502
13 The Structure and Metabolism of Carbohydrates
The best-investigated chitinolytic system is that
of the moth Manduca sexta. This consists of six
enzymes (E-I to E-VI), all of which have been
isolated and characterized in detail. E-IV, E-V
and E-VI from the moulting fluid and the integument are chitinases which can also degrade chitin
oligosaccharides to di- and trisaccharides at a
pH optimum of 6; their activity increases with
chain length. They have different molecular masses (75, 62 and 50 kDa, respectively) and amino
acid compositions, but are immunologically
related. The glycosidase E-I in the haemolymph
of larvae and pupae as well as in the moulting
fluid and integument cleaves di- to hexasaccharides to about the same extent as it cleaves pnitrophenyl-B-N-acetylglucosamine; E-III from
pupal haemolymph has similar properties. In contrast, E-II from larval and pupal haemolymph is
active almost exclusively against glycosides. E-I is
without doubt a moulting enzyme; the functions
of E-II and E-III probably lie more in the metabolism of proteoglycans, glycoproteins and glycolipids. Whereas E-I and E-III are immunologically similar, E-II again occupies a special position.
All three glycosidases have the same molecular
mass of 61 kDa, although E-I consists of one
polypeptide chain and E-II and E-III have two
chains [154]. The isolated chitinases (E-IV to
E-VI) are monomers; nevertheless, the chitinase
activity of the moulting fluid shows sigmoid kinetics (Hill constant n = 1.95), which suggests a
homotropic allosteric interaction. The reconstituted chitinolytic system produces more free Nacetylglucosamine than predicted from the activities of the isolated enzymes; the largest turnover
is achieved when chitinases and B-N-acetylglucosaminidases are mixed in a ratio of 6: l.
Thus, the chitinases apparently form nonproductive enzyme-substrate complexes with low
molecular weight substrates and these can be
degraded by the B-N-acetylglucosaminidases
[50, 85]. The chitinolytic systems involved in the
moulting of other insects, e.g. Platysamia, Bombyx, Drosophila, Musca and Tribolium, have also
been investigated [50, 158, 159]. A 215-kDa protein identified immunologically in Bombyx mori
is apparently a chitinase zymogen from which the
active 65-kDa enzyme is released the day before
moulting. The silkworm also appears to contain inactive precursors of the B-N-acetylglucosaminidase [155].
The majority of insect chitinases have molecular masses between 45 and 75 kDa; similar values
have been reported for other chitinases, e.g. from
calf serum (47 kDa), from the digestive juices of
the spider Cupiennius salei (48 kDa), and from
the hepatopancreas and the crystalline style of
the edible mussel (60 and 76 kDa, respectively)
[28, 50]. However, the enzyme from the brine
shrimp Artemia is reported to be a glycoprotein of
only 32 kDa [86]. The catalytic activities of the
chitinases vary greatly with the species. The three
chitinases from Manduca sexta produce oligomers
of N-acetylglucosamine; in contrast, the chitinases of the silkmoth Bombyx mori, the fly Stomoxys calcitrans and the spider Cupiennius salei
primarily release dimers. The spider chitinases
degrade chitin to chitobiose (88 %). chitotriose
(5 %) and free N-acetylglucosamine (7 %),
whereas, for example, the moulting enzyme of
Bombyx mori produces no trisaccharides. Almost
all chitinases produce free N-acetylglucosamine,
probably by the cleavage of chitotriose. Even the
chitinases from Manduca have at least a low
hydrolytic activity with chitotriose. The vertebrate chitinases produce no free N-acetylglucosamine [50]. One problem with the
investigations of chitinases is the difficulty of
avoiding contamination by bacterial and fungal
enzymes. A chitinase which is certainly not contaminated is secreted into the medium of Drosophila cell cultures [32].
The N-acetylglucosaminidases of the insects
present a very heterogeneous picture. The enzymes of Manduca sexta have a mass of about
61 kDa, whilst enzymes from other sources are
over 100 kDa. The enzyme of Bombyx mori is
a dimer with subunits of equal size, whilst that
of Stomoxys calcitrans is a heterodimer with
subunits of 67 and 43 kDa [50]. The N-acetylglucosaminidases or N-acetylhexos aminidases of
animals have in some cases digestive functions
but are mainly involved in the metabolism of
proteoglycans, glycoproteins and glycolipids.
