Ilan Friedberg

Permeabilization of Transformed Cells in Culture by External ATP

procedures include the use of ionophores, manipulation of osmotic conditions, treatment with ouabain or with agents that reduce the level of intracellular ATP, and also the use of inhibitors of Ca ++calmodulin systems.

Permeability change in transformed mouse fibroblasts caused by ionophores, and its relationship to membrane permeabilization by exogenous ATP.

SummaryElectrogenic ionophores have been found to induce membrane permeabilization in Swiss mouse 3T3 cells that had undergone spontaneous transformation (3T6 cells). Cells attached to plastic dishes were loaded with [3H] uridine, and then the medium was replaced by buffered salt solution at pH 7.8. The enhancement of membrane permeability was assayed by following the efflux of uridine nucleotides, normally impermeant substances. Titration with electrogenic ionophores, such as carbonylcyanidem-chlorophenylhydrazone (CCCP), SF-6847 and gramicidin D, markedly increased the membrane permeability within a very narrow range of ionophore concentration. Nonelectrogenic ionophores, such as monensin and nigericin, did not affect membrane permeability. Measurements of the distribution of the lipophilic cation tetraphenylphosphonium (TPP+) between the cells and their environment implied that the remarkable increase in permeability took place within a narrow range of membrane potential (Δψ). The data could be explaine by a Δψ threshold value, under which aqueous channels are opened in the plasma membrane. The effects exerted by electrogenic ionophores on the plasma membrane were found to be similar to those induced by exogenous ATP. In both cases rapid efflux of K+, influx of Na+ and reduction of Δψ preceded membrane permeabilization to low molecular weight, charged molecules, such as nucleotides. It is suggested that dissipation of Δψ induces conformational alterations in membranal components, and/or topological changes, such as aggregation of protein molecules, to form membranal aqueous channels. Electrogenic ionophores permeabilize both normal (3T3) and transformed (3T6) mouse fibroblasts, whereas ATP effects are specific for transformed cells. Thus, it is postulated that ATP actsvia specific sites on the surface of transformed cells.

Two distinct receptors for ATP can be distinguished in Swiss 3T6 mouse fibroblasts by their desensitization.

The stimulation of calcium efflux from Swiss 3T6 mouse fibroblasts by extracellular ATP was studied. It was found that the cells could be desensitized to ATP by a previous exposure to the nucleotide, lending support to the theory that this is a receptor mediated process. Another ATP-receptor mediated process in Swiss 3T6 cells, that is also subject to desensitization, causes the permeabilization of the plasma membrane to nucleotides and other normally impermeant compounds [Gonzalez et al., J. Cell. Physiol. 139:109 (1989)]. Here we demonstrate that selective desensitization of the ATP-dependent calcium mobilization pathway can be achieved without affecting ATP-induced permeabilization. Data are presented in support of the existence of multiple ATP-receptors (purinoceptors) in Swiss 3T6 cells.

Effects of salts and ionophores on proline transport in a moderately halophilic halotolerant bacterium

The effect of salt on proline uptake in a moderately halophilic halotolerant bacterium was studied. Cells were grown either on low salt or high salt media. A correlation was found between the salt concentrations in the growth media and the optimal concentration for uptake. The uptake rate was stimulated 2--3-fold by NaCl, as compared to KCl. The Km, V and activation energies values for proline uptake, as well as the external pH effect, were similar in low-salt-grown cells and high-salt-grown cells. This suggests that the halotolerance of the transport system is not due to alterations of the system during growth at various conditions, but rather to its intrinsic ability to function under extreme environmental conditions. The uptake was inhibited by cyanide and carbonyl cyanide m-chlorophenylhydrazone, but not by arsenate, indicating that the electrochemical proton gradient (delta mu- H+), generated by respiration, is the main driving force for proline transport. In low-salt-grown cells, at pH 6.0, partial inhibition was exerted by nigericin or valinomycin, whereas at pH 8.0 the uptake was inhibited by valinomycin only. Similar, although less pronounced effects were found in high-salt-grown cells. The data suggest that at pH 6.0 proline transport is driven by delta mu- H+ (composed of electrical potential (delta psi) and pH gradient), whereas at pH 8.0 delta psi is the main driving force. Procedures of pretreatment with EDTA were developed to enable the penetration of the ionophores into the cells.

