THE JOURNAL cm B~LO~ICAL CHEMISTRY
Vol. 254, No. 11, Issue of June 10, pp. 4901-4907, 1979
Printed in U.S.A.
An Endochitinase from Wheat Germ
ACTIVITY ON NASCENT AND PREFORMED CHITIN*
(Received for publication, November 6, 1978)
Jesus Molano,$ Itzhack Polacheck,g Angel Duran,l and Enrico Cabibll
From the National Institutes of Health, National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda,
A chitinase has been obtained in milligram amounts agglutinin, obtained by affinity adsorption on chitin (2), were
from wheat germ by affinity chromatography on chitin indeed found to be strongly inhibitory on a solubilized and
followed by chromatography on Sephadex G-50. The partially purified preparation (3) of chitin synthetase. Upon
purified enzyme was free from wheat germ agglutinin closer scrutiny, however, this effect was found to be due to a
and showed a single peak on sodium dodecyl sulfate- protein contaminant. After this protein was purified, we dis-
acrylamide gel electrophoresis. Isoelectric focussing covered that it was not a true inhibitor of the synthetase.
on acrylamide gel slabs gave rise, however, to several Rather, the decrease in chitin accumulation observed in its
bands, with isoelectric points ranging between 7.5 and presence resulted from destruction of the polysaccharide as it
9.2. The molecular weight of the enzyme, as determined was formed, i.e. the protein was a chitinase. A relatively
from sodium dodecyl sulfate-gel electrophoresis, was simple procedure was devised to prepare large amounts of the
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30,000, and that determined from sedimentation anal-
chitinase in homogeneous state. It was found that the enzyme
ysis was 33,000; therefore, the enzyme exists as a mono-
acts as an endochitinase, an activity that has been rarely
mer in solution. The enzyme releases oligosaccharides
2 to 4 units in length from chitin, ie. it is an endochiti- described (4, 5) and never purified to homogeneity. A kinetic
nase. When allowed to act on “nascent” chitin, that is, study of the enzyme showed that the chitinase was much
chitin that is being synthesized by a preparation of more active on nascent than on preformed chitin. This finding
solubilized chitin synthetase, the chitinase shows an may have interesting implications for the mechanism of cell
enhancement in activity of about 2 orders of magnitude. wall synthesis and degradation in plants and fungi and also
The products formed under these conditions consist of for the function of the chitinase in wheat germ as a possible
higher oligosaccharides than those obtained with “pre- defense against parasites.
formed” chitin substrate. It is proposed that the nascent
chitin is more susceptible to the enzyme because its EXPERIMENTAL PROCEDURES
chains have not yet coalesced with others to yield a Materials
tighter and more impervious structure. Such a situation
Sephadex G-25, G-75, and G-50 were obtained from Pharmacia,
may prevail during turnover of structural polysaccha- DEAE-cellulose paper was from Reeve Angel, and anion exchange
rides in uiuo; therefore, in vitro measurement of poly- resin AG 2-X8 was from Bio-Rad. Of the polysaccharides used,
saccharide breakdown concomitant to synthesis may chitosan was from Pfanstiehl, glycol chitosan was from Sigma, cellu-
reflect conditions in the cell better than the static meth- lose was from Reeve Angel, pustulan @l--f g-linked glucose polymer)
ods usually employed. was from Calbiochem, and laminarin (PI + 3-linked glucose polymer)
A method for the separation and measurement of was from ICN. Micrococcus lysodeikticus dried cells and p-nitro-
chitin, chitin oligosaccharides, and IJDP-N-acetylglu- phenyl-P-N-acetylglucosaminide were purchased from Sigma:
cosamine by chromatography on DEAE-cellulose paper Tritiated or unlabeled chitin and glycol chitin were prepared by
is also described. acetylation of chitosan and glycol chitosan (6,7) and (8), respectively.
Yeast glucan was obtained as already reported (9).
UDP-N-Acetylglucosamine was purchased from Sigma and UDP-
[U-‘4C]N-acetylglucosamine (300 mCi/mmol) was from Amersham/
Wheat germ agglutinin was recently found to inhibit growth Searle.
