Cocompartmentation of proteins and K+ within the living cell.

Abstract

Monolayer H-50 tissue culture cells were treated with Triton X-100 and Brij 58 nonionic detergents, and their electron microscopic morphology along with the release of the intracellular proteins and K+ were studied. Although Triton X-100 was more effective, both detergents removed the lipoid membranes within 5 min. The mobilization and solubilization of the cytoplasmic and nuclear proteins occurred much faster with Triton X-100 than with Brj 58. In Triton X-100-treated cells, the loss of K+ was complete within 2 min. The loss of K+ from the Br" 58-treated cells was complete only after 10 min and the mobilization of K+ showed sigmoid-type release kinetics. These results support the view that most of K+ and "diffusible" proteins are not freely dissolved in the cellular water, but they are cocompartmentalized inside the living cell.In the past two decades, experiments with nonionic detergents (Triton X-100, Nonidet P-40, etc.) have considerably improved our knowledge about the composition and structure of the living cell (1-9). The cellular components may be divided into detergent-resistant and detergent-soluble elements. The detergent-soluble elements are the membrane lipoids, several proteins, smaller organic molecules, and most of the inorganic ions (1,8,9). The detergent-resistant cellular components are the "detergent-resistant cytoskeleton" composed of microtubules, intermediate filaments and microfilaments, nuclear matrix, chromatin, DNA-and RNAcontaining filaments, etc. (2-7). The release kinetics of the different cellular components solubilized by detergents are not known, and only a few follow-up studies have ever been performed on the detergent-treated cells (3, 10, 11).The major goal of our experiments was to perform a careful study on the release kinetics of K+ and proteins mobilized from tissue culture cells with Triton X-100 and Brij 58 nonionic detergents. The encouragement for these studies came from earlier reports that indicated similarities in the lipo-or membranolytic effect of Triton X-100 and Brij 58 detergents but differences in protein mobilization (7, 11). As confirmed by electron microscopic observations, both detergents remove the lipoid membranes within 2-5 min; however, the release of K+ was much faster in Triton X-100-treated cells than in Brij 58-treated cells. In the case of the Brij 58, although the membranes were removed-i.e., the cells were " opened" in 2-5 min-the release of K+ took place after several minutes delay. The parallel studies of protein release in response to detergent treatment demonstrated K+ release correlated more closely with the release of proteins than with the removal of the lipoid membranes. These findings suggest that cellular structures other than the membrane are also significantly involved in the maintenance of the extra-and intracellular K+/Na+ gradients. In addition, these findings support the view that the intracellular K+ ions are not freely dissolved in the cell water (12-18) and suggest that a cocompartmentation exists within the living cel...

Role of plasma membrane and of cytomatrix in maintenance of intracellular to extracellular ion gradients in chicken erythrocytes

“…The major findings from the erythrocyte studies reported here are in agreement with the studies of Kellermayer et al (1984Kellermayer et al ( ,1986 in showing a restricted release of protein and K + from the Brij 58-opened cells. The chicken erythrocyte clearly demonstrated a Fig.…”

Evidence for cooperativity of protein dissolution in Brij 58 permeabilized L929 cells

“…The data shown in Figure 4 indicate that the profile of proteins released by Brij was very constant as a function of time and that there was no apparent sieving on the basis of size. The similarity of the profiles of proteins leaking from detergentpermeabilized cells has been noted before (Kelner et al, 1986;Kellermayer et al, 1986).…”

Bioluminescent Monitoring of ATP Release from Human Red Blood Cells Treated with Nonionic Detergent

“…The Brij 58-induced much slower protein release was observed in different cell types and even intracellular K + was found to express a retarded diffusion from the Brij 58-permeabilized cells, indicating the existence of a close contact between K + and the intracellular proteins (7,8). In our experiments, the detected very rapid ATP release kinetics of the Triton X 100-, and the much slower ATP mobilization rate of the Brij 58-treated cells were in good agreement with the nonionic detergent-induced protein and K + mobilization found in other cells (8).…”

“…The validity of such an approach has already been proved by the observation that nonionic detergent-induced removal of the lipid membrane of a cell is not immediately followed by the complete release of the intracellular "soluble" components (6,7). Analysis of the release kinetics of K + and proteins in the presence of nonionic detergents has already shown that this technique can provide information on the compartrnentation of K + in living cells (7,8).…”