Apart from chitobiose, they cleave other Bglycosidic derivatives of N-acetylglucosamine and
N-acetylgalactosamine, whereby their relative
activity against the individual substrate varies
greatly with the origin of the enzyme. The molecular masses of these enzymes span a wide range:
55 kDa in Tetrahymena, 125 kDa in Entamoeba,
100-200 kDa in various mussel species,
112-124 kDa in the nematode Turbatrix and
150-160 kDa for the enzyme from rat kidney
[21, 234, 268]. Even in closely related species
there are large differences in temperature stability and these can not be explained as adaptations;
for example, the enzyme activity of the mussel
Spisula solidissima is not affected at all after 1 h
at 45°C, but the activity is reduced in Mya
13 The Structure and Metabolism of Carbohydrates
The best-investigated chitinolytic system is that
of the moth Manduca sexta. This consists of six
enzymes (E-I to E-VI), all of which have been
isolated and characterized in detail. E-IV, E-V
and E-VI from the moulting fluid and the integument are chitinases which can also degrade chitin
oligosaccharides to di- and trisaccharides at a
pH optimum of 6; their activity increases with
chain length. They have different molecular masses (75, 62 and 50 kDa, respectively) and amino
acid compositions, but are immunologically
related. The glycosidase E-I in the haemolymph
of larvae and pupae as well as in the moulting
fluid and integument cleaves di- to hexasaccharides to about the same extent as it cleaves pnitrophenyl-B-N-acetylglucosamine; E-III from
pupal haemolymph has similar properties. In contrast, E-II from larval and pupal haemolymph is
active almost exclusively against glycosides. E-I is
without doubt a moulting enzyme; the functions
of E-II and E-III probably lie more in the metabolism of proteoglycans, glycoproteins and glycolipids. Whereas E-I and E-III are immunologically similar, E-II again occupies a special position.
All three glycosidases have the same molecular
mass of 61 kDa, although E-I consists of one
polypeptide chain and E-II and E-III have two
chains [154]. The isolated chitinases (E-IV to
E-VI) are monomers; nevertheless, the chitinase
activity of the moulting fluid shows sigmoid kinetics (Hill constant n = 1.95), which suggests a
homotropic allosteric interaction. The reconstituted chitinolytic system produces more free Nacetylglucosamine than predicted from the activities of the isolated enzymes; the largest turnover
is achieved when chitinases and B-N-acetylglucosaminidases are mixed in a ratio of 6: l.
Thus, the chitinases apparently form nonproductive enzyme-substrate complexes with low
molecular weight substrates and these can be
degraded by the B-N-acetylglucosaminidases
[50, 85]. The chitinolytic systems involved in the
moulting of other insects, e.g. Platysamia, Bombyx, Drosophila, Musca and Tribolium, have also
been investigated [50, 158, 159]. A 215-kDa protein identified immunologically in Bombyx mori
is apparently a chitinase zymogen from which the
active 65-kDa enzyme is released the day before
moulting. The silkworm also appears to contain inactive precursors of the B-N-acetylglucosaminidase [155].
The majority of insect chitinases have molecular masses between 45 and 75 kDa; similar values
have been reported for other chitinases, e.g. from
calf serum (47 kDa), from the digestive juices of
the spider Cupiennius salei (48 kDa), and from
the hepatopancreas and the crystalline style of
the edible mussel (60 and 76 kDa, respectively)
[28, 50]. However, the enzyme from the brine
shrimp Artemia is reported to be a glycoprotein of
only 32 kDa [86]. The catalytic activities of the
chitinases vary greatly with the species. The three
chitinases from Manduca sexta produce oligomers
of N-acetylglucosamine; in contrast, the chitinases of the silkmoth Bombyx mori, the fly Stomoxys calcitrans and the spider Cupiennius salei
primarily release dimers. The spider chitinases
degrade chitin to chitobiose (88 %). chitotriose
(5 %) and free N-acetylglucosamine (7 %),
whereas, for example, the moulting enzyme of
Bombyx mori produces no trisaccharides. Almost
all chitinases produce free N-acetylglucosamine,
probably by the cleavage of chitotriose. Even the
chitinases from Manduca have at least a low
hydrolytic activity with chitotriose. The vertebrate chitinases produce no free N-acetylglucosamine [50]. One problem with the
investigations of chitinases is the difficulty of
avoiding contamination by bacterial and fungal
enzymes. A chitinase which is certainly not contaminated is secreted into the medium of Drosophila cell cultures [32].
The N-acetylglucosaminidases of the insects
present a very heterogeneous picture. The enzymes of Manduca sexta have a mass of about
61 kDa, whilst enzymes from other sources are
over 100 kDa. The enzyme of Bombyx mori is
a dimer with subunits of equal size, whilst that
of Stomoxys calcitrans is a heterodimer with
subunits of 67 and 43 kDa [50]. The N-acetylglucosaminidases or N-acetylhexos aminidases of
animals have in some cases digestive functions
but are mainly involved in the metabolism of
proteoglycans, glycoproteins and glycolipids.
Apart from chitobiose, they cleave other Bglycosidic derivatives of N-acetylglucosamine and
N-acetylgalactosamine, whereby their relative
activity against the individual substrate varies
greatly with the origin of the enzyme. The molecular masses of these enzymes span a wide range:
55 kDa in Tetrahymena, 125 kDa in Entamoeba,
100-200 kDa in various mussel species,
112-124 kDa in the nematode Turbatrix and
150-160 kDa for the enzyme from rat kidney
[21, 234, 268]. Even in closely related species
there are large differences in temperature stability and these can not be explained as adaptations;
for example, the enzyme activity of the mussel
Spisula solidissima is not affected at all after 1 h
at 45°C, but the activity is reduced in Mya