the preparation of purified cell walls, “cell residues” (2000 x g pellet), prepared as described above, were suspended in 25 mM KH2PO~-KOH buffer and disrupted by shaking with glass beads in a Bronwill disintegrator for 20 s. The cell wall fraction was then recovered and washed three times with buffer by centrifugation at 2000 X g for 5 min. Samples of this cell wall preparation were subjected to a total of three cycles of breakage and washing. After each cycle, chitin synthetase activity was tested.

General Methods

Protein was measured by the method of Lowry et al. (12). Total hexosamine content was determined by the Elson-Morgan procedure (13). Paper chromatograms were irrigated with Solvent A, isoamyl alcohol-pyridine-water (1 .O : 1 .O : 0.8)) Solvent B, 95% ethanol-l N-acetic acid (7 :3), or Solvent C, 95% etha- nol-l M ammonium acetate, pH 3.8 (7:3). Radioactive com- ponents of the chromatograms were located by a Vanguard Autoscanner 880. Radioactivity was measured by liquid scin- tillation counting using a Packard Tri-Carb liquid scintillation spectrometer. The radioactivity in aqueous samples was counted in vials containing 15 ml of scintillation fluid A (60 g of naphthalene, 100 ml of methanol, 20 ml of ethylene glycol, 0.2 g of 1,4-bis[2-(4-methyl-5-phenyloxazolyl)]benzene, and 4 g of 2,5-diphenyloxazole made to 1000 ml with p-dioxane). Paper strips containing radioactive compounds were counted in vials containing 20 ml of scintillation fluid B (0.1 g of 1,4-bis[2-(4- methyl-5-phenyloxazolyl)]-benzene and 2.0 g of 2,5-diphenyl- oxazole dissolved in 1000 ml of toluene).

Chemicals

Uridine diphosphate N-acetyl-n-glucosamine, and D-glUCOSa-

mine g-phosphate were obtained from Sigma Chemical Com- pany. Other biochemicals were obtained from Calbiochem.

Preparation of l‘%-o-GIucosamine &Phosphate

Radioactive GlcN, labeled with 14C at the position 1 (New England Nuclear Corporation), was phosphorylated by a modi- fication of the method of Distler, Merrick, and Roseman (14). The reaction mixture, containing 27.5 mg of 14C-glucosamine hydrochloride (specific activity 1 to 2 Ci per mole), ATP (300 pmole), sodium barbital (30 pmole), MgClz (200 pmole), yeast hexokinase (83 units), and 250 ~1 of a 1% insulin solution, was adjusted to pH 8.0 with 1 M KOH and incubated at 30” for 1 hour. During the course of the reaction the pH was maintained at 7.5 to 8.0 by the addition of 1 M KOH. Subsequently the mixture was evaporated under reduced pressure and the entire

sample was chromatographed on a Dowex 1 (acetate form) column (20 x 1 cm). The column was eluted with a linear gradient (250 ml) of acetic acid, from 0.05 to 1.0 M; the eluate was collected in 5.0-ml fractions. Fractions containing the radioactivity, which eluted as a single peak, were pooled and evaporated to a small volume. Acetic acid was removed by extraction with ether in a liquid-liquid extractor. More than 92% of the initial radioactivity was recovered as W-glucosamine 6-phosphate (14C-GlcN-6-P).

Preparation of N-.&etyl-14C-o-glucosamine B-Phosphate

Selective N-acetylation of 14C-glucosamine g-phosphate was accomplished by a modification of the method of Distler et al. (14). The reaction mixture, containing 100 pmole of 14C-GlcN- 6-P (specific activity 1 to 2 Ci per mole), methanol (0.6 ml), and sodium bicarbonate (4.0 mg), in a total volume of 5.1 ml, was cooled in an ice bath to 4”. Acetic anhydride (0.14 ml) was added to the stirred mixture over a period of 1 hour while the pH of the reaction mixture was maintained at 6 to 7 .by the addition of 1 M KOH. The solution was then applied to a col- umn (20 x 1 cm) of Dowex 50 (Hf form) to adsorb any un- reacted substrate and the acetylated product was washed from the column with 100 ml of water. This solution was then evapo- rated under reduced pressure to 2 ml and applied to a column (20 x 1 cm) of Dowex 1 (acetate form). After washing with 100 ml of water, the column was irrigated with 0.05 M sulfuric acid. Fractions (5.0 ml) were collected, and those containing the radioactivity, which eluted as a sharp peak, were pooled and evaporated under reduced pressure to 4.0 ml. This con- centrate was then extracted continuously with ether to remove acetic acid. Excess barium chloride and silver carbonate were then added to the solution and the precipitates so formed were removed by centrifugation. Finally, the solution was decat- ionized in a Dowex 50 (H+ form) column. About 66% of the original radioactivity was recovered as N-acetyl-14C-n-glucosa- mine 6-phosphate (W-GlcNAc-6-P).

The acetylation procedure described above was also used for the preparation of N-acetyl-14C-n-glucosamine (14C-GlcNAc) from i4C-n-glucosamine.

