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Model Amphipathic Peptides 73
its D-amino acid analog, but not after incubation with its nonamphipathic counterpart
II and a double D-amino acid with impaired amphipathicity; 8 this suggested an
amphipathicity-dependent cellular uptake. Internalization of the synthetic peptide I
and its D-analog was observed even at 0°C and after energy depletion 8 analogous
to that reported for the natural Antennapedia sequence 9 indicating a nonendocytic
mode of uptake. Further studies confirmed these surprising initial observations and
provided evidence for the involvement of various nonendocytic mechanisms in this
process. 10
4.2 MECHANISTIC ASPECTS OF THE CELLULAR UPTAKE OF MAPs
4.2.1 QUANTITATION OF INTERNALIZED MAPs
MAPs exhibit strong membrane lytic properties at concentrations above 4 µM; 10 thus
cellular uptake experiments must be confined to concentrations below this value in
order to avoid bias of the results by pore formation. The resulting peptide levels in
the cell lysates nevertheless proved sufficient for HPLC analysis by fluorescence
detection. The surface-bound portion of cell-associated MAPs found in the cell
lysates, however, exceeded that actually internalized by about tenfold. 10 Therefore
precise discrimination of both peptide fractions was required as a precondition for
quantitative deductions. Such discrimination was achieved by treatment of the peptide-exposed,
washed cells with diazotized 2-nitroaniline. 10 This reagent, previously
shown to modify surface-bound primary amino compounds while leaving those
within the cell intact, 11 has the crucial advantage of being highly reactive even at
0°C, so that bias of the results by counteracting efflux processes, strongly attenuated
or suppressed at this temperature, is minimized. By contrast, acid washing of the
peptide-loaded cells (5 min at 0°C with HAc/NaCl, 0.2 M/0.05 M), a procedure
commonly used to strip surface-bound peptides, failed to remove I from the cell
surface (loss