The participation of phosphate in the formation of a carrier for the transport of Mg++ and Mn++ ions into yeast cells

During the absorption of phosphate by yeast, the cells acquire the capacity to absorb Mn⁺⁺ and Mg⁺⁺, a capacity which is retained even after phosphate is no longer present in the medium. Cells pretreated with phosphate and then washed, slowly lose their ability to absorb Mn⁺⁺, the rate of loss depending on the temperature and on the metabolic state. The fermentation of sugars induces a very rapid loss of absorptive capacity, whereas the respiration of ethyl alcohol, lactate, or pyruvate has little effect. Inhibitor studies with sodium acetate, redox dyes, and arsenate, reveal parallel effects on Mn⁺⁺ absorption, and on phosphate absorption. It is concluded that the synthesis of a carrier for the transport of Mg⁺⁺ and Mn⁺⁺ involves a phosphorylation step closely coupled with reactions involved in the absorption of phosphate.     

The active transport of phosphate into the yeast cell

Phosphate can distribute in the cell wall space, but is not bound to an appreciable extent at the cell surface in non-metabolizing yeast. During metabolism of sugars, phosphate is actively transported into the yeast cell by a mechanism specifically involving glycolysis reactions. The movement of phosphate is in the inward direction only (no appreciable efflux), and it can proceed against a concentration gradient of 100 to 1. It is dependent on external phosphate concentrations in an asymptotic relationship, but is independent of the cellular orthophosphate concentration. The pH optimum for the phosphate uptake of 6.5 is shifted to the acid side by potassium. At certain values of pH a stimulation of 700 per cent by potassium can be observed. The nature of the effects of K⁺ and H⁺ are discussed.     

Phosphorylation of the red blood cell membrane during the active transport of C++

The phosphorylation of red blood cell membrane fragments (RBCMF) during Ca++ transport was investigated. When red cell membrane fragments are incubated with [gamma-32P]ATP under the experimental condition which minimizes the phosphorylation of Na+-K+-ATPase, RBCMF are labeled in the presence of Mg++ without Ca++. When Ca++ is added, the labeling decreases due to dephosphorylation of RBCMF. The initial reaction of phosphorylation is reversed in the presence of excess ADP. The treatment of RBCMF with n-ethylmaleimide (NEM) does not interfere with the initial phosphorylation reaction, but blocks the dephosphorylation in the presence of Ca++. These data suggest that the enzymatic sequence of the Ca++ transport mechanism may be very similar to that of the Na+ transport mechanism.     

Interaction of metallic cations with DNA VI. Specific binding of Mg++ and Mn++

The binding of Mg²⁺ and Mn²⁺ by DNA by a divalent cation specific electrode and by ultracentrifugation. Both techniques give similar results for the stoichiometry of the reaction. An oscillating densiemete allowed us to detect small changes of volume accompanying the binding. The reaction was also followed by circular dichroism measurements. Interpretation of the results is only possible if one assumes an electrostate site-binding of Mg²⁺ to phosphate group, and a chelation Mn²⁺ between the phosphate group and the N₇ of the guanine. Physical modifications accompanying these two types of binding are discused and compared to the role of these cations in some biological systems involving DNA.     

The influence of Mg++ and Zn++ on polypurine-polypyrimidine multistranded helices

We have studied by X-ray diffraction fibres of complexes of polypurine-polypyrimidine with divalent cations. In the presence of Mg++, poly(dC) and poly(dG) form a very stable triple helix at neutral pH, based on G-G-C triplexes, whereas Zn++ prevents its formation, both at neutral and acidic pH. The poly(dC) . poly(dG) complex with Zn++ is of the B form, but its X-ray diffraction pattern shows an unusual intensity distribution. This is probably due to the fact that counterions occupy defined positions on the helix. The A form has not been observed. With poly[d(A-G)].poly [d(C-T)] a different triple helical structure is formed, both with Zn++ and Mg++. Direct, X-ray diffraction evidence for these triple helices is provided here for the first time.     