Preparation of UDPJ4C-GlcNAc

A culture of M. rouxii, grown for 16 hours, was harvested and disrupted in a Hughes press. The disrupted mycelia and ex- pressed cell extract were resuspended in 10 ml of ice-cold Tris- HCl buffer (pH 7.5) and centrifuged at 2000 X g to remove cel1 residues. The supernatant was diluted with buffer to 20 m1 (4.7 mg of protein per ml). This cell-free extract was then added to an incubation mixture containing UTP (250 mg); inorganic pyrophosphatase (350 units), glucosamine 1,6-diphosphate (Cl.001 M), MgClz (0.01 M), 14C-GlcNAc-6-P (60 pmole; specific activity 1 to 2 Ci per mole) made up to a final volume of 106 ml with 0.01 M Tris-HCI buffer (pH 7.5) containing 0.001 M EDTA. After incubation for 2 hours at 30”, the reaction was terminated by the addition of 20 ml of 0.2 M acetic acid and the entire mix- ture was evaporated to 10 ml under reduced pressure. The concentrated mixture was then washed with water onto a Dowex 1 (formate form) column (20 x 1 cm). Eluate was collected in 5.0-ml fractions and analyzed for protein while the elution of ultraviolet light-absorbing substances was monitored continu- ously. The column was washed with water (250 ml) until the eluate was free from protein. A linear gradient (600 ml) of

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ammonium formate from 0.01 to 2.0 M was then applied to the column. The main peak of radioactivity, which eluted at a concentration of 0.35 to 0.45 M ammonium formate, was col- lected and evaporated to 2.5 ml. Final isolation of UDP-W- GlcNAc (42 pmole total) was achieved by descending chromatog- raphy on Whatman No. 3MM paper. Chromatograms were first irrigated for 24 to 48 hours with 95% ethanol, The radio-

active material, which migrated slowly as a well defined, ultra- violet light (230 to 270 mp)-absorbing band was excised, eluted, and rechromatographed three times in Solvent C and then three times in Solvent B (see “General Methods”).

Preparation of UDP-G~CN-~~C-AC

GlcNAc labeled with 14C in the carbonyl group (GlcN-‘4C-AC) was used to prepare GlcNJ4C-AC-6-P (4 pmole) by the phos- phorylation method described above. The product was isolated by paper chromatography and was converted to UDP-GlcN- 14C-AC (specific activity 10.0 Ci per mole). Reaction conditions and concentrations of reactants used were essentially as de- scribed in Table I. The final product was purified by paper chromatography and diluted with unlabeled UDP-GlcNAc as required.

Preparation of N-Acetyl-aH-D-glueosamine

Generally labeled aH-GlcN, purchased from the Volk Radio- chemical Company (specific activity 3.7 mCi per pmole), was N-acetylated (15). The resulting aH-GlcNAc was purified by passage through a Dowex 50 (Hf form) column followed by paper chromatography with Solvent B.

Preparation of UDP-aH-GlcNAc

High specific activity tritiated UDP-GlcNAc was prepared using living cells of M. roux% An 8-hour culture (50 ml inocu- lated with 300,000 spores per ml) was centrifuged; the cells were washed with 0.01 M KHzPOd and resuspended in 5 ml of 0.1% glucose plus 0.2% uridine. aH-GlcNAc (500 PCi) was added to the cell suspensions and incubated at 30” for 10 min with occa- sional shaking. Cells were washed three times by centrifugation with cold 0.01 M KHzPOd and then extracted in 10 ml of 50% aqueous ethanol in a boiling water bath for 10 min. The mix-

ture was centrifuged and the supernatant plus subsequent wash- ings with 50% ethanol were combined and evaporated to dry- ness. About 135 $Zi were thus recovered. The residue was redissolved in water and chromatographed twice on Whatman No. 3MM with Solvent B. Over 90% of the spotted radio- activity had the chromatographic mobility of UDP-GlcNAc. On the basis of extinction coefficient at 260 rnp its specific ac- tivity was calculated to be 266 /&i per pmole. This is a mini- mum estimate since the sample may have contained other non- radioactive sugar nucleotides which could not be separated by paper chromatography.

Chitin Xyntktase Assay

Unless specified otherwise, the standard incubation mixture contained 0.01 to 0.02 pmole of 14C-labeled substrate, 10 pmoles of GlcNAc, 5 InM MgCh, 25 mM KHzPOd-KOH buffer (pH 6.5), 0.1 to 0.3 ml of cell extract, and other additions in a final volume of 0.5 ml. Mixtures were incubated at 22” for periods up to 1 hour as indicated and then the reaction was terminated by the addition of 20 ~1 of glacial acetic acid. Samples (0.1 to 0.3 ml) of the incubated mixture were spotted on strips of Whatman

TABLE I Synthesis of uridine diphosphate IV-acetyl-o-glucosamine

Data are expressed as disintegrations per min incorporated into either UDP-14GlcNAc from W-GlcNAc-6-P or UDP-GlcN- ‘%-AC from GlcN-‘%-AC. Additions of ATP (2.0 rmole) and UTP (2.0 pmole) were made as indicated to the incubation mix- tures containing 0.02 pmole of ‘%-labeled substrate, 5 mmole of MgClz, 0.1 ml of dialyzed supernatant II to a total volume of 0.5 ml in 0.05 M Tris-HCl buffer (pH 7.5). Mixtures were incu- bated at 22” for 1 hour and then inactivated by the addition of 20 pl of glacial acetic acid.

