208
5 Plasma Proteins, Yolk Proteins and Metal-Binding Proteins
lies of the suborder Zoarcoidei: Stichaeidae
(pricklebacks), Cryptacanthodidae (wrymouths),
Pholididae (gunnels) and Anarhichadidae (wolf
fish). The variety and distribution of the three
AFP classes suggests that they arose independently and relatively recently: classes I and II in
the Arctic and class III in the Antarctic. The
effective selection pressure was probably the
appearance of ice in surface waters. With the
resulting unavoidable contact with ice, it became
necessary for the surface-water fish to cool their
body fluids below the freezing point of --0.8 °C;
this was not a requirement for the deep-sea fish.
The fact that the AFPs of all species show marked
heterogeneity suggests that they are always
encoded by multi-gene families. In fact, the sea
raven Hemitripterus american us has 12-15 AFP
genes, the winter flounder Pseudopleuronectes
american us 30-40 genes, and different populations of the ocean pout Macrozoarces americanus
between 30-40 and about 150 genes. The AFPs
are only produced during the cold season; this
seasonal regulation of AFP biosynthesis in the
winter flounder involves a reduction during the
winter in the formation or release of a specific
inhibitor by the hypophysis [58].
Like AFPs, AFGPs have a specific effect on
the freezing point but not on the melting point
(thermal hysteresis). The large differences
between the AFGPs and AFPs and within the
AFPs prompts the question whether the effect of
all these proteins involves the same mechanism;
the answer is still uncertain [58]. The biosynthesis
of both AFGPs and AFPs takes place in the liver.
It is interesting to ask why the relatively small
molecules of the antifreeze proteins are not lost in
the urine. The antarctic Nototheniidae have aglomerulous kidneys in which there is no filtration.
Pseudopleuronectes and Rhigophila do have glomeruli but filtration of the AFPs is apparently
prevented [67].
Antifreeze proteins which give rise to thermal
hysteresis have been detected in a series of terrestrial arthropods: beetle species from six different
families, a cockroach, a bug, a scorpion fly and
even a spider [65, 275]. However, only a few of
these have been characterized in any detail.
Seven antifreeze proteins of 9-17 kDa have been
isolated from the larvae of the flour beetle Tenebrio molitor, and the concentration of these proteins increases to 10 mg/ml during cold acclimatization. The proteins vary greatly in amino acid composition, containing only a little alanine but large
numbers of polar amino acids; one such protein
contains 28 % cysteine. The antifreeze proteins
from the caterpillars of Choristoneura fumifrana
are of 13-16 kDa and also contain little alanine
and up to 6 % cysteine [65]. In many insect species frost resistance is achieved not by the prevention of haemolymph freezing by special proteins
or other substances but by the converse mechanism of ice nucleation: specific haemolymph proteins (ice nucleators) lead to rapid ice formation in
the extracellular fluid under freezing conditions,
thereby preventing lethal intracellular ice formation. There has as yet been little biochemical
characterization of the ice nucleators. In the hornet Vespula maculata there is a protein with 20 %
glutamate residues, and in the crane-fly Tipula trivittata several specific proteins and lipoproteins
are involved [190].
5.10 Metallothioneins
The metallothioneins (MTs) were discovered in
1957 in the liver and kidneys of mammals exposed
to cadmium; they have subsequently been
detected not only in the liver, kidneys and other
organs of all vertebrate classes but also in a
variety of invertebrates (annelids, crustaceans,
insects, molluscs and echinoderms), in the ciliate
Tetrahymena and in higher plants, lower fungi and
yeast. They are therefore probably ubiquitous in
the eukaryotes. In contrast, the metal-binding
proteins of the prokaryotes do not appear to be
related to the mammalian MTs. The MTs are small
polypeptides of less than 10 kDa which contain
23-33 % cysteine residues and have the capacity
for complex formation with 4-12 metal ions. The
cysteine residues all take part in metal binding and
form no intra- or intermolecular disulphide bridges. The MTs lack the aromatic amino acids phenylalanine and tyrosine and therefore, unlike most
proteins, they show no absorption at 280 nm [93].
