IV. INVESTIGATION OF C. DIPHTHERIAE
81
These observations show that toxin inactivates EF2 (transferase 11) by
ADP ribosylation in the living animal as well as in cell-free extracts. The
intact toxin molecule is enzymically inactive in vitro, and the presence of
certain degradation products accounts for the enzymic activity of most
toxin preparations; activation signifies reduction of one of the disulphide
bridges and hydrolysis of a peptide bond. This change unmasks the
24000 daltons heat stable fragment A, on which the enzymic activity
is located. It is suspected (Uchida et al., 1971) that such modifications of the
toxin occur at the cell membrane where fragment A is split off and gains
access to the cell cytoplasm, inactivating transferase I1 intracellularly.
The remaining fragment B (38000 daltons) has no known enzymic
activity but seems to be required for attachment of the toxin to the sensitive
cell membrane. A single molecule of fragment A is sufficient to convert the
entire cell content of EF2 to its inactive derivative within a few hours.
By mutations of the structural gene tox it has become possible to prepare
diphtherial lysogenic strains producing mutant, non-toxic proteins of
various types. Some possess an altered fragment A and thus have become
enzymically inactive, others contain a normal fragment A which cannot
gain access to the cell cytoplasm because of an altered B fragment. There
is a further exciting problem to be underlined in this connection.
It is known that formol toxoid immunises and prevents the lesions
produced by diphtherial toxin. It reacts specifically with diphtherial
antitoxin to form toxoid-antitoxin precipitates. In vivo the antibodies
neutralise toxin. Toxoid is incapable of blocking adsorption of toxin to
HeLa cells. Until the discovery of the molecular structure of diphtherial
toxin it was assumed that formol conversion of toxin to toxoid renders the
toxic site of toxin non-toxic, but leaves it antigenic. According to the new
data, it seems that the toxoid induces antibodies, which block adsorption of
the carboxy-terminal B fragment of the toxin to the animal cell.
3 . General properties of diphtherial toxin
Studies 'on diphtherial toxin have been promoted by its availability in
large amounts and by the accuracy and sensitivity of the methods for its
bioassay, originally worked out by Ehrlich (1897).
Diphtherial toxin is a typical thermolabile exotoxin, readily inactivated
by heat (5 min at 70°C) as well as by acid solutions of pH below 6 and by
sunlight. The isoelectric point is 4.1. The toxin should therefore be stored
in the cold and in the dark, but even so, it slowly and gradually loses its
toxicity, without parallel loss of its serological activity. The toxins produced by all naturally occurring strains appear to be immunologically
identical..
Immunisation with toxoid, plain or purified and adsorbed, results in
4
81
These observations show that toxin inactivates EF2 (transferase 11) by
ADP ribosylation in the living animal as well as in cell-free extracts. The
intact toxin molecule is enzymically inactive in vitro, and the presence of
certain degradation products accounts for the enzymic activity of most
toxin preparations; activation signifies reduction of one of the disulphide
bridges and hydrolysis of a peptide bond. This change unmasks the
24000 daltons heat stable fragment A, on which the enzymic activity
is located. It is suspected (Uchida et al., 1971) that such modifications of the
toxin occur at the cell membrane where fragment A is split off and gains
access to the cell cytoplasm, inactivating transferase I1 intracellularly.
The remaining fragment B (38000 daltons) has no known enzymic
activity but seems to be required for attachment of the toxin to the sensitive
cell membrane. A single molecule of fragment A is sufficient to convert the
entire cell content of EF2 to its inactive derivative within a few hours.
By mutations of the structural gene tox it has become possible to prepare
diphtherial lysogenic strains producing mutant, non-toxic proteins of
various types. Some possess an altered fragment A and thus have become
enzymically inactive, others contain a normal fragment A which cannot
gain access to the cell cytoplasm because of an altered B fragment. There
is a further exciting problem to be underlined in this connection.
It is known that formol toxoid immunises and prevents the lesions
produced by diphtherial toxin. It reacts specifically with diphtherial
antitoxin to form toxoid-antitoxin precipitates. In vivo the antibodies
neutralise toxin. Toxoid is incapable of blocking adsorption of toxin to
HeLa cells. Until the discovery of the molecular structure of diphtherial
toxin it was assumed that formol conversion of toxin to toxoid renders the
toxic site of toxin non-toxic, but leaves it antigenic. According to the new
data, it seems that the toxoid induces antibodies, which block adsorption of
the carboxy-terminal B fragment of the toxin to the animal cell.
3 . General properties of diphtherial toxin
Studies 'on diphtherial toxin have been promoted by its availability in
large amounts and by the accuracy and sensitivity of the methods for its
bioassay, originally worked out by Ehrlich (1897).
Diphtherial toxin is a typical thermolabile exotoxin, readily inactivated
by heat (5 min at 70°C) as well as by acid solutions of pH below 6 and by
sunlight. The isoelectric point is 4.1. The toxin should therefore be stored
in the cold and in the dark, but even so, it slowly and gradually loses its
toxicity, without parallel loss of its serological activity. The toxins produced by all naturally occurring strains appear to be immunologically
identical..
Immunisation with toxoid, plain or purified and adsorbed, results in
4