Substrate (0.02 pm&) Additions Sugar

nucleotide formed

W-GlcNAc-6-P (39,500 dpm). . . None 14C-GlcNAc-6-P (39,500 dpm). . . . ATP 14C-GlcNAc-6-P (39,500 dpm). . . . . UTP GlcN-W-AC (39,500 dpm) . . . None GlcN-W-AC (39,500 dpm). ATP GlcN-14C-AC (39,500 dpm). . UTP GlcNJ4C-AC (39,500 dpm). . ATP + UTP 14C-GlcN (41,000 dpm). ATP + UTP

am

243 461

25,349 137 222 192

26,122 217

No. 3MM paper and irrigated for 24 hours by descending flow- using Solvent A, B, or C. The radioactivity which remained at the origin of chromatograms was taken as a measure of- chitin synthesis. Accordingly, the chromatogram origins were’ excised and placed in vials containing 20 ml of nonaqueous scin- tillation fluid B and counted for radioactivity.

Puri$c&m. of Chitinase

Commercial chitinase (25 mg) was dissolved in 0.2 M sodium acetate buffer (pH 5.0) and centrifuged at 2000 x g for 10 min. Chitinase was recovered from the supernatant by ammonium sulfate precipitation (70 to 80% (w/v)). The enzyme was collected by centrifugation (2000 X g), redissolved in 0.2 M sodium acetate (2.0 ml), and dialyzed against 500 ml of 0.2 M sodium acetate.

Autoradiographic Studies

Cellular Localization of Chitin Synthetase-Cell residues pre- pared by freeze-thawing as described above were washed with cold buffer (0.5 M KH~POI-KOH containing 0.01 Y MgC12; pH 6.5) and incubated at room temperature for 1 hour with 0.06 pmoles (16 PCi) of UDP-aH-GlcNAc in the presence of 100 pmoles of GlcNAc and 2.5 pmoles of ATP in a total volume of 0.5 ml. The incubation was stopped by addition of 0.5 ml of 2 M HCI. The cell residues were washed with water by centrifuga- tion and then extracted with boiling water for 10 min. After staining with Congo red the cells were affixed onto microscope slides and autoradiographs prepared as described earlier (16).

Chitin Deposition in V&F-TO locate the site of deposition of cell wall aminopolysaccharides (chitin + chitosan), living cells of M. rouxii were incubated with aH-GlcNAc. The procedure was similar to that described earlier (16). About 50,000 spores were germinated aerobically in Medium A for 7.5 hours. The

culture was centrifuged and most of the supernatant fluid was discarded leaving the cells suspended in about 2 ml. To this suspension aHYGlcNAc (111 PCi; specific activity 3.7 mCi per pmole), labeled in the glucosamine moiety, was added; after 5 min of incubation the cells were killed by addition of 2 ml of 2

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TABLE II

Incorporation of radioactivity from z&dine diphosphate N-acetylglucosamine into chitin

Data are expressed as disintegrations per min incorporated in 1 hour at 22” into chromatographically immobile polymers from either UDP-I%-GlcNAc (0.02 rmole) or UDP-GlcN-I%-AC (0.02 rmole) in incubation mixtures containing dialyzed supernatant II (0.1 ml), mitochondrial fraction (0.1 ml), microsomal fraction (0.1 ml), 5 mM MgCla, and 50 mM Tris-HCI buffer (pH 7.5). Where indicated, GlcNAc (10 pmoles) and UDP-GlcNAc (1 pmole) were added.

Substrate Additions Radioac- tivity in-

corporated

UDPJ%-GlcNAc (40,000 dpm)

UDP-GlcN-%-AC (21,000 dpm)

None GlcNAc UDP-GlcNAc +

GlcNAc None GlcNAc UDP-GlcNAc +

GlcNAc

d9m 660

4,370 210

410 2,220

160

M HCl. After washing with water, the cells were extracted with 3 ml of alkaline ethanol (aqueous 1 M NaOH: 95% ethanol, 1:2, v/v) in boiling water for 5 min. The resulting cell ghosts were stained with Congo red, heat-fixed onto slides, and dipped in Kodak nuclear emulsion NTB-3. Slides were exposed for several days and then developed.

RESULTS

Synthesis of U&line Diphosphate N-Acetyl-o-glucosamine-The soluble cytoplasm of M. rouxii has the eneymic capacity to syn- thesize uridine diphosphate N-acetyl-n-glucosamine. This is shown in Table I in which three different compounds, 14C-GlcN, GlcN-i4C-AC, and i4C-GlcNAc-6-P, were tested as substrates with dialyzed supernatant II as enzyme source. Only the acetylated substrates gave rise to a sugar nucleotide (14C-UDP- GlcNAc). No corresponding sugar nucleotide was formed from i4C-GlcN. Both UTP and ATP were necessary for the forma- tion of UDP-GlcNAc from GlcNAc, but only UTP was required when the substrate was GlcNAc-6-P. A small amount of i4C-acetvlglucosamine l-phosphate (0.993 pmole) was also de- * tected together with the sugar nucleotide.

Incorporation of Radioactivity from Uridine Diphosphate Acetyl-o-gkosamine into Chitin-Uridine diphosphate N-acetyl- n-glucosamine labeled in the glucosamine moiety (UDP-14C- GlcNAc), or the acetyl moiety (UDP-GlcN-14C-AC), was in- cubated with a reconstituted cell-free extract of M. rouxii con- taining mitochondrial, microsomal, and soluble fractions (Table II). Only a small amount of radioactivity was incorporated into chitin (chromatographically immobile material). However, in the presence of free N-acetyl-n-glucosamine the incorporation of

6 7 8 9

PH

FIG. 1 (left). Effect of pH on chitin synthesis from either UDP-%-GlcNAc (-) or UDP-GlcN-“C-AC (- - -) in the pres- ence of GlcNAc. Conditions and concentrations of reactants used were as indicated in Table I, except that pH was varied with KHaP04-KOH (0) or Tris-HCI (0) buffers at a final concentra- tion of 25 111~.

