@article {1103,
	title = {Cytotoxic ribonucleases: the dichotomy of Coulombic forces.},
	journal = {Biochemistry},
	volume = {46},
	year = {2007},
	month = {2007 Sep 11},
	pages = {10308-16},
	abstract = {<p>Cells tightly regulate their contents. Still, nonspecific Coulombic interactions between cationic molecules and anionic membrane components can lead to adventitious endocytosis. Here, we characterize this process in a natural system. To do so, we create variants of human pancreatic ribonuclease (RNase 1) that differ in net molecular charge. By conjugating a small-molecule latent fluorophore to these variants and using flow cytometry, we are able to determine the kinetic mechanism for RNase 1 internalization into live human cells. We find that internalization increases with solution concentration and is not saturable. Internalization also increases with time to a steady-state level, which varies linearly with molecular charge. In contrast, the rate constant for internalization (t1/2 = 2 h) is independent of charge. We conclude that internalization involves an extracellular equilibrium complex between the cationic proteins and abundant anionic cell-surface molecules, followed by rate-limiting internalization. The enhanced internalization of more cationic variants of RNase 1 is, however, countered by their increased affinity for the cytosolic ribonuclease inhibitor protein, which is anionic. Thus, Coulombic forces mediate extracellular and intracellular equilibria in a dichotomous manner that both endangers cells and defends them from the potentially lethal enzymatic activity of ribonucleases.</p>
},
	keywords = {Biomechanics, Cell Death, Endocytosis, Fluorescent Dyes, Humans, K562 Cells, Kinetics, Mutant Proteins, Pancreatic, Protein Structure, Ribonuclease, Secondary},
	issn = {0006-2960},
	doi = {10.1021/bi700857u},
	author = {Johnson, R Jeremy and Chao, Tzu-Yuan and Lavis, Luke D and Raines, Ronald T}
}
