An Endochitinase from Wheat Germ by fdh56iuoui


									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,
                  Mayland   20205

    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.,
                                                                                                        Tokyo,       Japan.
    * 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-
                                 Enzyme        Purification
                                                                                                   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).

                                                                                                                0    6
                                                                                                                c    5
                      10   20       30    40     50       60       70
                                     TUBE NUMBER                                                                $!   4
                                                                                                                                                  Wheat           germ chitinase
   FIG. 3. Chromatography              of wheat       germ     chitinase         and agglutinin                 ?i
                                                                                                                3    3
on Sephadex    G-50 column.

                                                                                                                                                                        \     Cytochrome   c
                                                                                                                               0.2       0.4           0.6            0.8          1.0
                                                                                                                                         RELATIVE        MOBILITY
                                                                                                      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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                                                                           epH       7


                                                                                                      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
the gel.
    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
                                                                                                                          Threonine                                                 22”
    The amino acid analysis (Table II) showed 48 acidic and
                                                                                                                          Serine                                                    24”
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
                                                                                                                          Valine                                                    14
resis was negative; with anthrone, the amount of sugar was
                                                                                                                          Methionine                                                 36
below the limit of detectability    (5 pg/lOO pg of protein).                                                             Isoleucine                                                 9
                                                                                                                          Leucine                                                   13
                            Kinetics                                                                                      Tyrosine                                                  14
                                                                                                                          Phenylalanine                                             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~
                                                                                                                   al                                                                        ORIGIN
     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

                                                                                                         m 20
                                                                                                         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
                              TABLE III
   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
under   “Methods.”
                Substrate                         Products                                             0           0.1          1         10               100          lwo
                                                                                                                         SUGAR CONCENTRATI@N        ImMl
              (GlcNAc)s                  None
                                                                                          FIG. 11. Relative       affinity     of different      N-acetylglucosamine           oligo-
              (GlcNAc)a                  None”
                                                                                      saccharides      for chitinase.       The reaction       mixture      was the same as in
              (GlcNAc)r                  (GlcNAc)s6
                                                                                      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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the presence of higher oligosaccharides         in the digest with this             Commun. 58, 13-19
substrate, i.e. finding less of a steric hindrance, the enzyme               3. Duran,      A., and Cabib, E. (1978) J. Biol. Chem. 253,4419-4425
can reach further down the chain and excise larger fragments.                4. Pow&g,         R. F., and Irzykiewicz,       H. (1965) Comp. Biochem. Phys-
                                                                                    iol. 14, 127-133
   These results may have a more general significance. During                5. Otakara,       A. (1964) Agric. Biol. Chem. 28,811-818
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this connection, we have recently found in yeast a membrane-                10. Ruplei,      j. A. (1964) Biochim. Biophys. Acta 83,245-255
bound chitinase that is capable of degrading the polysaccha-                11. Park, J. T., and Johnson,              M. J. (1949) J. Biol. Chem. 181, 149-
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   The effect found with nascent and preformed chitin may                   12. Sonnino,       S., Carminatti,      H., and Cabib, E. (1966) Arch. Biochem.
also be relevant to the function of the chitinase in wheat germ.                    Biophys. 116,26-33
It has been hypothesized        (4) that chitinases present in seeds        13. Lis, H., and Sharon,           N. (1972) Methods Enzymol. 28,360-365
                                                                            14. Shapiro,      A. L., Vtiuela,      E., and Maizel,   J. V., Jr. (1967) Biochem.
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such as fungi. Their action would be most effective if exerted              15.   Moore,      S., and Stein, W. H. (1963) Methods Enzymol. 6,819-831
on the apex of a hypha, since this is the part of the invading              16.   Moore,      S. (1963) J. Bill. Chem. 238,235-237
organism that usually enters the cell and also the region where             17.   Edelhoch,       H. (1967) Biochemistry 6, 1948-1954
all fungal growth occurs (24). It is interesting         then that the      18.   Brunnert,      H. (1965) J. Chromatog. 17,621-623
apical area seems to be the most susceptible portion of the                 19.   Trevelyan,       W. E., Procter,   D. P., and Harrison,   J. S. (1950) Nature
hypha to attack by hydrolytic          enzymes. Polacheck and Ro-           20.   Lowry,     0. H., Rosebrough,        N. J., Farr, A. L., and Randall,    R. J.
senberger (25) reported that in Aspergillus         the newly synthe-                 (1951) J. Biol. Chem. 193,265-275
sized areas of the cell wall (apical zone) were preferentially              21.   He&in,       S., Feingold,     D. S., and Schramm,       M. (1955) Methods
hydrolyzed by autolysins. This finding may be related to the                         Enzymol. 1,231-257
observation (24) that the apical portion of the cell wall is less           22.   Trevelyan,       W. E., and Harrison,     J. S. (1952) Biochem. J. 50,298-
organized than the lateral areas. The polysaccharides             at the             303
                                                                            23.   Keller, F. A., and Cabib, E. (1971) J. Biol. Chem. 246, 160-166
apex, therefore, may have a structure resembling the nascent                24.   Burnett,     J. H. (1976) Fundamentals ofMvcology, 2nd Ed, pp. 61-
state, which would make them more susceptible to the action                          101, Edward       Arnold    and Crane Ru&ak;       New York
of lytic enzymes.                                                           25.   Polacheck.       Y.. and Rosenbereer.       R. F. (1975) J. Bacterial. 121,
   To our knowledge,       this is the first report in which the                     332-337’

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