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monitored by NMR, was unchanged, although it should be noted that the structure of the loop containing
Arg14 was not well defined in either native or trypsinised AP-A. That the backbone structure was
largely unaffected was confirmed by the observation that the NH and CαH chemical shifts were
unaltered except in the immediate vicinity of the cleavage (which is the site of a reverse turn in AP-A
[46]) and the N-terminal regions of the loop and the second strand of the sheet. It is possible that
perturbations of functionally important groups near the start of the loop may be responsible for the lack
of activity of the trypsinised derivative, and now that a high-resolution structure is available for AP-A a
more detailed comparison with the cleavage product would be useful. Other possible explanations for
the lack of activity are that the position of the Arg14 side chain relative to other key residues in the
molecule is more important than suggested by chemical modification and site-directed mutagenesis data,
or that the introduction of additional charges associated with the new termini affects activity.
Endoproteinase LysC cleaved AP-A between Lys37 and Ala38 to yield a derivative with cardiotonic
activity an order of magnitude lower than that of the parent molecule (Monks SA and Norton RS,
unpublished results). This reduction in activity could be a consequence of local conformational
perturbations. Treatment of AP-B with carboxypeptidase B removed Lys49, resulting in only a two-fold
reduction in cardiotonic activity (Monks SA and Norton RS, unpublished results).
C. Sequence Comparisons
Among the Type 1 toxins shown in Figure 2, the Bc and Bg toxins (from the genus Bunodosuma) form a
subgroup with characteristic differences from the Anemonia and Anthopleura toxins at residues 5, 12–13
and 37–42. In addition, the Bg toxins also have Asp7 rarrow.gif Lys and Gly27 rarrow.gif Arg
substitutions. A potent toxin in mice [26], the cardiac stimulatory activity of Bg II has not been reported.
The potent activity of Bg II was ascribed to its abundance of positive charges [26]. Ignoring the
histidines, which at least in AP-A and ATX II would be predominantly in their neutral forms at
physiological pH [57], Bg II has six positively charged side chains and only one negatively charged side
chain, whereas, AP-A has three and two respectively, and AP-B has five and two. As discussed below,
however, it may be that an abundance of positive charge is associated with a lack of discrimination
between the neuronal and cardiac sodium channels, as found for the scorpion α-toxins.
Comparison of the activities of Af I and Af II is useful because of their close similarity. Differences
between Af I and Af II are Ala3 rarrow.gif Pro, Asn12 rarrow.gif Ser, Thr21 rarrow.gif Ile, and an
additional Gly at the N-terminus. Inspection of the sequences in Figures 2 shows that the identity of
residue 3 correlates with that of residue 21, Ala or Ser at 3 co-occurring with Thr21, and Pro 3 with
Ile21
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