The transport of proteins into yeast mitochondria. Kinetics and pools

By double isotope pulse-labeling of yeast cells, we determined the kinetics of labeling at 9 degrees C of total mitochondrial membrane, mitochondrial matrix, and cytosolic proteins, the alpha, beta, and gamma subunits of F1 ATPase, and glyceraldehyde-3-phosphate dehydrogenase. We find that none of the mitochondrial proteins show a lag in the incorporation of label compared to cytosolic proteins. These results argue against the existence in the cytosol of large pools of mitochondrial proteins awaiting transport into the organelle. Cycloheximide addition during the pulse stops [35S]methionine incorporation into mitochondrial membrane and cytosolic proteins rapidly (approximately 1 min) and with identical kinetics. Compared to cytosolic protein, however, there is a persistent incorporation of label into mitochondria after a chase with cold methionine (t1/2 approximately 1.5 min at 9 degrees C) which cannot be accounted for solely by chain completion. We conclude that this continued incorporation reflects some transport process in addition to a completion of a round of translation. When cells are labeled during a synchronous "restart" of protein synthesis, where ribosome run-off from mRNA was first induced either by incubating cells for 4 h at 0 degrees C or by treatment with 5 mM aurintricarboxylic acid, the initial rate of incorporation of label into mitochondrial protein now lags behind that of cytosolic proteins. From these results and those in the accompanying report (Ades, I.Z., and Butow, R.A. (1980) J. Biol. Chem. 255, 9918-9924) we propose that the translation of mRNA specific for mitochondrial proteins takes place in the cytoplasm and that at least a portion of the polysomes are then transported and bind to the outer mitochondrial membrane, followed by completion of translation and transfer of the newly synthesized polypeptides into the mitochondria. From a consideration of all of the available data on protein transport into mitochondria in yeast, we conclude that cytoplasmic polysomes bound to the outer mitochondrial membrane function in the transport of proteins into mitochondria by a process not necessarily mutually exclusive of post-translational transport.     

The effect of Mg ++ on the conformation of the Ca ++ -binding component of troponin

Mg⁺⁺ like Ca⁺⁺ induces a conformational change in the Ca⁺⁺-binding component of troponin. However, this change is only 36 % of the change in fluorescence intensity and 80 % of the change in optical rotation induced by Ca⁺⁺. The apparent binding constant of Mg⁺⁺ to the Ca⁺⁺-binding component is 5 × 10³ M⁻¹, much smaller than that of Ca⁺⁺. Circular dichroism measurements show that these changes are simple helix-coil transitions. Unlike the Ca⁺⁺-induced conformational change, the Mg⁺⁺-induced change cannot be propagated to other muscle proteins, and therefore has no physiological meaning.     

Passive and active transport in the parietal cell

1. Models are presented for (a) HK ATPase acting in the presence of K and Cl conductances; (b) a pH regulatory system where Na/H exchange is regulated directly by second messenger and the anion exchanger is activated secondarily to the rise in cell pH; (c) vesicle fusion and K and Cl conductances activation in the gastric parietal cell. 2. It is suggested that H transport involves protonation and deprotonation of histidine groups as well as the motion of these groups relative to the membrane barrier. 3. The HK ATPase would have a voltage generating and voltage sensitive step in the forward direction. 4. Given net electroneutrality the K transport reaction would also be charge translocating and voltage sensitive.     

The active transport of oxygen and carbon dioxide into the swim-bladder of fish

The active transport of oxygen and carbon dioxide into the swim-bladder of fish is discussed. The rete mirabile is a capillary network which is involved in the gas secretion into the bladder. The rete is regarded as a counter-current multiplier. Lactic acid which is produced in the gas gland generates in the rete single concentrating effects for oxygen and carbon dioxide; i.e., for equal partial pressures the concentrations of the gases in the afferent rete capillaries are higher than those in the efferent ones. The single concentrating effects were calculated from measurements of sea robin blood (Root, 1931). The multiplication of these effects within the rete for different rete lengths and different transport rates was numerically evaluated. The calculated O₂ and CO₂ pressures in the bladder are in good agreement with the experimental results of Scholander and van Dam (1953). The descent velocities at equilibrium between bladder pressure and hydrostatic pressure are discussed for fishes with different rete lengths.     

Arsenic-lipid complex formatinon during the active transport of arsenate in yeast

In studying formation of an arsenic-lipid complex during the active transport of (74)As-arsenate in yeast, it was found that adaptation of yeast to arsenate resulted in cell populations which showed a deficient inflow of arsenate as compared to the nonadapted yeast. Experiments with both types of cells showed a direct correlation between the arsenate taken up and the amount of As-lipid complex formed. (74)As-arsenate was bound exclusively to the phosphoinositide fraction of the cellular lipids. When arsenate transport was inhibited by dinitrophenol and sodium azide, the formation of the As-lipid complex was also inhibited. Phosphate did not interfere with the arsenate transport at a non-inhibitory concentration of external arsenate (10(-9)m). The As-adapted cells but not the unadapted cells were able to take up phosphate when growing in the presence of 10(-2)m arsenate.     

The effects of external Ca++ and Mg++ on the voltage sensitivity of desensitization in Electrophorus electroplaques

Desensitization onset was studied in voltage-clamped Electrophorus electroplaques during prolonged exposure to bath-applied carbamylcholine. The time-course of desensitization was described by a first-order rate constant kappa obs, which increased exponentially with membrane hyperpolarization from -20 to -90 mV. When Ca++ was increased from 2 to 10 mM, the voltage sensitivity of kappa obs decreased; kappa obs decreased for voltages more negative than -40 mV, and increased slightly at voltages more positive than -40 mV. 10 mM Mg++ had a less pronounced effect and the voltage sensitivity of kappa obs was unchanged. The equilibrium level of desensitization, estimated from the carbamylcholine-dependent conductance which remained after desensitization was apparently complete, also increased with hyperpolarization.