Of the proteins used, myoglobin, ovomucoid, papain, and cyto-
of some fungi (1). Since this lectin binds very strongly to chitin chrome c were from Sigma, ovalbumin and pepsin were from Worth-
(2), a major component of the fungal cell wall, we investigated ington, bovine serum albumin was from Aldrich, and wheat germ
the possibility that its effect on growth might be mediated by agglutinin was from Miles.
an inhibition of chitin synthetase. Some preparations of the Polyoxin D was a generous gift of the Kaken Chemical Co., Ltd.,
* The costs of publication of this article were defrayed in part by Chitin oligosaccharides were prepared by a modification of Ru-
the payment of page charges. This article must therefore be hereby pley’s procedure (10). Chitin (tritiated or unlabeled), 100 mg, was
marked “advertisement” in accordance with 18 U.S.C. Section 1734 dissolved in 10 ml of concentrated HCl and the mixture was incubated
solely to indicate this fact. at 40°C for li% h. The solution was brought to dryness in a rotary
$ Present address, Departamento de Bioquimica, Ciudad Sanitaria evaporator at 40°C. The residue was subjected to two cycles of
“La Paz”, Madrid-34, Spain. solution in 20 ml of water and evaporation to dryness to remove most
§ Present address, National Institutes of Health, National Institute of the HCl. The final residue was redissolved in 7.5 ml of water and
of Allergy and Infectious Diseases, Laboratory of Clinical Investiga- neutralized with 0.2 M Na&Os. Some insoluble material was removed
tions. This work was performed during tenure of long term Fellowship by centrifugation and the supernatant fluid was applied to two Seph-
ALTF 1-1977 of the European Molecular Biology Organization. adex G-25 columns connected in tandem (fist column, 2.5 X 90 cm;
1 Present address, Universidad de Salamanca, Facultad de Cien- second column, 4 x 100 cm), previously equilibrated with distilled
cias, Departamento de Microbiologia, Salamanca, Spain. water. Fractions of 10 ml were collected at a flow rate of 34 ml/h.
]I To whom correspondence concerning this paper should be ad- Sugars in the eluate were detected by measuring either radioactivity
dressed. or reducing power (11). Fractions from each peak, from the di- to the
Wheat Germ Chitinase
hexasaccharide, were pooled and evaporated under reduced pressure.
The peaks corresponding to the tetra-, penta-, and hexasaccharide
were rechromatographed in the same system for further purification.
Chitin synthetase was obtained from yeast protoplasts, solubilized,
and purified as previously reported (3). This preparation was found
to contain some chitinase activity,’ which was removed by adsorption
on chitin, as follows. Purified chitin synthetase (0.25 ml) was applied
to a small chitin column (bed volume, 0.5 ml), previously equilibrated
with 25 mM Tris-chloride, pH 7.5, containing 5 rmu MgS04 and 0.1%
digitonin. The effluent was again applied to the column; the operation
was repeated a third time, whereafter the column was washed with
0.5 ml of the equilibrating buffer. Effluent and washing were com-
Wheat germ was purchased from Sigma and defatted as follows.
One kilogram of wheat germ was suspended into 2500 ml of petroleum
ether and the suspension was shaken for 15 min at 2°C. The solvent
was decanted and another extraction was carried out in the same way.
The defatted wheat germ was spread out on aluminum foil and dried
at room temperature under a hood.
Chitinase Assay-Chitinase was measured by the liberation of
soluble radioactivity from tritiated chitin as previously described (7).
In some cases it was determined by the liberation of reducing power
(11). When the action of chitinase on nascent and preformed chitin
was studied, the procedure described in the following section was
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Determination of Chitin, Oligosaccharides, and Unreacted UDP-
GlcNAc in Mixtures Containing Chitinase and Chitin Synthetase-
In experiments with “nascent chitin” (see “Results”), it was necessary
to measure independently chitin and chitin oligosaccharides produced
in the reaction mixture. Ascending chromatography on DEAE-cellu-
lose paper provided an efficient separation of these substances. Never-
theless, some problems were found because of the multiple chroma-
tographic developments that were required: the DEAE-cellulose pa- FIG. 1. Frame used for chromatography on DEAE-cellulose paper.