FIG. 2 (right). Time course of chitin formation by cell walls (-) and microsomes (- - -). Radioactivity incorporated into chitin from UDPJhC-GlcNAc in the presence of 10 mM GlcNAc is expressed as a percentage of, the total radioactivity incorporated in 60 min. Standard incubation mixtures (see “Materials and Methods”) containing cell walls were buffered with either 25 mM KHzPOd-KOH, pH 6.5 (0) or 25 mM Tris-HCI, pH 7.5 (0). Mix- tures containing microsomes (A) were buffered with 25 mM KHzPOd-KOH, pH 6.5.

TABLE III

Chitin syn.thesis by cell fractions of M. rouxii

Samples (0.1 ml) of cell fractions containing about 1 to 6 mg of protein were added to reaction mixtures containing 0.02 pmole of substrate as indicated, 10 pmoles of GlcNAc, 5 mru MgCla, and 25 mM phosphate buffer (pH 6.5) in a final volume of 0.5 ml. Cell fractions were prepared as described in “Materials and Methods,” except that the supernatant was not dialyzed before use. Data are expressed as radioactivity incorporated into chitin per mg of protein per hour. Total activity was calculated on the basis of the relative protein content of each cell fraction: cell residues 33% ; mitochondria 2%; microsomes 6y0 ; supernatant 51%. -

Cell fraction Substrate

Cell residues. Mitochondria. Microsomes. Supernatant

Cell residues.

Mitochondria.

Microsomes.

Supernatant

-

Radioac- Total tivity in- activ-

CorporateI ity

Qm % UDP-14C-GlcNAc (38,506 dpm) 24,740 85.0 UDP-i4C-GlcNAc (38,500 dpm) 15,810 3.3 UDP-I%-GlcNAc (38,500 dpm) 16,395 10.2 UDP-14CGlcNAc (38,500 dpm) 290 1.5

UDP-GlcN-“C-AC (19,800 dpm)

UDP-GlcNJ4C-AC (19,800 dpm)

UDP-GlcNJ4C-AC (19,806

dpm) UDP-GlcN-‘%-AC (19,800

dpm)

7,330 86.4

4,600 3.3

4,080 8.7

90 1.6

radioactivity was greatly enhanced. Essentially the same properties of chitin. Treatment of the excised paper strips for

amount of polymer was synthesized from either substrate. As 30 min at 100” with 1 M acetic acid or 1 M KOH removed only

expected, when a large excess of unlabeled UDP-GlcNAc was 10 to 15% of the radioactivity. The optimum pH for chitin

added to the incubation mixtures it effectively competed with synthesis was found to be in the range of 5.8 to 6.2 (Fig. 1). the labeled material and the incorporation of radioactivity into Chitin Synthesis by Different Cell Partick---Chitin synthetase chitin was nearly nullified. activity was found distributed among the different particulate

The chromatographically immobile material had the solubility fractions of M. routii (Table III). The specific activities (pro-

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tein basis) were highest for the cell residues and lowest for the soluble supernatants. Those of the mitochondrial and micro- somal fractions were intermediate. On the basis of total ac- tivity, it was calculated that nearly 85% of the chitin synthetase remained in the cell residues. About 10% was found in the microsomes, whereas the mitochondrial and soluble fractions contain only traces of activity. Similar findings on chitin syn- thetase distribution were obtained when either UDP-14C-GlcNAc or UDP-GlcNJ4C-AC was employed as glycosyl donor.

Chitin Synthetase Activity of Cell Walls-The high values of specific and total chitin synthetase activities of cell residues of M. rouxii suggested that a great deal of the enzyme was in- timately associated with the cell wall. To confirm this supposi- tion, cell walls were prepared and tested for chitin synthetase activity at different stages of purification (Table IV). Repeated breaking and washing failed to remove the chitin synthetase activity from the cell walls. On the contrary, the specific ac- tivity increased as the purification progressed, probably reflect- ing the removal of contaminating protein. Microscopic exami-

nation of the cell walls revealed no evidence of cytoplasmic contamination.

Kinetics of Chitin Formation by Cell Walls-Cell walls (con- taining 3.4 mg of protein), prepared by 6 cycles of breakage and washing, were incubated in mixtures containing 0.1 pmole of UDPJ4C-GlcNAc, 5 pmoles of GlcNAc, 5 mM MgC& in a total volume of 0.5 ml in either 25 mM KHsPOrKOH buffer (pH 6.5) or 25 my Tris-HCl buffer (pH 7.5). After incubation for

varying times the enzymes were inactivated with 50 ~1 of glacial acetic acid and the formation of chromatographically immobile 14C-labeled compounds was assayed. After 1 min of incubation, the formation of chromatographically immobile 14C-labeled material increased linearly with time for up to 1 hour. The rate of incorporation was consistently higher in incubations of less than 1 min. Over-all, the radioactivity incorporated in 1 min accounted for approximately 10% of the total amount of radio- activity incorporated in 1 hour, regardless of the buffer used in the incubation mixture (Fig. 2). This biphasic character of the rate curve was in contrast with that observed for microsomes and mitochondria. Both of these particulate fractions incorpo- rated radioactivity at a rate which was linear throughcut the whole period of incubation (Fig. 2).