Phosphate transport in Micrococcus lysodeikticus

Phosphate accumulates in Micrococcus lysodeikticus cells against a concentration gradient, by an energy-dependent process. The phosphate transport is derepressed during phosphate deprivation. The depression process is inhibited by chloramphenicol. The apparent Km of phosphate transport is 4.3 micronM. The activation energy of the transport is 21 kcal per mol in the temperature range of 0-29degrees C, and 4.9 kcal per mol between 29 and 40degrees C. The rate of the transport increases in presence of K+ and Mg2+. Arsenate is a competitive inhibitor of phosphate transport, having an apparent Ki of 6.0 micronM. Sulfhydryl reagents, respiratory inhibitors and uncouplers of oxidative phosphorylation inhibit phosphate transport.

COMBINED EFFECTS OF ATP AND ITS ANALOGS ON THE MEMBRANE PERMEABILITY IN TRANSFORMED MOUSE FIBROBLASTS

Extracellular ATP (0.6 mM) induces a marked decrease in the membrane potential, followed by an increase in cell membrane permeability in transformed mouse fibroblasts. The effects of the ATP analogs, p[CH2]ppA and p[NH]ppA (0.6 mM), on the membrane potential and permeability are much less pronounced. ATP at 0.05 mM has no effect by itself, but markedly increases the analog‐induced membrane potential dissipation and permeability. The data suggest that ATP‐induced membrane permeation is composed of two processes: One is common to ATP and its analogs and appears to be a receptormediated process. The second is unique for ATP, effective even at low concentration (0.05 mM), and might be mediated by cell surface enzymes, for which ATP, but not its analogs, serves as a substrate.

The effect of ionophores on phosphate and arsenate transport in Micrococcus lysodeikticus.

Phosphate (pi) has recently been shown to accumulate within Micrococcus lysodeikticus cells against its concentration gradient by an energy-dependent process [l] . The main driving force of active transport systems, according to the chemiosmotic hypothesis [2-41, is the proton electrochemical gradient across the cell membrane, the proton motive force (AFH’). Two components, membrane potential (A

Membrane-bound ATPase in chloroplasts of Euglena gracilis

) and proton gradient (ApH) contribute to the proton motive force according to the relation:

Polysomes in Escherichia coli during amino acid starvation: Structural change observed by electron microscopy

Membrane-bound ATPase activities in chloroplasts of Euglena were examined. Ca2+- and Mg2+-dependent activities were relatively high in membrane preparations and could not be further activated by a number of procedures. The enzyme was found to be highly specific for purine nucleotides and was inhibited by the usual inhibitors of photophosphorylation. Km values of Ca2+ and Mg2+ ATPase for ATP were 2.5 and 2.1 mM, respectively. Both activities were competitively inhibited by ADP and inorganic phosphate. A relationship was found between Ca2+- or Mg2+-dependent ATPase activities and chloroplast completeness. The possibilities that these activities result from one enzyme depending on Ca2+ or Mg2+ or from two different enzymes are discussed.

Localization of oxidized 3,3′-diaminobenzidine deposits in chloroplasts

Earlier experiments have shown that ribosomes are released from polysomes when they encounter ‘a codon for which no charged tRNA is available [ 1, 21. It was further shown that these ribosomes then reinitiate and resume translation [3,4]. Inhibition of RNA synthesis by the addition of rifampin [5] resulted in the elimination of polysomes from amino acid starved cells [3]. These findings indicate that during amino acid starvation polysomes are broken down and reconstituted continually. The rate of breakdown of polysomes in starved cells was slower than in growing cells [3,6] suggesting that when a ribosome reaches a codon specifying a missing amino acid there is a delay before its release from the mRNA. It is conceivable that the rate with which new ribosomes go on the mRNA at the point of initiation of translation, and the rate of translocation are independent of the rate at which ribosomes are released from mRNA at the codon for which no charged tRNA is available. This possibility might result in a ‘piling up’ of ribosomes on mRNA during amino acid starvation