per crumpled badly after drying and, since the top part of the paper The frame (F) of standard Pyrex glass rod, is shaped as an inverted
was excised after the fit development, it was necessary to use a U. The arms, 20 cm long, form right angles with the middle bar, 18
system in which paper strips of different length could be-accommo- cm wide. The ends of the long arms are shaped in the form of hooks
dated. The solution was found in an adiustable frame (Fie. 1) built (M. The straight rod (R) is introduced through one of the hooks,
from glass rods. Since the paper is stretched on the frame during both then threaded through cuts made in the paper strips and finally
development and drying, crumpling is avoided. The length of the pushed through the second hook. Rubber rings (B) are then slipped
frame is adjustable, allowing papers of different length to be accom- over the rod ends, to keep it in place. The paper strip end is attached
modated. with a stainless steel chromatography clip (C) to the movable rod
For the “nascent chitin” experiments, the reaction mixture con- (R’). R’ rests on the finger grips of other chromatography clips (C’)
tained 18 mM Tris-chloride (pH 7.5), 3.6 mM MgSOa, 32 mM N-acetyl- to which it is attached with rubber bands. The clips (C’) can be slid
o-glucosamine, 0.18 mg/ml of phosphatidylserine, 0.06% digitonin, 1 up and down the arms of the frame, to adjust to the length of the
mM UDP-[r4C]GlcNAc (specific activity, 1.65 X lo6 cpm/nmol), 1 paper strips used. The whole assembly is placed in a thin layer
milliunit/ml of solubilized and partially purified yeast chitin synthe- chromatography tank, which contains the appropriate solvent in the
tase (see Ref. 3 and “Materials”) and 0.2 pg/ml of purified chitinase. bottom. After development, the assembly is taken out and hung in an
After varying incubation times at 30°C, the reaction was stopped by oven at 60°C for 5 min to dry out. Although in the vhotosravh two
immersing the tubes in a boiling water bath for 7 min. Portions of 200 separate paper strips are shown for clarity, in most expe&nents it
$ of each reaction mixture were evaporated to dryness in a rotary was found more convenient to cut the strips from a single piece of
evaporator under reduced pressure. The residues were dissolved in 40 DEAE-cellulose paper, so that they would all be attached to the same
~1 of water and transferred to the origin of the chromatographic strip. basal portion through which rod R was threaded. In this way up to
After drying the paper with a hair dryer, the assembly was placed in eight 1.5-cm wide strips could be accommodated in the frame at the
a tank with water as solvent. The oligosaccharides in the reaction same time.
mixture traveled with the solvent front, whereas chitin and the
unreacted UDP-GlcNAc remained at the origin. When the solvent sion. Both the resin and chitin remained in the fdter, whereas oligo-
front approached 1 cm from the strip end, development was stopped saccharides were collected in the filtrate and counted. A similar
and the papers were dried at 60°C for 5 min. A segment of 3 cm from method was previously used for the assay of GDP-glucose hydrolase
the top was cut out and placed in a scintillation vial containing 10 ml (12). Reaction mixtures and incubation conditions were the same as
of Aquasol (New England Nuclear). described in the previous section. After stopping incubation by heat-
Glass rod R’ was now moved down as needed and the paper strips ing, 50 ~1 of the mixture were added to 150 al of an aqueous suspension
again were fastened to it with clips. The second chromatographic of AG 2-X8 resin (67 mg/ml) in the acetate form. After shaking in a
development, carried out with 0.2 M NaCl containing 0.1 M HCl as wrist shaker for 10 min, the suspension was faltered into a scintillation
solvent, resulted in migration of UDP-GlcNAc from the origin; the vial through a Gelman A/E glass fiber filter. The tube and filter were
chitin remained immobile and was measured by cutting out a 3-cm washed with three 0.2~ml portions of water. To the combined filtrates,
long segment encompassing the origin and counting it as above. If 15 ml of Aquasol were added and the samples were counted.
desired, a 3-cm long zone, ending at the solvent front, may also be cut Chitin Synthetase Assay-Chitin synthetase was assayed by incor-
out for determination of UDP-GlcNAc. poration of N-acetylglucosamine into chitin as described in a previous
Determination of Oligosaccharide Liberation from Nascent Chi- report (3).
tin with the Use of Anion Exchange Resin-In experiments similar Agglutination Assay-In a porcelain well, 30 ~1 of a 2% (v/v)
to those of the previous section, a simpler procedure could be applied suspension of trypsin-treated human red blood cells (13) in 0.9% NaCl
when only the incorporation of radioactivity into oligosaccharides was containing 10 mM sodium bicarbonate were mixed with 10 ~1 of the
measured. The method consisted of adsorbing the unreacted UDP- sample to be tested, which had been diluted in the same buffer. Serial
GlcNAc with an anionic exchange resin and then filtering the suspen- dilutions were made, and agglutination was estimated visually, on a
scale from 0 to 4+. The number of units/ml of a sample was defined
’ I. Polacheck, J. Correa, and E. Cabib, unpublished results. as the inverse of the dilution that yielded an agglutination of 2+.