The biphasic nature of the rate curve for the synthesis of chitin by cell wall preparations of M. rouxii recalls the findings of Villemez and Clark (17) on the rate of incorporation of man- nose, from GDP-mannose, and glucuronic acid, from UDP- glucuronic acid, into polymers by particulate enzymes from Phaseolus aureus. The biphasic character was due to the forma- tion of lipid intermediates early in the course of the incubation and their subsequent incorporation into a polysaccharide was demonstrated. Our attempts to isolate soluble lipid inter- mediates in chitin synthesis of M. rouxii have been unsuccessful

(W. Identijicatim of Biosynthesized Chitin-For characterization

of the polymer formed from UDP-GlcNAc by M. rouxii, the microsomal enzyme was used. Ten reaction mixtures, each con- taining 0.02 pmole of UDP-‘4C-GlcNAc, 10 pmoles of GlcNAc, 5 mM MgC&; 0.1 ml of microsomal suspension (5.3 mg of protein),

25 mM phosphate buffer (pH 6.5), in a total volume of 0.5 ml, were incubated at 22” for 1 hour and inactivated with 20 ~1 of glacial acetic acid. The mixtures were pooled and dialyzed against four changes of distilled water (700 ml). The non-

TABLE IV Chitin synthetase activity of cell walls

Cell residues prepared by freezing and thawing were subjected to the indicated number of breakage cycles. The cell wall frac- tions recovered after each cycle were assayed for chitin synthe- tase activity using the standard incubation mixture buffered with 25 mM KHzPOd-KOH (pH 6.5). Data are expressed as radioac- tivity incorporated into chitin from UDP-“C-GlcNAc (40,000 dpm) per mg of protein per hour.

Number of ballistic-disruption cycles Radioactivity incorporated

-em 0 21,430 1 32,160 2 31,115 3 32,255

dialyzable material was centrifuged at 2000 x g for 10 min to yield a radioactive sediment and a clear radioinactive super- natant. The radioactive sediment was washed twice with water and suspended in 5.0 ml of water. Samples (0.5 to 1.0 ml) of this suspension were used for the following treatments.

1. The radioactive suspension was heated with 1 M acetic acid (5.0 ml) for 30 min at 100” and centrifuged at 2000 x g for 10 min to yield a sediment containing 84.7% of the initial radio- activity. The supernatant, which was slightly turbid, was evaporated under reduced pressure to 0.1 ml and chromato- graphed using Solvent C. At least 95% of the radioactivity on the chromatogram was immobile.

2. The radioactive suspension was incubated with purified chitinase (5 mg of protein) in 0.2 M sodium acetate (pH 5.0) in a total volume of 1.0 ml for 8 hours at 30”. The reaction was terminated by the addition of 100 ~1 of glacial acetic acid and centrifuged at 2000 x g for 10 min. The pellet was washed three times with water. The precipitate and supernatant were then counted for radioactivity. Of the original radioactivity, only 11.1% remained insoluble; the rest was shown to be solely N-acetyl-n-glucosamine by paper chromatography.

3. The radioactive suspension was hydrolyzed in 5.0 ml of 6 M hydrochloric acid at 120” for 4 hours. The hydrolysate was neutralized with lead carbonate and centrifuged. Lead traces were removed by passing a stream of hydrogen sulfide and removing the resulting precipitate by centrifugation. At least 95% of the initial radioactivity was recovered in solution. Paper chromatography revealed only glucosamine.

4. Samples (0.5 ml) of the radioactive suspension were mixed with concentrated hydrochloric acid (1.0 ml) and were partially hydrolyzed by heating at 60” for 10 and 20 min. The hydroly- sates were neutralized as described above, then selectively N-acetylated (15) with acetic anhydride, and any incompletely acetylated substances were removed by exchange on Dowex 50 (H+ form) columns (20 x 1 cm). About 70% of the original radioactivity was recovered and the solutions were evaporated to 0.1 ml under reduced pressure. Paper chromatography, using Solvent A, revealed four major peaks in the lo-min hydrolysate (Fig. 3). Hydrolysis for 20 min increased the proportion of radioactivity in the faster moving peaks. Peaks I and II were tentatively identified as N-acetyl-n-glucosamine and its dimer (diacetylchitobiose), respectively, on the basis of their chromato- graphic mobilities. The radioactive compounds in Peak III were eluted, evaporated to 1.0 ml, reduced with sodium boro-

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- 6OOOr

0 IO 20 30

DISTANCE FROM ORIGIN (cm)

FIG. 3. Distribution of radioactivity in a paper ohromatogram of a partial acid-hydrolyzate of biosynthesized W-chitin. The W-chitin synthesized by microsomes was partially hydrolyzed with HCI, acetylated with acetic anhydride, and chromatographed on paper using Solvent A.

TABLE V

Net synthesis of chitin

For the initial incubation the standard reaction mixture con- tained 0.02 pmole of UDPJ4-C-GlcNAc, 0.1 ml of mitochondrial fraction, 0.1 ml of microsomal fraction, and other additions as described in “Materials and Methods.” In the second incuba- tion 0.1 pmole of either UDP-W-GlcNAc or UDP-GlcNAc was added.

First incubation (15 min) I

Second incubation (45 min)

Substrate

A. UDP-W-GlcNAc (39,700 dpm) . .

B. UDP-W-GlcNAc (39,700 dpm) .

Radio- activity in chitin

Additions

10,470 UDP-GlcNAc. . . . .

