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Biomedical subjects

I Friedberg

Publications and source records attributed to I Friedberg.

At least 37 records · Page 2Linked to original sources
PubMed1985

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

Electrogenic 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 carbonylcyanide m-chlorophenylhydrazone (CCCP), SF-6847 and gramicidin D, markedly increased the membrane permeability within a very narrow range of ionophore concentration. Non-electrogenic 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 (delta psi). The data could be explained by a delta psi 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 delta psi preceded membrane permeabilization to low molecular weight, charged molecules, such as nucleotides. It is suggested that dissipation of delta psi 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 acts via specific sites on the surface of transformed cells.

Adenosine Triphosphate↗
PubMed1984

On the role of protein phosphorylation in the ATP-dependent permeabilization of transformed cells.

Incubation of transformed mouse fibroblasts with external ATP in alkaline medium low in divalent cations causes an increase in the permeability of the plasma membrane to nucleotides and other small molecules. Previous suggestions that the phosphorylation of a 44,000 dalton membrane protein is involved in this permeabilization process have been pursued. Fractionation of cells that had been incubated with [gamma-32P]ATP revealed that the labeled 44K phosphoprotein was found in both the membrane and mitochondrial fractions. Incubation of fractions isolated from unlabeled cells with [gamma-32P]ATP resulted in substantial formation of 32P-44K in the mitochondrial fraction and less incorporation in the membrane fraction. The 44,000 dalton protein was identified as the alpha-subunit of mitochondrial pyruvate dehydrogenase by partial proteolytic mapping and immunological cross-reactivity with antibodies prepared against bovine pyruvate dehydrogenase. The phosphorylation of this protein in whole cells by externally added ATP is suppressed by inclusion in the incubation medium of carboxyatractyloside (CAT) and EDTA. These substances have no effect on ATP-dependent permeabilization, indicating that the phosphorylation of pyruvate dehydrogenase is not involved in this process.

Adenosine Triphosphate↗
PubMed1984

Cellular responses to external ATP which precede an increase in nucleotide permeability in transformed cells.

Transformed mouse fibroblasts, such as 3T6, exhibit an increase in plasma membrane permeability to nucleotides and other normally impermeant molecules when incubated with external ATP in an alkaline medium low in divalent cations. Increased nucleotide permeability, induced by external ATP, occurs after a 3- to 5-min lag period. Prior to this event, there is a dramatic Na+ influx and K+ efflux, a significant reduction in the levels of intracellular ATP and organic phosphates, and a reduction in the plasma membrane potential. Accordingly, we postulate that these cellular responses to external ATP play a role in the efflux of nucleotides. Ouabain, a specific inhibitor of the plasma membrane (Na+,K+)-ATPase, acts together with low concentrations of external ATP to increase nucleotide permeability in 3T6 cells. This effect occurs at concentrations of ouabain and ATP which alone do not increase nucleotide permeability. In addition, ouabain and low concentrations of ATP alone have little effect on the level of intracellular ATP. This is in contrast to energy inhibitors and uncouplers which appear to enhance nucleotide permeability by lowering the intracellular ATP concentration. Ouabain alone causes a threefold increase in intracellular Na+ levels and a similar reduction in intracellular K+ levels under our experimental conditions, supporting the idea that ion fluxes are involved in the mechanism of permeabilization.

Adenosine Triphosphate↗
PubMed1984

Respiratory control in Micrococcus lysodeikticus.

The respiration rate of Pi-deprived cells of Micrococcus lysodeikticus is markedly increased by Pi, and returns to the original level following Pi consumption. The stimulation of the respiration was found to be specific for Pi and arsenate. Although succinate and valinomycin enhanced the respiration of both Pi-grown and Pi-deprived cells, only the latter could be further stimulated by Pi. The effect of Pi on the respiration rate was found to be concentration dependent. The control of respiration by Pi is due to its rapid uptake and its subsequent polymerization to polyphosphate via ATP. Both of these processes are coupled to proton influx into the cell, and thus stimulate the proton efflux and the respiration rate.

Adenosine Triphosphate↗
PubMed1983

The role of the proton-motive force and electron flow in light-driven solute transport in Rhodopseudomonas sphaeroides.

The initial rate of uptake of alanine was studied in whole cells of Rhodopseudomonas sphaeroides under conditions such that the proton-motive force generated by light-induced cyclic electron transfer, was composed of the membrane potential only. The proton-motive force was varied by varying the light intensity or by pretreatment of the cells with the uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone. At constant light intensity a threshold delta psi is required before alanine uptake occurs. Above this threshold value the rate of alanine uptake increases with the delta psi. The threshold value of the delta psi is higher the lower the light intensity. Under conditions of constant delta psi the rate of alanine uptake increases linearly with the light intensity. At low delta psi values a threshold light intensity is required which is higher the lower the delta psi. These results demonstrate that both a proton-motive force and electron transfer are required for alanine uptake.

Alanine↗
PubMed1982

Energy coupling of facilitated transport of inorganic ions in Rhodopseudomonas sphaeroides.