Wheat Germ Chitinase 4903
Ultracentrifugation-Meniscus depletion sedimentation equilib- was 600 ml/h and 17-ml fractions were collected. Fractions
rium experiments were performed at 3-5°C in a Spinco model E corresponding to the main protein peak (Fractions 110 to 147
ultracentrifuge. Before centrifugation, the protein sample was di- in Fig. 2) were adjusted immediately to pH 8.3 to 8.5 with a
alyzed against 0.05 M potassium borate at pH 8.5, containing 0.15 M
NaCl. Rotor speed varied between 24,000 and 40,000 rpm. A value of mixture containing 2 M Tris and 3.3 M NaOH. Chitinase and
0.71 for the partial specific volume was calculated from the amino red blood cell agglutinating activity were eluted together in
acid composition. The solvent density was taken as 1.0. this column (Figs. 2 and 4). Fractions 126 to 143 were pooled
Gel Ebctrophoresis-SDS’-polyacrylamide gel electrophoresis and concentrated at 2°C in an Amicon filtration assembly
was carried out as described by Shapiro et al. (14). Isoelectric focusing fitted with a PM-10 filter, to a volume of 13.5 ml. A small
on polyacrylamide slabs was performed according to the instructions
of the manufacturer (LKB Products). Bovine serum albumin (pI4.8), precipitate was removed by centrifugation in the cold for 15
hemoglobin (pI6.9), and cytochrome c (~19.8) were used as standards. min at 24,000 X g.
The pH was also measured with a surface pH electrode. Coomassie Step 4: Chromatography on Sephadex G-50 column-This
brilliant blue was used as the protein stain. chromatography was carried out at 2°C. The concentrated
Amino Acid Analysis-Amino acid analysis was performed with a chitin column eluate, 13 ml, was applied to a Sephadex G-50
Beckman model 120C analyzer. Hydrolysis with hydrochloric acid (fine) column (2.5 x 95 cm) previously equilibrated with 5 mM
was carried out essentially as described by Moore and Stein (15).
Cysteic acid and methionine sulfone were determined after performic hydrochloric acid. Elution was carried out with the same
acid oxidation (16). Tryptophan was measured spectrophotometri- solvent. In this column, the chitinase emerged first, followed
tally (17) and nitrogen by Kjeldahl digestion and nesslerization. by the agglutinating activity (Figs. 3 and 4). Immediately after
Miscellaneous-Paper chromatography was carried out with measuring the absorbance at 280 nm, Fractions 30 to 60 were
Whatman No. 1 paper. Chromatograms were developed for 40 h with brought to pH 8.5 with 1.5 M Tris, containing 0.5 M NaOH.
isoamyl alcohokpyridine:water (1:1:0.8) (18). The chromatograms
After measuring chitinase and agglutinating activity, Frac-
were scanned for radioactivity with a Vangard model 930 autoscanner.
Reducing sugars were detected on the chromatograms with the silver tions 37 to 46 were pooled. The enzyme was stored in this
nitrate reagent (19). The position of the different oligosaccharides solution, in the presence of 0.02% azide.
was ascertained by the use of external and internal standards. The final yield of enzyme in the preparation of Table I was
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Protein was determined according to Lowry et al. (20) and reducing 18% and the puritlcation was about 300-fold.
sugars were determined by the method of Park and Johnson (ll), The purified enzyme was stable for at least several weeks
except when cellulose was used as a possible substrate; in this case
the copper reagent (21) was employed. Total hexose was determined when kept either at 2°C or at -20°C.
with anthrone (22). Radioactivity was measured in a Beckman LS
8100 scintillation spectrometer. Physical and Chemical Characterization of Chitinase
The purified enzyme gave rise to a single band upon SDS-
acrylamide gel electrophoresis (Fig. 4). In contrast with this
result, several bands were found after subjecting the prepa-
ration to isoelectric focussing on polyacrylamide gel slabs (Fig.
Step 1: Extraction-All operations were carried out at 5). The possible reasons for this behavior will be considered
about 2”C, except where indicated otherwise. Defatted wheat under “Discussion.”
germ (see “Materials”), obtained from 1 kg of untreated Determinations of molecular weight by SDS-acrylamide
material, was suspended in 4 liters of 20 mu sodium bicarbon- electrophoresis (Fig. 6) and by equilibrium sedimentation (Fig.
ate and the mixture was shaken for 1 h at 2°C. After elimi- 7) yielded values of 30,000 and 33,000, respectively. The ap-
nating large particles by passage through a Buchner funnel,
the residue was extracted again in the same fashion with 2.5 TABLE I
liters of solution. The combined filtrates were centrifuged for
Purification of wheat germ chitinase
20 min at 11,000 X g, and the pellet was discarded.
