When the radioactive isotope of potassium, K 4~, became available, it was possible to observe the normal exchange of potassium between the intracellular phase of erythrocytes and their extracellular medium. The in vivo work of Hahn, Hevesy, and Rebbe (1) in 1939 and of Cohn (2) in 1941 showed that some exchange of potassium does occur in mammalian erythrocytes. Dean and Fenn and their collaborators (3,4) showed that the red cell membrane was permeable in vitro and in vivo to potassium and calculated exchange rates.The following experiments were undertaken to determine the rate of transfer of potassium across the human erythrocyte membrane in a stable in vitro system. For this purpose human red cells were incubated in diluted plasma contain~g radioactive potassium as a tracer. The change with time of the specific activity of the extracellular potassium was determined and the rate of transfer of potassium between the intracellular and extracellular phases was calculated.
MethodsThe rocker-dilution technique developed by Geiman et al. (5) for the cultivation of malarial parasites was used. This permits the incubation of red cells for 52 hours with minimal change in pH. Centrifugation, washing, and excessive agitation of the red cells prior to incubation were avoided because of the demonstrated effects of mechanical procedures on the permeability of red cells (6).Two parts of freshly drawn, heparinized, whole blood were diluted with three parts of an inorganic medium having the composition shown in Table I. This medium has a potassium concentration of 4.42 millimoles per liter and a freezing point of -0.56°C. About 1 millicurie of radioactive potassium was present per liter of medium. When in later experiments the amount of potassium chloride was changed, the sodium chloride was changed by an equivalent amount in order to keep the medium isotonic. Glass *
The TraT protein is a surface-exposed lipoprotein, specified by plasmids of the IncF group, that mediates serum resistance and surface exclusion. The structure and function of the TraT protein determined by plasmid R6-5 was probed by genetic insertion of a foreign antigenic determinant, the C3 epitope of polio virus, at residues 61, 125, 180, 200 or 216 of the protein. The chimaeric proteins were transported to the outer membrane and, in three cases, immunoassays with an anti-C3 monoclonal antibody indicated that the C3 epitope was exposed on the cell surface. Three of the hybrids, with insertions at residues 125, 180 and 200, assembled into the trypsin-resistant oligomeric form characteristic of the wild-type protein, which suggested that these regions are not involved in TraT subunit:subunit interactions. Additionally, the hybrid protein carrying the C3 epitope at position 180 functioned in a genetic suppression assay and retained partial surface-exclusion activity. Thus, its localization, folding and organization does not appear to be grossly altered from that of the wild-type protein. Applications of the protein for the transport of foreign antigenic determinants to the cell surface are discussed.
The TraT protein is a highly cell-surface-exposed lipoprotein specified by F-like plasmids that confers serum resistance and blocks the conjugative transfer of plasmids to cells bearing identical or closely related plasmids, a process known as surface exclusion. The TraT protein specified by the antibiotic-resistance plasmid R6-5 was purified to apparent homogeneity. When added to mating mixtures, TraT blocked the transfer of plasmids belonging to Surface Exclusion Group IV (Sfx IV) but had no significant effect on the transfer of plasmids belonging to other groups. Additionally, the purified protein has a protective effect on bacterial cells incubated in serum, indicating that it does not have to be located on the cell surface to mediate serum resistance. To localize regions of the protein that were responsible for surface exclusion specificity, the amino acid sequence of the TraT protein specified by CoIB2-K98 (Sfx II) was determined by cloning and sequencing of the corresponding gene. Comparison of the derived sequence with those of the F and R100-1 proteins indicated that surface exclusion specificity of TraT is determined by single alterations in a five-amino-acid region (residues 116-120). This was confirmed by segment swapping experiments in which the specificity of the R6-5 TraT protein (Sfx IV) was switched to that of the CoIB2-K98 protein (Sfx II). Our results suggest that the region defined by residues 116-120 is located on the external face of the outer membrane and interacts specifically with the donor cell in surface exclusion.
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