Ionophore Carbonyl Cyanide M-chlorophenylhydrazone Research Articles

L‐Aspartate Transport into Plasma Membrane Vesicles Derived from Rat Brain Synaptosomes

Aspartate uptake by membrane vesicles derived from rat brain was investigated. The uptake is dependent on a Na+ gradient ([Na+] outside greater than [Na+] inside). Active transport of aspartate is strictly dependent upon the presence of sodium and maximal extent of transport is reached when both Na+ and Cl- ions are present. The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. The uptake of aspartate is stimulated by a membrane potential (negative inside), as demonstrated by the effect of the ionophore carbonyl cyanide m-chlorophenylhydrazone and anions with different permeabilities. The presence of ouabain, an inhibitor of (Na+ + K+)-ATPase, does not affect aspartate transport. The kinetic analysis shows that aspartate is accumulated by two systems with different affinities, showing Km and Vmax values of similar order to those found in slightly "cruder" preparations. Inhibition of the L-aspartate uptake by D-aspartate and D- and L-glutamate indicates that a common carrier is involved in the process, this being stereospecific for the D- and L-glutamate stereoisomers.

Glycine transport into plasma-membrane vesicles derived from rat brain synaptosomes.

1. Transport of glycine has been demonstrated in membrane vesicles isolated from rat brain, using artificially imposed ion gradients as the sole energy source. 2. The uptake of glycine is strictly dependent on the presence of Na+ and Cl- in the medium, and the process can be driven either by an Na+ gradient (out greater than in) or by a C1- gradient (out greater than in) when the other essential ion is present. 3. The uptake of glycine is stimulated by a membrane potential (interior negative), as demonstrated by the effects of the ionophores valinomycin and carbonyl cyanide m-chlorophenylhydrazone and anions of different permeabilities. 4. The kinetic analysis shows that glycine is accumulated by two systems with different affinities. 5. The presence of ouabain, an inhibitor (Na+ + K+)-activated ATPase, does not affect glycine transport. 6. The existence of a high-affinity, Na+-dependent glycine-uptake system in membrane vesicles derived from rat brain suggests that this amino acid may have a transmitter role in some areas of the rat brain.

Alkali Cation/Sucrose Co-transport in the Root Sink of Sugar Beet

The mechanism of sucrose transport into the vacuole of root parenchyma cells of sugar beet was investigated using discs of intact tissue. Active sucrose uptake was evident only at the tonoplast. Sucrose caused a transient 8.3 millivolts depolarization of the membrane potential, suggesting an ion co-transport mechanism. Sucrose also stimulated net proton efflux. Active (net) uptake of sucrose was strongly affected by factors that influence the alkali cation and proton gradients across biological membranes. Alkali cations (Na(+) and K(+)) at 95 millimolar activity stimulated active uptake of sucrose 2.1- to 4-fold, whereas membrane-permeating anions inhibited active sucrose uptake. The pH optima for uptake was between 6.5 and 7.0, pH values slightly higher than those of the vacuole. The ionophores valinomycin, gramicidin D, and carbonyl cyanide m-chlorophenylhydrazone at 10 micromolar concentrations strongly inhibited active sucrose uptake. These data are consistent with the hypothesis that an alkali cation influx/proton efflux reaction is coupled to the active uptake of sucrose into the vacuole of parenchyma cells in the root sink of sugar beets.

Active transport of l-proline by membrane vesicles isolated from rat brain

1. (1) Active transport of l-proline has been demonstrated in membrane vesicles isolated from rat brain. The process is dependent on external Na + and is observed only under conditions when artificial Na + gradients ( C out > C in ) are imposed across the vesicle's membrane. 2. (2) The transport process, the K m value of which has been determined to be 140 μM, is strongly inhibited (75–85%) by nigericin. The process is stimulated by valinomycin and, in the absence of a pH gradient, inhibited (about 50%) by the proton ionophore carbonyl cyanide m-chlorophenylhydrazone. These data are consistent with the concept that proline transport is electrogenic. It seems optimal in the presence of a membrane potential (interior negative). In contrast, transport was not affected by ouabain and only slightly inhibited by arsenate. Optimal transport is obtained in the presence of external Cl −. This dependency is only partial, but even persists when K +-loaded vesicles are used in the presence of valinomycin. In addition, the process is inhibited by alkaloids like veratridine and aconitine and this inhibition is prevented by tetrodotoxin. The results provide direct evidence for Na +-coupled active transport by rat brain synaptic plasma membrane vesicles. 3. (3) The proline carrier has been solubilized from the membrane vesicles using cholate. The solubilized transporter was reconstituted in the presence of soybean phospholipids and potassium phosphate using the cholate dialysis technique. The reconstituted proteoliposomes catalyzed Na +-dependent proline transport, which was 6–10-fold stimulated by valinomycin.