Step 2: Precipitation at pH 4.5-The crude extract (see
Table I) was brought to pH 4.5 with 2 M acetic acid. After ml 76 units/mg
standing at room temperature for 15 min, the suspension was Crude extract 4500 2610 54,000 100 0.048
centrifuged in the cold for 20 min at 11,000 X g. The pellet pH 4.5 supernatant 4450 2540 38,300 97 0.066
was discarded and the supernatant liquid was adjusted to pH Chitin eluate 300 660 180 25 3.7
8.5 with 4 N NaOH. Sodium azide was added to a final Senhadex G-50 eluate 65 474 32 18 14.60
concentration of 0.02%. All solutions used in subsequent steps a One unit of enzyme is defined as the amount that catalyzes the
contained sodium azide in the same concentration to prevent liberation of 1 pmol of oligosaccharide (calculated as N-acetylgluco-
growth. samine)/min at 3O’C.
Step 3: Chromatography on Chitin Column-chromatog-
raphy on a chitin column was performed at room temperature
(-25°C). The acid precipitation supernatant (4450 ml) was
added to a chitin column (chitin obtained by reacetylation of
chitosan: see “Methods”), 6.2 cm in diameter and 20 cm long,
previously equilibrated with 20 mu sodium bicarbonate. Pas-
sage of the extract through the column takes 9 to 10 h, with
retention of about 70% of chitinase activity. After washing
successively with 1.3 liters of 20 InM sodium bicarbonate and
with 1.8 liters of 20 mu sodium acetate at pH 5.3, a linear
gradient was applied with 1 liter of 20 mu acetic acid, pH 3.3,
in the reservoir and 1 liter of 20 mu sodium acetate, pH 5.3,
in the mixing flask. Upon completion of the gradient, an
additional 1.5 liters of 20 mu acetic acid was applied. During
the elution, subsequent to passage of the sample, the flow rate FIG. 2. Chromatography of wheat germ chitinase and agglutinin
on chitin column. Fraction 1 corresponds to the start of the pH
’ The abbreviation used is: SDS, sodium dodecyl sulfate. gradient.
Wheat Germ Chitinase
9. The optimum temperature was 3O”C, with a broad maxi-
mum between 25 and 4O’C. The K,,, for chitin (reacetylated
chitosan; see “Materials”) was 2 mM (chitin concentration
calculated as N-acetylglucosamine).
10 20 30 40 50 60 70
TUBE NUMBER $! 4
Wheat germ chitinase
FIG. 3. Chromatography of wheat germ chitinase and agglutinin ?i
on Sephadex G-50 column.
\ Cytochrome c
0.2 0.4 0.6 0.8 1.0
FIG. 6. Determination of molecular weight of chitinase by SDS-
polyacrylamide gel electrophoresis. BSA, bovine serum albumin;
WGA, wheat germ agglutinin.
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FIG. 7. Sedimentation equilibrium of purified chitinase in the ul-
FIG. 4 (left). SDS-polyacrylamide gel electrophoresis of eluates tracentrifuge. The rotor speed was 26,000 rpm.
from chitin column (A) and Sephadex G-50 column (B). CH, chitinase;
WGA, wheat germ agglutinin. In B, 15 pg of protein were applied to TABLE II
FIG. 5 (right). Isoelectric focussing of purified chitinase (30 pg of Amino acid composition of wheat germ chitinase
protein) on polyacrylamide gel. For experimental details see “Meth- Values are the average or extrapolation of three determinations
ods.” (24-, 48-, and 72-h hydrolyses). They were calculated on the basis of
a molecular weiaht of 30.000.
