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Transportans 57
especially the endoplasmic reticulum (Figure 3.1B, E). The combined image of
peptide and Con A-TRITC localization in Bowes melanoma cells shows a preferential
targeting of biotinyl-transportan to membranous structures, both at 0°C and
37°C (Figure 3.1C, F). Furthermore, we suggest the nucleoli to be the intranuclear
structures where transportan concentrates, since the staining of the nucleoli with
acridine orange coincided with localization of the peptide. 5
The mechanism of cell entry of transportan remains unknown. Translocation of
transportan into cells is probably not mediated by specific receptors in the plasma
membrane, since the internalization of biotinyl-transportan cannot be blocked by
preincubation of cells with tenfold excess of its “parent” compounds: galanin,
mastoparan, or galparan. Other indirect evidence points to nonreceptor-mediated cell
entry of transportan: we have not yet found a cell line that does not internalize the
peptide. Moreover, for transportan, protein-mediated uptake can be excluded because
extensive cross-linking of proteins on the cell surface does not abolish internalization
of the peptide. 5
In order to study the role of endocytosis in the internalization of transportan,
hyperosmolar solution conditions and lowered temperature were used to suppress
endocytosis. Clathrin-dependent endocytosis is inhibited by more than 90% in hyperosmolar
solution through blockage of clathrin-coated pit formation. 13 In 0.45 M
sucrose solution, Bowes cells shrink and change morphology slightly, but internalization
of biotinyl-transportan is not markedly affected. Lowering temperature below
18°C abolishes endocytosis, but cellular translocation of transportan is not abolished
even at 4 or 0°C. Thus, the cell entry of transportan is not an active cellular process
and not directly fueled by ATP energy.
Transportan penetrates into cells rapidly; in a suspension of human melanoma
Bowes cells, the half maximal intracellular concentration is reached in 3 to 5 min
at 37°C as estimated from uptake experiments with radioactively labeled ([ 125 I]-
Tyr 9 ,N ε13- biotinyl)-transportan. The translocation into cells attached on plastic is
slower, but still in the same time scale. Moreover, transportan not only enters cells,
but also concentrates there. At first, using radioactively labeled transportan, we
estimated that the intracellular transportan concentration at saturation exceeds the
extracellular by at least twofold. More careful estimation of the cell volume revealed
that the extent of intracellular accumulation of transportan is at least 20-fold. 14
However, great care has to be taken when interpreting uptake data as cellular
penetration only, since plasma membrane adsorption and uptake by endocytotic
mechanisms also contributed in our uptake experiment using either radioactively or
fluorophore-labeled transportan.
3.4 PROPERTIES OF TRANSPORTAN
The N-terminal part of transportan corresponds to the fragment of the neuropeptide
galanin that is the main determinant of binding to type 1 galanin receptors (GalR1).
Hence, binding of biotinyl-transportan to galanin receptors in Bowes cell membranes
was expected. Biotinyl-transportan showed high affinity toward galanin receptors
(K D = 17 nM), exceeding that of the galanin fragment (1–13) several-fold, and of