UDP-“C-GlcNAc 10,690 (195,500 dpm). .

Radioac- ti vity

in chitin

13,210

25,062

hydride (19), hydrolyzed with 4 M hydrochloric acid for 2 hours at loo”, and neutralized as described previously. One-half of the hydrolysate was chromatographed directly on paper using Solvent A. The remainder was N-acetylated with acetic anhy- dride (15) and chromatographed on paper in Solvent A. On both chromatograms the radioactive components of the hydroly- zate migrated as two peaks. About 73% of the radioactivity in the nonacetylated hydrolyzate was located in a peak with the same chromatographic mobility as D-glucosamine. The remain- ing 27% of the radioactivity was located in a slower moving peak which was probably D-glucosaminitol. In the acetylated hydrolysate, 69% of the radioactivity migrated as a peak with the same chromatographic mobility as N-acetyl-D-glucosamine. The remaining radioactivity cochromatographed with a sample of N-acetyl-D-glucosaminitol, prepared as described by Barker

et al. (19). These percentage values suggest that the oligomers in Peak III have an average chain length of 4 residues.

5. Samples (0.2 ml) of the radioactive suspension were incu- bated with 5 mg of various hydrolytic enzymes in 1.0 ml of 0.2 M sodium acetate (pH 5.0) for 6 to 12 hours at 30”. Paper chromatography of the incubated mixtures revealed that the radioactive polymer was not appreciably hydrolyzed by chymo- trypsin, cellulase, or lysozyme. In all cases, a small amount of the total radioactivity (0.3 to 2%) became chromatographi- tally mobile.

The radioactive products synthesized by the cell residues and mitochondrial fractions were not fully identified, but it was shown in both cases that the radioactive products were similar to that synthesized by the microsomal fraction. At least 80% of the radioactivity incorporated by the cell residues, and about 90% of the radioactivity incorporated by the mitochondria was hydrolyzed to N-acetyl-D-glucosamine on treatment with chiti- nase. More than 85% of the radioactivity incorporated into the cell residues and about 80% of the radioactivity incorpQrated into the mitochondria wag insoluble either in hot 1 M acetic acid, or in hot 1 M KOH, but was hydrolyzed by hot 6 M hydro- chloric acid to glucosamine.

Net Synthesis of Chit&-This was demonstrated by a modifica- tion of the procedure used by Glaser and Brown (3). Two standard reaction mixtures, containing 0.1 ml of mitochondrial fraction and 0.1 ml of microsomal fraction, were incubated for 15 min at 22’ (Table V). One half of each incubation mixture was used to measure the radioactivity incorporated into chitin. The remainder was supplemented with 0.1 pmole of either un- labeled UDP-GlcNAc or UDPJ4C-GlcNAc and reincubated for a further 45 min. The mixtures were then reassayed for chroma- tographically immobile radioactivity. As shown in Table V, the second incubation with unlabeled UDP-GlcNAc did not lower the radioactivity incorporated into chitin during the first incu- bation. In the control experiment (B) in which UDP-W-Glc- NAc was employed in the second incubation, a large increase in the radioactive product was detected, proving that the enzyme was still active. These results suggest that the enzyme in the mitochondrial and microsomal particles catalyzed a net synthesis of chitin rather than an exchange reaction between acetylglucos- amine units of chitin and those of UDP-GlcNAc.

Effect of Culture Age on Chitin Synthetme Level-Cultures of 1M. routii were grown for various time intervals. The harvested material was suspended in 20 ml of Tris-HCl buffer (pH 7.5) and transferred to a 100 ml Erlenmeyer flask. The cell suspen- sion was then rapidly frozen by immersing the base of the flask in a solid Cot-acetone mixture for 5 min. After thawing at 20” the cell suspension was homogenized in a Sorvall Omni-Mixer. The freeze-thaw cycle was repeated three times. Microscopic examination of the suspension showed that more than 95% of the cells were ruptured and had emptied their contents into the suspending buffer. The remaining 5% appeared to have intact cell walls but showed no internal organization. Samples of the disrupted cell suspension, containing 3 to 5 mg of protein, were then added to mixtures containing 5 mM MgClz, 10 pmoles of GlcNAc, 0.01 pmole of UDPJ4C-GlcNAc, and 25 mM Tris-HCl buffer (pH 7.5) made up to a final volume of 0.5 ml. The mixtures were then incubated for 15 min at 22” and assayed for chitin synthesis by the standard procedure. Total chitin synthetase activity (expressed per ml of culture) increases throughout the growth of the culture (Fig. 4). In contrast, the

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TABLE VI

4005

I I 5 10 15 200

TIME (hoursI PH

Fro. 4 (left). Effect of culture age on chitin synthetase level and hexosamine content. Radioactivity incorporated into chitin from UDP-W-GlcNAc by enzyme from 1.0 ml of culture (0-O) or by 1 mg of cell protein (O-O) was measured in mixtures containing 20 rnM GlcNAc (see “Results”). Total hexosamine content of cultures (A- - -A).

FIG. 5 (right). Effect of pH on chitin formation from “C-Glc- NAc-6-P in the presence of UTP (4 111~) and GlcNAc (20 mM). Radioactivity incorporated into chitin was measured in incubation mixtures in which the pH was varied with either 25 rnM KHzPOc KOH (O-0) or 25 mM Tris-HCl (O-O) buffers.

values obtained for specific activity of chitin synthetase (ex- pressed on a protein basis) reached a maximum after 8 hours of growth. As seen in Fig. 4, the values obtained for the total chitin synthetase activity of the cultures correlated with their hexosamine contents. Cultures were judged to be growing exponentially between 2 and 12 hours of incubation on the basis of the increase in protein concentration per ml of culture.