Amino acid Number of residues/
parent molecular weight obtained by filtration through Seph- molecule of enzyme
adex G-75 was 29,000. There was no suggestion of lack of Lysine 8
homogeneity in the sedimentation results (Fig. 7). From all Histidine 4
these data, it may be concluded that the enzyme exists in Arginine 14
solution as a monomer. Aspartic acid 28
The amino acid analysis (Table II) showed 48 acidic and
only 22 basic amino acids/mol, despite the high isoelectric Glutamic acid 20
point (between 7.5 and 9.2, depending on the various bands Proline 15
seen in isoelectric focussing). No evidence was found for the Glycine 52
presence of carbohydrate in the chitinase. Staining with pe- Alanine 27
riodic acid-Schiff reagent after SDS-acrylamide electropho- Half cystine 12b
resis was negative; with anthrone, the amount of sugar was
below the limit of detectability (5 pg/lOO pg of protein). Isoleucine 9
Kinetics Tyrosine 14
Plots of product formation versus time were curved (not Tryptophan 4’
shown) as previously observed in the case of Streptomyces a Values obtained by extrapolation to zero time.
chitinase (7). The enzyme had a broad pH optimum around * Determined after performic acid oxidation and acid hydrolysis.
pH 6 with 75% of the maximal activity at pH 3 and 53% at pH ’ Determined spectrophotometrically.
Wheat Germ Chitinase 4905
Mechanism of Action and Specificity DISACCHARIDE TRISACCHARIDE TETRASACCHARIDE PENTAsACCHARI0~
Three oligosaccharide peaks, from disaccharide to tetrasac-
charide, were observed by paper chromatography after diges-
tion of tritiated chitin with wheat germ chitinase (Fig. 8). The (/2h
tetrasaccharide predominated at short incubation times, but
the smaller compounds accumulated over longer periods. No
monosaccharide was found, even after 24 h. Clearly, the 2h
enzyme acts as an endochitinase. Under these conditions, the
enzyme seemed unable to liberate larger fragments, as sup-
ported by the fact that no increase in reducing power was
found in the insoluble product that remained after partial
digestion (not shown).
A different pattern resulted from experiments in which the
action of chitinase on nascent chitin chains was studied. In
these experiments, the enzyme was added to a reaction mix- TEmA!%wHARlM
ISACCHARIOE TRISACWARIDE PENTASACCHARI
ture containing UDP-[“C]N-acetylglucosamine and solubi- - r_j
lized chitin synthetase; the chitinase degraded chitin as it was
produced by the synthetase. The amount of final products
(oligosaccharides) and of remaining chitin was evaluated after
chromatography on DEAE-cellulose paper (see “Methods”).
16 10 6 0
I 1 Oh’ I cm
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FIG. 10. Paper chromatography of oligosaccharides produced by
the action of chitinase on nascent chitin. The same reaction mixture
used in Fig. 9B (open and closed circles) with the addition of 0.02%
sodium azide, was employed. After incubation at 30°C for the times
indicated in the different panels, the reaction was stopped by heating
in a boiling water bath for 7 mm and the unreacted UDP-GlcNac and
chitin were eliminated by treatment with anionic resin and filtration,
as described under “Methods.” The filtrate was evaporated under
reduced pressure and transferred to the paper. For details on chro-
matographic development and scanning of radioactivity, see “Meth-
When the chitinase was omitted, almost all of the radioactivity
was found in chitin (Fig. 9A), whereas in the presence of the
hydrolase the polysaccharide was completely degraded to
water-soluble compounds (Fig. 9B).
FIG. 8. Paper chromatography of oligosaccharides released by chi-
tinase from tritiated chitin. For experimental details, see “Methods.” In other experiments, chitin was fit allowed to accumulate
The incubation time is indicated in the different panels. in the synthetase reaction mixture for 4.5 h; further
reaction was blocked with polyoxin D (23) and chitinase was
added. The subsequent liberation of oligosaccharides was
I I I E
extremely slow (Fig. 9B, solid triangles). These experiments
demonstrate that chitinase is much more active on nascent
chitin than on preformed chitin. Not only the rate of reaction
but also the nature of the products changed when the enzyme
attacked nascent chains. As shown in Fig. 10, oligosaccharides
higher than the pentasaccharide predominated after short
incubations and, even after 25 h, large amounts of pentasac-
charide were present.
Analogous experiments with Streptomyces chitinase (7)
showed a similar, although smaller, enhancement in activity
when nascent chitin was the substrate (not shown). With this
30 60 so enzyme, however, diacetylchitobiose was the product under
30 60 90 120
TIME IMinutes) all conditions.
FIG. 9. Liberation of oligosaccharides with chitinase acting an To further establish the specificity of the enzyme, chitin
either nascent or preformed chitin. In Panel A, the reaction mixture oligosaccharides were directly used as substrates (Table III).
was as described under “Methods,” but the chitinase was omitted. As expected from the results of Fig. 8, diacetylchitobiose was
o----<>, chitin formed; W, oligosaccharide formed. In Panel B,
completely resistant to the enzyme and the trisaccharide was
two different but simultaneously performed experiments are depicted.