Mammalian MTs consist of 61-62 amino acids,
including 20 cysteines, 6-8 lysines and 7-10 serines but no histidine; the N-terminus is Nacetylated. The polypeptide chain is divided into
two domains, each with a cluster of cysteines
which are able to bind four cadmium, four zinc or
five to six copper ions; cluster A in the Cterminal a-domain contains 11 cysteines and cluster B in the N-terminal ~-domain 9 cysteines
(Fig. 5.4a). Each divalent metal ion is bound tetrahedrally with four cysteine residues. The affinity of the MTs for different metals varies considerably, being 1000-fold higher for cadmium than for
zinc, and a further 100-fold higher for copper
5 Plasma Proteins, Yolk Proteins and Metal-Binding Proteins
lies of the suborder Zoarcoidei: Stichaeidae
(pricklebacks), Cryptacanthodidae (wrymouths),
Pholididae (gunnels) and Anarhichadidae (wolf
fish). The variety and distribution of the three
AFP classes suggests that they arose independently and relatively recently: classes I and II in
the Arctic and class III in the Antarctic. The
effective selection pressure was probably the
appearance of ice in surface waters. With the
resulting unavoidable contact with ice, it became
necessary for the surface-water fish to cool their
body fluids below the freezing point of --0.8 °C;
this was not a requirement for the deep-sea fish.
The fact that the AFPs of all species show marked
heterogeneity suggests that they are always
encoded by multi-gene families. In fact, the sea
raven Hemitripterus american us has 12-15 AFP
genes, the winter flounder Pseudopleuronectes
american us 30-40 genes, and different populations of the ocean pout Macrozoarces americanus
between 30-40 and about 150 genes. The AFPs
are only produced during the cold season; this
seasonal regulation of AFP biosynthesis in the
winter flounder involves a reduction during the
winter in the formation or release of a specific
inhibitor by the hypophysis [58].
Like AFPs, AFGPs have a specific effect on
the freezing point but not on the melting point
(thermal hysteresis). The large differences
between the AFGPs and AFPs and within the
AFPs prompts the question whether the effect of
all these proteins involves the same mechanism;
the answer is still uncertain [58]. The biosynthesis
of both AFGPs and AFPs takes place in the liver.
It is interesting to ask why the relatively small
molecules of the antifreeze proteins are not lost in
the urine. The antarctic Nototheniidae have aglomerulous kidneys in which there is no filtration.
Pseudopleuronectes and Rhigophila do have glomeruli but filtration of the AFPs is apparently
prevented [67].
Antifreeze proteins which give rise to thermal
hysteresis have been detected in a series of terrestrial arthropods: beetle species from six different
families, a cockroach, a bug, a scorpion fly and
even a spider [65, 275]. However, only a few of
these have been characterized in any detail.
Seven antifreeze proteins of 9-17 kDa have been
isolated from the larvae of the flour beetle Tenebrio molitor, and the concentration of these proteins increases to 10 mg/ml during cold acclimatization. The proteins vary greatly in amino acid composition, containing only a little alanine but large
numbers of polar amino acids; one such protein
contains 28 % cysteine. The antifreeze proteins
from the caterpillars of Choristoneura fumifrana
are of 13-16 kDa and also contain little alanine
and up to 6 % cysteine [65]. In many insect species frost resistance is achieved not by the prevention of haemolymph freezing by special proteins
or other substances but by the converse mechanism of ice nucleation: specific haemolymph proteins (ice nucleators) lead to rapid ice formation in
the extracellular fluid under freezing conditions,
thereby preventing lethal intracellular ice formation. There has as yet been little biochemical
characterization of the ice nucleators. In the hornet Vespula maculata there is a protein with 20 %
glutamate residues, and in the crane-fly Tipula trivittata several specific proteins and lipoproteins
are involved [190].
5.10 Metallothioneins
The metallothioneins (MTs) were discovered in
1957 in the liver and kidneys of mammals exposed
to cadmium; they have subsequently been
detected not only in the liver, kidneys and other
organs of all vertebrate classes but also in a
variety of invertebrates (annelids, crustaceans,
insects, molluscs and echinoderms), in the ciliate
Tetrahymena and in higher plants, lower fungi and
yeast. They are therefore probably ubiquitous in
the eukaryotes. In contrast, the metal-binding
proteins of the prokaryotes do not appear to be
related to the mammalian MTs. The MTs are small
polypeptides of less than 10 kDa which contain
23-33 % cysteine residues and have the capacity
for complex formation with 4-12 metal ions. The
cysteine residues all take part in metal binding and
form no intra- or intermolecular disulphide bridges. The MTs lack the aromatic amino acids phenylalanine and tyrosine and therefore, unlike most
proteins, they show no absorption at 280 nm [93].
Mammalian MTs consist of 61-62 amino acids,
including 20 cysteines, 6-8 lysines and 7-10 serines but no histidine; the N-terminus is Nacetylated. The polypeptide chain is divided into
two domains, each with a cluster of cysteines
which are able to bind four cadmium, four zinc or
five to six copper ions; cluster A in the Cterminal a-domain contains 11 cysteines and cluster B in the N-terminal ~-domain 9 cysteines
(Fig. 5.4a). Each divalent metal ion is bound tetrahedrally with four cysteine residues. The affinity of the MTs for different metals varies considerably, being 1000-fold higher for cadmium than for
zinc, and a further 100-fold higher for copper