Chitin Synthesis from N-Acetyl-&%ucosamine &Phosphate- Cell-free extracts of M. rouxii were shown to synthesize chitin from N-acetyl-n-glucosamine 6-phosphate. A reconstituted cell-free extract containing mitochondria, microsomes, and soluble supernatant was incubated as indicated in Table VI. Chromatographically immobile polymers were synthesized from GlcN-I%-AC-6-P provided that UTP and free N-acetyl-n- glucosamine were also present. UTP was not replaced by ATP, CTP, or GTP. Also, it was not possible to replace GM-14C- AC-6-P with 14C-GlcN-6-P. These results indicate that the formation of chitin from GlcNAc-6-P can be accomplished in a single incubation and that it occurs via the formation of UDP- GlcNAc. The effect of pH on the incorporation of radioactivity from 14C-GlcNAc-6-P into polymers was examined in the presence of UTP and GlcNAc over the range of 5.8 to 8.5 using KHzPOd- KOH (25 mM) and Tris-HCl (25 mM) buffers. Different pH optima were obtained, 7.2 and 8.2, respectively, depending on the buffer used (Fig. 5).

A&radiographic Localization of Chitin Synthetase- The position of chitin synthetase activity along the cell walls was ascertained by incubating a washed sample of cell residues of M. rouxii with UDP-aH-GlcNAc of high specific activity. Autoradiographs of these largely empty cells demonstrated that the radioactivity was preferentially incorporated at the tips of both main hyphae and side branches (Fig. 6A). In a parallel test, a large excess of unlabeled UDP-GlcNAc (4 pmoles) was added to the incubation mixture. Autoradiographs of these control hyphae showed only a random, background pattern of labeling, thus confirming that the apical deposits of silver grains represent regions of incorporation of N-acetylglucosamine from UDP-GlcNAc, i.e. chitin synthetase sites.

Significantly, this apical pattern of chitin synthetase distribu-

Incorporation of radioactivity from N-acetyl-o-glucosamine 6- phosphate into chitin

Data are expressed as radioactivity incorporated into chitin from either r4C-GlcNAc-6-P (0.02 pmole) or 14C-GlcN-6-P (0.02 pmole) in 1 hour at 22’. Where indicated, additions of GlcNAc (10 pmoles), UTP (2.0 pmoles), ATP (2.0 rmoles), CTP (2.0 rmoles), and GTP (2.0 pmoles) were made to incubation mixtures containing 0.1 ml of dialyzed supernatant II, 0.1 ml of mito- chondrial fraction, 0.1 ml of microsomal fraction, 5 mM MgC12, and 25 mM Tris-HCl buffer (pH 7.5) in a final volume of 0.5 ml.

Substrate

W-GlcNAc-6-P (40,000 dpm) W-GlcNAc-6-P (40,000 dpm) 14C-GlcNAc-6-P (40,000 dpm) W-GlcNAc-6-P (40,000 dpm) 14C-GlcNAc-6-P (40,000 dpm) “C-GlcNAc-6-P (40,060 dpm) W-GlcNAc-6-P (40,060 dpm) W-GlcN-6-P (40,000 dpm) I%-GlcN-6-P (40,000 dpm) W-GlcN-6-P (40,000 dpm) W-GlcN-6-P (40,060 dpm)

Additions

None GlcNAc UTP GlcNAc + ATP GlcNAc + CTP GlcNAc + GTP GlcNAc + UTP GlcNAc + ATP GlcNAc -I- CTP GlcNAc + GTP GlcNAc + UTP

Radioac- tivity in- corporated

am

14 21 69 28 28 20

2,590 13 20 13 59

FIG. 6. Autoradiographic localization. A, chitin synthetase in hyphal walls of M. rouxii. Disrupted hyphae incubated with UDP-aH-GlcNAc. B, sites of cell wall synthesis (chitin + chito- san) in living hyphae of M. rouxii incubated with a short pulse of *H-GlcNAc.

tion found in vitro parallels the apical pattern of chitin (plus chitosan) deposition in &JO. The latter (Fig. 6B) was also demonstrated autoradiographically by incubating whole living cells with a short pulse of aH-GlcNAc. After exhaustive extrac- tion, the resulting cell ghosts showed a distinct pattern of apical labeling. As shown earlier (16), the radioactivity represents largely, if not entirely, newly synthesized chitosan and chitin.

DISCUSSION

Our data show that cell extracts of M. rouxii contain enzymes which catalyze the conversion of N-acetyl-n-glucosamine into uridine diphosphate N-acetyl-n-glucosamine and its subsequent transfer into chitin. Evidence was obtained for the presence in the soluble cytoplasm of M. routii of an ATP-dependent kinase, which phosphorylates GlcNAc at position 6, and a uridine

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4006 Chitin Synthesis by Mucor rouxii Vol. 246, l\o. 12

diphosphstc N-acetyl-n-glucosamine pyrophosphorylase (20), which accomplished the fir1ul symhesis of the UJ>P-GlcNAc. Since GlcNAc-6-P served as a substrate for thr synthesis of UDPGlcNAc but ordy GlcSAc-l-l’ is known to be a glycosyl donor in the pyrophosphorvlar;e reaction (2O), phosphomutation would have been necessary. The formation of a compound with the paper chromatographic mobility of GlcNAc-l-l’ during the synthesis of UDP-GlcSAc, supports the presence of phospho- ncet.ylglucosamine mutate as previously described for .Veurospora crassa (21, 22). In A’. crassa the mutate requires glucose 1,6- diphosphate as a cofactor. The activating influence of this compound was not assessed in the present study but it was always included in the incubation mixtures used for preparation of UDP-GlcNAc.

