ABSTRACT
Protein transduction domains (PTDs) have proven to be an invaluable tool to transduce a wide variety of cargo's including peptides across the plasma membrane and into intact tissue. The PTDs are able to deliver biologically active molecules both in vitro and in vivo. This study describes many new polybasic PTDs of which some are just as potent as the PTDs derived from extracellular RNAses or other published PTDs. Large differences in potency became apparent when the PTDs are coupled to particular cargoes. Therefore, the unique characteristic of a PTD may only become apparent when it is selected for a particular application. Rules for optimization of PTDs for particular applications are now emerging and open the way for a new generation of drug delivery agents. Because fixation artifacts and irreversible membrane binding may cause misinterpretation of the amount of internalization of polybasic peptides, we have developed an enzyme transduction assay based on the intracellular loading of a cell permeable substrate. In this assay, a fluorescent signal is generated by internalized enzyme in intact cells and not by membrane-bound or extracellular enzyme.
Subject(s)
Molecular Biology/methods , Peptides/metabolism , Amino Acid Sequence , Animals , Binding Sites , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cattle , Cells, Cultured , Fluorescein-5-isothiocyanate/chemistry , Fluorescein-5-isothiocyanate/metabolism , Fluoresceins/chemistry , Fluoresceins/metabolism , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Galactosides/chemistry , Galactosides/metabolism , Gene Products, tat/genetics , Gene Products, tat/metabolism , Heparin/metabolism , Horseradish Peroxidase/genetics , Horseradish Peroxidase/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Molecular Sequence Data , Peptide Mapping , Peptides/analysis , Peptides/genetics , Protein Engineering/methods , Protein Structure, Tertiary , Protein Transport , Streptavidin/chemistry , Streptavidin/metabolism , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , beta-Galactosidase/genetics , beta-Galactosidase/metabolismABSTRACT
Escherichia coli cells were synchronized by the elutriation technique. The pattern of penicillin-binding proteins (PBPs) in synchronously growing cells was determined with an iodinated derivative of ampicillin in intact cells as well as in isolated membranes. This was done under nonsaturating conditions as well as under conditions in which the PBPs were saturated with [125I]ampicillin. No evidence was found for fluctuations in the PBP pattern: the PBPs seem to be present in a constant ratio throughout the division cycle. The E. coli cells exert their control on shape maintenance and cell wall growth apparently not on the level of concentration of PBPs in the cell but rather on activation of existing components.