In one of them, the complete mixture described under “Methods,” only slightly attacked. Higher oligosaccharides were suscep-
including chitin synthetase and chitinase, was used. The symbols tible to the chitinase.
(open and closed circles) have the same meaning as in Panel A. In a The following polysaccharides were inactive as substrate
parallel experiment, the reaction mixture lacking chitinase was incu- when tried at the concentrations indicated in parenthesis:
bated for 4.5 h at 30°C, and chitin synthesis was stopped by adding glycol chitin (3.6 ml/ml) yeast glucan (12.5 mg/ml), pustulan
polyoxin D to a final concentration of 20 gg/ml. At this point the
chitin concentration was 3.8 amol/ml. After adding chitinase as above, (12.5 mg/ml), laminarin (12.5 mg/ml), cellulose (2.5 mg/ml),
incubation was continued and the formation of oligosaccharides was and Micrococcus Zysodeikticus mucopeptide (10 mg of dried
monitored (A- - -A). cells/ml). In each case, degradation was assayed by determi-
4906 Wheat Germ Chitinase
Hydrolysis of individual ‘H-labeled chitin oligosaccharides by
wheat germ chitinase
Each oligosaccharide was incubated for 20 hat 30°C in the presence
of 0.04 unit of wheat germ chitinase, in 0.05 M potassium phosphate
at pH 6.3. The concentration of each substrate, calculated as N-
acetylglucosamine, was as follows: disaccharide, 7 mM; trisaccharide,
5 mM; tetrasaccharide, 2.2 mM; pentasaccharide, 1.7 mM, and hexa-
saccharide, 0.8 nm. After incubation, the products were subjected to
paper chromatography and scanning of radioactivity as described
Substrate Products 0 0.1 1 10 100 lwo
SUGAR CONCENTRATI@N ImMl
FIG. 11. Relative affinity of different N-acetylglucosamine oligo-
saccharides for chitinase. The reaction mixture was the same as in
Fig. 9B (open and closed circles), except that the specific activity of
(GlcNAc)s (GlcNAc)z, (GlcNAc)a
UDP-[‘%]GlcNAc was one-fourth as large, the concentration of chi-
(GlcNAc)a (GlcNAc)z, (GlcNAc)a
tinase was 3.1 pg/ml, and different amounts of each oligosaccharide
’ Traces of GlcNAc and (GlcNAc)n. were added, as indicated. N-Acetylglucosamine was omitted from the
’ Also traces of GlcNAc and (GlcNA& reaction mixtures, except for the determination of N-acetylglucosa-
mine affinity itself, where different concentrations were included, as
indicated. In the absence of chitinase, free N-acetylglucosamine has
nation of reducing power. Chitosan was degraded at a rate relatively little effect on the activity of solubilized chitin synthetase
about 10% that of chitin; since chitosan is only partially (3). Chitin formation was measured as previously reported (3) and is
deacetylated, this small degradation may be due to stretches expressed as percentage of the amount synthesized in a control
experiment in which chitinase was omitted. Although all experiments
of the polysaccharide chains that conserve the acetyl groups.
were carried out with the same preparations
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of chitinase and chitin
Whether the chitinase could act on glycoproteins as an synthetase, some variations were found in the zero sugar concentra-
endo-&acetylglucosaminidase was investigated using ovalbu- tion points of the curves, each of which was determined on a diierent
min (40 mg/ml) and ovomucoid (120 mg/ml) as substrates: in day.
the case of ovalbumin, the liberation of trichloroacetic acid-
soluble, anthrone-reacting material was measured; for ovo- DISCUSSION
mucoid, the products of the reaction were chromatographed By the procedure described in this paper, wheat germ
on a Sephadex G-25 column and the formation of anthrone- chitinase can be easily obtained in large amounts in homoge-
reacting material emerging after the void volume was moni- neous form. One kilogram of wheat germ yields about 30 mg
tored. In both cases, the results were completely negative. of purified enzyme. Whereas a single protein band was ob-
Measurements of P-N-acetylglucosaminidase activity in the served after SDS-acrylamide electrophoresis independently of
purified enzyme withp-nitrophenyl-P-N-acetylglucosaminide the amount of protein used, isoelectric focussing gave rise to
as substrate also yielded negative results, in contrast with a several bands. The reason for this discrepancy is not clear,
high activity of this enzyme in the crude extract. but it may residue in a varying percentage of amide groups in
An attempt was made to measure the relative affinity of the aspartic and glutamic residues. This would also explain
the different oligosaccharides for chitinase by determining why the enzyme forms have on the average a high isoelectric
their inhibitory power on the hydrolysis of tritiated chitin by point, despite the predominance of acidic over basic amino
the enzyme. However, very little, if any, inhibition was ob- acid residues.