When incubated in the l)rcscnce of UTP and a cell-free extract. containing both soluble and particulate fractions, radioactivity from 1%GlcNAc-6-l’ was incorporated into chitin. The opti- mum pH for this incorporation was significantly different from that of chitin synthesis from UL)P-*YY-GlcNAc. This indicates different pH optima for UDP-GlcSAc pyrophosphorylasc (20) and chitin synthetasc and suggests that for maximum efficiency of chitin synthesis in tiuo, some degree of cell compartmentaliza- tion would be necessary if, indeed, these enzymes operate at their plf optima in tiuo.

The following lines of evidence supported the conclusion that chitin, the @-(1 + 4)-linked polymer of in-aret.~l-u-gluroalminc, was the main, if not the sole, polymer biosynthesized from Ul)P- GlcNAc. (a) Chitin was chromatograI)hicnlly immobile and insoluble in hot alkali; insolubility in hot acetic acid showed that the product was not chitosan (1). (5) Complete hydrolysis with hydrochloric acid gave onl,v one radionct.ivr product, namely glucosamine. (c) Of the several hydrolytic enzymes tested, only chitinase caused substantial breakdown of the reaction product, and only one monomer, namely, S-ncetyl-r-glucosa- mine, was formed in appreciable amounts. Proteolysis failed to render the product chromatographically mobile though it caused substantial breakdown of the microsomrs. (d) Isolation of radioactive oligomers after partial acid hydrolysis of the radioactive product confirms that chains of N-acetyl-nglucos- mine residues had been synthesized. (e) Incubation of l)re- formed radioactive chitin with unlabeled UDP-GlcNAc and active enzyme did not diminish the radioactivity in the polymer. It wCJllS unlikely, therefore, that the radioactivity in the polymer arose bv a simple glycosyl interchange between the trrn1inal units of preformed chitin chains nrld the N-acctyl-u-glucosa- mint in CDP-GlcNhc.

Tl1e chitin syrithetasc of nf. rouxii is strongly and specifically activated by free N-acetyl-n-glucosamine (18). This activation has also been recorded for the chitin synthetases from ot.her fungi

(3, 5-7) but not from crustaceans (8). In previous studies of fungal chitin synthetase, enzymic

activity was reported to be mainly in mitochondrial (5-7) or microsomal fractions (3, 5-7). In none of these studies were the cell walls examined. A novel feature of the present study of $f. rouxii is the finding of high specific chitin synthetase act,ivity in the cell ~a.11 fraction. AIoreover, calculation of total chitin synt.hetnse activity in broken cell suspensions revealed that most of the act.ivitp remained in the cell residues, thus suggesting an intimate association with the cell walls. This was confirmed with a l)urifed cell wall fraction; repeated breaking and washing

of purified cell w-alls did not decrrose their chitin synthetasc activity, indicating that the enzyme was firmly bound to t.he wall fabric. The chitin syuthetasc found in microsomes, the other cellular fraction of Ai. rouxii with significant, activity, possibly represents nascent enzyme prior to its migration to the cell wall, whereas, the cell wall-bound synthetase represents enzyme at its site of operation which is chiefly in the hyphal tip region. This supposition is supported by autoradiographic studies showing that the incorporation of radioactivity from T!DP-3H-GlcNA~ into chitin by cell walls occurs principally in the hyphal tip region in agreement with observations on wall synthesis with living cells (16).

The nearly identical amounts of chitin synthesizcld from UDP- GlcNAc labeled in either the glucosamine or aretyl moieties indicate that the polymer synthesized was made of .Y-ncetyl-n- glucoswmiue residues, i.e. chitin, and excluded the possibility that chitosan, the corresponding p-(1 --f 4)-polymer of n-glucosa- mine, had been synthesized conrurrcntly to any measurable exter1t.. Chitosan is a much more nbuudant component.of the cell wall of U. rouxii than chitin (I), but its biosynthesis remains unclear. Total chitin synthetase activity increased rapidly at the onset of hyphal emergeuce, reached a maximum at the end of the phase of exponential growth, and thereafter increased only slightly. In contrast, the specific activity of chitin syn- thetase showed marked fluctuations, decreasing first, increasing rapidly during exponential growth, aud fir~all~ decreasing. I’rob- ably these fluctuations reflect marked changes in the over-all rute of protein synthesis affecting the protein content of the ccl1 against which t.he values of chitin sy11thetase specific activity are calculated. Barring artifacts of in L&O studies (see Refer- ence 23), these fluctuations underscore changes in biochemical priorities during the development of the fungus.

Ackno~bdglnent---~~fr thank Eleanor Lippman for her skillful collaboration in all esperimrnts involving autoradiography.

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Ian McMurrough, A. Flores-Carreon and S. Bartnicki-GarciaMucor rouxii

Pathway of Chitin Synthesis and Cellular Localization of Chitin Synthetase in

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