Cellular uptake and in situ binding of a peptide agonist for calmodulin.
We have used the method of inverted hydropathy to develop peptides that interact with EF hands of calmodulin (CaM). Previously we have shown these peptides specifically interact with their desired target in a productive manner, in that they activated CaM in the absence of Ca(2+). Therefore, we sought to determine whether these peptides would enter cells, remain intact, and interact with CaM in the interior of the cell. Using several techniques we have demonstrated cellular uptake, stability, and an intracellular interaction with CaM with fluorescein-labeled and radiolabeled peptides in Jurkat T cells. The results suggest that these peptides may be useful in the study and the manipulation of Ca(2+)-mediated pathways in cells.
Manion
M K
MK
Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
Villain
M
M
Pan
Z G
ZG
McDonald
J M
JM
Blalock
J E
JE
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000819:agonists* / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D019793:Fluorescein / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D015398:Signal Transduction; D013347:Subcellular Fractions; D013696:Temperature; D013997:Time Factors
D002147:Calmodulin; D003432:Cross-Linking Reagents; D010455:Peptides; D010727:Phosphoric Diester Hydrolases; D002118:Calcium; D019793:Fluorescein