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4
CONTENTS
0-8493-1141-1/02/$0.00+$1.50
© 2002 by CRC Press LLC
Model Amphipathic
Peptides
Johannes Oehlke, Burkhard Wiesner,
and Michael Bienert
Abstract....................................................................................................................72
4.1 Introducing History........................................................................................72
4.2 Mechanistic Aspects of the Cellular Uptake of MAPs.................................73
4.2.1 Quantitation of Internalized MAPs....................................................73
4.2.2 Cellular Uptake of I at 37 and 0°C in Comparison
with That of Its All-D Analog and That of the Antennapedia
Sequence XV......................................................................................73
4.2.3 Effects of Factors Known to Affect Endocytosis
or Transport Protein Function on the Cellular Uptake of I ..............74
4.2.4 Uptake of MAPs into Various Cell Types from Different
Species and Organs............................................................................76
4.3 Investigation of the Structural Requirements for Cellular Uptake of MAPs .....76
4.3.1 Influence of Hydrophobicity, Hydrophobic Moment,
and Size of the Hydrophilic Face on Cellular Uptake
of Helical Peptides.............................................................................76
4.3.2 Influence of Molecular Size, Charge, and Primary Structure...........78
4.3.3 Monitoring of Cellular Uptake of MAPs by an On-Line CLSM
Protocol That Avoids Bias of the Results by Wash-Out ...................81
4.4 Ability of MAPs to Shuttle Polar Bioactive Compounds into Cytosol
and Nucleus of Mammalian Cells .................................................................84
4.5 Other Model Amphipathic Peptides Applied in Uptake Studies ..................86
4.6 Experimental ..................................................................................................87
4.6.1 Cell Culture........................................................................................87
4.6.2 Uptake Experiments...........................................................................87
4.6.3 HPLC Analysis...................................................................................88
4.6.4 Confocal Laser Scanning Microscopy (CLSM)................................88
4.6.4.1 Fluorescence Imaging at 37°C, On-Line ...........................88
4.6.4.2 Measurement of Axial Response of the Confocal System ....89
4.6.4.3 Calculation of Contributions from Out-of-Focus Planes
to Intracellular Fluorescence Intensity ...............................89
References................................................................................................................90
71