Energization of glucose transport by Pseudomonas fluorescens.

We have measured the capacity of Pseudomonas fluorescens to transport the glucose analog 2-deoxy-d-glucose and the amino acids l-alanine and alpha-aminoisobutyric acid under conditions in which the cells could generate (i) both a membrane proton motive force and high-energy phosphate compounds, (ii) a proton motive force but not high-energy phosphate compounds, and (iii) neither a proton motive force nor high-energy phosphate compounds. This was done by depleting cells of adenosine triphosphate stores by treatment with sodium arsenate and then suspending them in a phosphate-free medium, where they could generate a proton motive force but not phosphate bond energy, or in a phosphate-containing medium, where they could generate both a proton motive force and phosphate bond energy. Inclusion of the proton-conducting ionophore carbonyl cyanide-m-chlorophenyl hydrazone under either condition precluded the generation of both a proton motive force and phosphate bond energy. The amino acids l-alanine and alpha-aminoisobutyric acid were transported independently of phosphate bond energy and required only a proton motive force. 2-Deoxy-d-glucose was transported only under conditions in which phosphate bond energy could be generated. These results are consistent with the findings of others that Pseudomonas aeruginosa produces an inducible shock-sensitive glucose-binding protein and conform to the generalization that binding protein-associated transport systems are energized by phosphate bond energy.

Active Transport of Calcium in Membrane Vesicles from Mycobacterium phlei

Active transport of calcium ions has been demonstrated in inside-out membrane vesicles from Mycobacterium phlei mediated by respiratory linked substrates as well as by ATP hydrolysis. The uptake of calcium exhibited an apparent Km of 80 microM and V of 16.6 nmol calcium uptake x min-1 x mg protein-1. A fortyfold concentration gradient for calcium ions was calculated for both the ATP-induced and the respiration-induced transport of calcium. Removal of coupling-factor-latent ATPase resulted in the complete loss of ATP-driven Ca2+ transport whereas the respiration-driven uptake was reduced by 40-50%. The uptake of calcium was inhibited by the proton conducting ionophores carbonylcyanide m-chlorophenylhydrazone and Gramicidin-D. The accumulated calcium was freely exchangeable with external calcium and was rapidly released by the addition of inhibitors of energy transduction, proton-translocating uncouplers or the ionophore A23187. The uptake of the weak base, methylamine, upon the oxidation of respiratory-linked substrates or the hydrolysis of ATP showed the generation of a protein gradient (inside acidic) which was partially collapsed on the addition of calcium ions. These results suggest that a Ca2+/H+ antiport mechanism may be responsible for the transport of calcium.

Active transport of L-glutamate by membrane vesicles isolated from rat brain.

Membrane vesicles, isolated after osmotic shock of synaptosomal rat brain fractions, actively accumulate L-glutamate. This process requires the presence of external sodium ions and internal potassium ions and is driven by artifically imposed ion gradients as the sole energy source. Either an Na+ gradient (out is greater than in) or a K+ gradient (in is greater than out) or both can be utilized to concentrate L-glutamate inside the vesicles. Transport is enhanced by valinomycin or by external thiocyanate ions and is about 50% inhibited by the proton ionophore carbonyl cyanide m-chlorophenylhydrazone. This transport thus appears to be stimulated by a membrane potential (interior negative). The glutamate transporter, the Km of which has been determined to be 3 micrometer, is specific for L-glutamate. The transport process is unaffected by ouabain but is strongly inhibited by p-hydroxymercuribenzoate as well as by nigericin, which collapses the energizing ion gradients across this membrane. Unlike the sodium dependent, but potassium independent active accumulation of gamma-aminobutyric acid in these vesicles (Kanner, B.I. (1978) Biochemistry 17, 1207) active L-glutamate uptake is not dependent on the presence of small monovalent anions in the external medium. The results provide direct evidence for Na+-coupled electrogenic active L-glutamate transport by rat brain membrane vesicles. The dependence on internal potassium ions is discussed.