Within the scope of a study on the effects of changes in medium composition on the proton motive force in Rhodopseudomonas sphaeroides, the energy coupling of sodium, phosphate, and potassium (rubidium) transport was investigated. Sodium was transported via an electroneutral exchange system against protons. The system functioned optimally at pH 8 and was inactive below pH 7. The driving force for the phosphate transport varied with the external pH. At pH 8, Pi transport was dependent exclusively on delta psi (transmembrane electrical potential), whereas at pH 6 only the delta pH (transmembrane pH gradient) component of the proton motive force was a driving force. Potassium (rubidium) transport was facilitated by a transport system which catalyzed the electrogenic transfer of potassium (rubidium) ions. However, in several aspects the properties of this transport system were different from those of a simple electrogenic potassium ionophore such as valinomycin: (i) accumulated potassium leaked very slowly out of cells in the dark; and (ii) the transport system displayed a threshold in the delta psi, below which potassium (rubidium) transport did not occur.

Biological Transport, Active↗
PubMed1980

Effects of salts and ionophores on proline transport in a moderately halopholic 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.

Arsenates↗
PubMed1980

Electrochemical proton gradient in Micrococcus lysodeikticus cells and membrane vesicles.

Using the distribution of weak acids to measure the pH gradient (delta pH; interior alkaline) and the distribution of the lipophilic cation [3H]tetraphenylphosphonium+ to monitor the membrane potential (delta psi; interior negative), we studied the electrochemical gradient or protons (delta mu- H+) across the membrane of Micrococcus lysodeikticus cells and plasma membrane vesicles. With reduced phenazine methosulfate as electron donor, intact cells exhibited a relatively constant delta mu- H+ (interior negative and alkaline) of -193 mV to -223 mV from pH 5.5 to pH 8.5. On the other hand, in membrane vesicles under the same conditions, delta mu- H+ decreased from a maximum value of -166 mV at pH 5.5 to -107 mV at pH 8.0 and above. This difference is related to a differential effect of external pH on the components of delta mu- H+. In intact cells, delta pH decreased from about -86 mV (i.e., 1.4 units) at pH 5.5 to zero at pH 7.8 and above, and the decreases in delta pH was accompanied by a reciprocal increase in delta psi from -110 mV at pH 5.5 to -211 mV at pH 8.0 and above. In membrane vesicles, the decrease in delta pH with increasing external pH was similar to that described for intact cells; however, delta psi increased from -82 mV at pH 5.5 to only -107 mV at pH 8.0 and above.

Benzoates↗
PubMed1977

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.

Arsenates↗
PubMed1976

Membrane-bound ATPase in chloroplasts of Euglena gracilis.

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.

Adenosine Triphosphatases↗
PubMed1976

Membrane-associated, energy-linked reactions in Bdellovibrio bacteriovorus.

Disrupted cells of Bdellovibrio bacteriovorus exhibited adenosine triphosphatase activity, 60 to 80% of which was in the soluble fraction. Dicyclohexylcarbodiimide did not inhibit the adenosine triphosphatase activity in membrane particles. The particles did not show energy-linked transhydrogenase activity. The activity of non-energy-linked transhydrogenase as well as the rate of oxygen consumption were higher in membrane particles of the host-independent strain than in the host-dependent strains. The uptake of amino acid uptake was inhibited by cyanide and by carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. Valinomycin, in the presence of K+, did not inhibit the uptake, and only partial inhibition was exerted by arsenate and dicyclohexylarbodiimide. Sulfhydryl reagents inhibited amino acid uptake.

Adenosine Triphosphatases↗
PubMed1975

Biogenesis of chloroplast membranes in Chlamydomonas reinhardi: Chloroplast-controlled transfer of cytoplasmic proteins to the developing chloroplast membranes as visualized by quantitative radioautography.

The light-induced formation of the photosynthetic membranes (greening) in y-1 mutant of Chlamydomonas reinhardi requires synthesis of new proteins which become incorporated into the growing membranes. It has been shown previously (Eytan and Ohad, 1970) that proteins synthesized by both chloroplast and cytoplasmic ribosomes concur in the formation of functional photosynthetic membranes, indicating the presence of a mechanism permitting the specific transfer of membrane proteins synthesized in the cytoplasm into the chloroplast. Transfer of such proteins cannot yet be identified by the usual biochemical techniques unless they become part of the growing photosynthetic membranes. However, it is possible to follow their synthesis and translocation between the different cellular compartments by use of quantitative electron microscopic radioautography. In the present work, the radioautographic grain distribution among chloroplast, chloroplast membrane, nucleus, mitochondria, and the remainder of the cytoplasm (cytosol) was carried out following short radioactive pulse-labeling and chase during greening of dark-grown mutants in the presence or absence of protein synthesis inhibitors. The results indicate that transport of some of the proteins of cytoplasmic origin to their final location within the chloroplast is at least partially controlled by concomitant synthesis of proteins by the chloroplast ribosomes.

Arginine↗