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Kinetics of Uptake of Cell-Penetrating Peptides 287
The advantage of this approach is the simultaneous fitting of the curves for all
five initial concentrations (A 0) of [ 125 I]–biotinyl–transportan. Consequently, values
obtained for the rate constants (k 1 = 0.019 min –1 , k 2 = 0.15 min –1 , k 3 = 0.058 min –1 ,
k 4 = 0.27 min –1 , and k 5 = 0.0039 min –1 ) are equally valid for all curves. The ratio
between constants for uptake of TP (k 1) and its release (k 2) and the ratio of volumes
outside and inside the cells are in accordance with a maximal accumulation of
[ 125 I]–biotinyl–transportan detected in the cells (see above). On the other hand, the
ratio of the constants for uptake (k 4) and release (k 5) of the degradation products
corresponds to the ratio of intracellular (2 to 5 µl) and extracellular (100 µl) volumes.
This suggests that degradation products do not accumulate in the membranes and
that transportan could be degraded in the cells.
Not only the maximal fraction of TP that internalizes into the cells (9 to 16%
of total amount of peptide; see above), but also the distribution of TP between
membranes and inner cellular solutions is of particular interest, although difficult to
determine. Additional experiments by the authors 8 revealed that the fraction released
from the outer surface of the cells after treating cells with the acidic solution was
less than 2%, pointing to a very small amount of TP that was loosely bound to the
outer membrane surface. 8,9 Even less is known about the interaction of TP with the
inner hydrophobic leaflet of the membrane. Molecular modeling revealed a possibility
of a stable interaction between the lipid bilayer and TP, 9 and membrane-specific
staining procedure disclosed the presence of TP in practically all membrane structures
of the cell. 8
Very little is known regarding differences in the specificity of TP for various
types of the cell membranes. However, intracellular distribution of TP coincides
with staining of high mannose-containing membrane proteins by FITC-conjugated
concavalin A. It is interesting that, after longer incubations (more than 1 h), TP
concentrates in the nuclear membrane and nuclei, where it localizes predominantly
in the nucleoli. 8 More detailed studies of the localization of TP have not been carried
out yet; neither has the kinetics of internalization of TP in particular cell structure
been undertaken.
13.4.2 TRANSPORTAN ANALOGUES
A number of transportan analogs have been synthesized. These analogues can be
divided into two classes: first, the analogues denoted TP2–TP6 where specific parts
in the original TP sequence were replaced by other sequences, 7 and second, the TP
analogues, denoted TP7–TP15, derived from TP by deletion of groups of amino
acids from various parts of the original TP sequence. 9
As described in detail in Chapter 3, TP is a chimeric peptide composed of two
natural peptides: galanin (1–13) fragment of neuropeptide galanin in N terminus and
mastoparan, a component of wasp toxin, in C terminus. In analogues TP2–TP6, each
part of TP was selectively modified in order to reveal its role in the cell internalization.
7 The differences between TP and TP2 peptides are in the C-terminal where
transportan 2 contains the inactive mastoparan analogue, Mas 17. 20 In TP3, the
galanin part of the peptide was exchanged by the synthetic vasopressin antagonist