served. The experience gained with nascent chitin suggested The enzyme is specific for chitin. Even glycol chitin, a
another way of obtaining the same information. A chitin material that has been used as a substrate for other chitinases
synthetase reaction mixture was prepared, with the addition (5) is not attacked by the wheat germ enzyme. The pattern of
of just enough chitinase to hydrolyze all the chitin being product formation, as analyzed by paper chromatography,
produced. Portions of this mixture were supplemented with indicates that the enzyme acts as an endochitinase. It differs
increasing amounts of an oligosaccharide and the formation sharply from the Streptomyces chitinase (23) which yields
of chitin was measured. If the oligosaccharide competes effec- diacetylchitobiose as the only product. From the results ob-
tively with the nascent chitin for the chitinase, one would tained both with chitin and with individual oligosaccharides,
expect a decrease in the degradation of the chitin and, there- it may be concluded that the smallest substrate that the
fore, an increase in its accumulation when the oligosaccharide enzyme can attack is a trisaccharide. It should be noted,
concentration rises. This expectation was borne out by the however, that the trisaccharide is a marginal substrate. Even
results (Fig. 11). N-Acetylglucosamine was a very poor com- after prolonged incubation,. only traces of diacetylchitobiose
petitor. Diacetylchitobiose was better by 2 orders of magni- and acetylglucosamine were observed. After a 24-h incubation
tude, and the higher oligosaccharides showed increased affin- of tritiated chitin, the smallest fragment detected was the
ity up to the pentasaccharide. These results are in general disaccharide. From these results, it appears that the specificity
agreement with those of Table III, in that both the effective- of the wheat germ chitinase is similar to that of the two
ness as substrate and the affinity for the enzyme increase with enzymes isolated from beans (4).
oligosaccharide size. The results obtained for the relative affinity of the different
Finally, the ability of the enzyme to act as a transglycosylase oligosaccharides for the enzyme are in general agreement with
was investigated. In a set of experiments chitin was incubated their behavior as substrates. Thus, acetylglucosamine is a very
with tritiated acetylglucosamine tetrasaccharide and the en- poor ligand in comparison with all the other compounds. The
zyme. After incubation, the reaction mixture was subjected to affinity increases with chain length, but it seems to stabilize
paper chromatography. No oligosaccharide higher than the after the pentasaccharide.
tetrasaccharide was detected (not shown). Analogous experi- A striking and unexpected result was the much greater
ments with unlabeled hexasaccharide as donor and tritiated activity of the enzyme when acting on nascent rather than
tetrasaccharide as acceptor also yielded a negative result. preformed chitin. From the data of Fig. 9B, using the steady
Wheat Germ Chitinase
state portion of the lower curve, one can calculate that the breakdown of a structural polysaccharide has been examined
enzyme is about 80 times as active on nascent as on preformed in vitro concomitant with its synthesis. It would seem of
chitin. This is a minimal value, because the steady state interest to repeat this type of measurement with other hydro-
concentration of nascent chitin was almost undetectable, lytic enzymes, whenever the corresponding synthetase is avail-
whereas that of preformed chitin was about 4 mM (calculated able, in order to gain a better insight into the real capabilities
as N-acetylglucosamine) . of the degrading enzymes in viuo.
How can this difference be explained? We propose that
preformed chitin has acquired the very tight hydrogen-bonded Acknowledgments-We are greatly indebted to Dr. L. Kohn for
structure that is characteristic of this polysaccharide. As a the ultracentrifugal analysis, to Dr. A. Hefetz for the isoelectric
consequence of steric hindrance, few positions are available in focussing, to Mr. G. Poi for the amino acid analysis, and to Ms. P.
Parisius for the Kjeldahl determination. We also wish to express our
this structure for successful binding and hydrolysis by the thanks to Drs. G. Ashwell, J. Correa, W. B. Jakoby, L. Shematek, and
enzyme. A similar hypothesis was previously advanced to M. Slater for useful discussions and criticism.
explain the curved plots of product formation uersus time
yielded by Streptomyces chitinase (7). On the other hand, the REFERENCES
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