Leucine transport in plasma membrane vesicles of Saccharomyces cerevisiae

Yeast plasma membrane vesicles were obtained by the fusion of liposomes with purified yeast membranes by means of the freeze thaw-sonication technique. Beef heart mitochondria cytochrome-c oxidase was incorporated into the vesicles. Addition of substrate (ascorbate/TMPD/cytochrome c) generated a membrane potential negative inside, and an alkaline pH gradient inside the vesicle, that served as the driving force for leucine transport. Both delta pH and delta psi could drive leucine transport. When delta pH was increased in the presence of valinomycin and potassium, at the expense of delta psi, leucine uptake increased by 10%.     

Glucose transport in vesicles reconstituted from Saccharomyces cerevisiae membranes and liposomes.

Glucose transport activity was reconstituted into liposomes by the freeze-thaw-sonication procedure from unextracted Saccharomyces cerevisiae membranes and preformed phospholipid liposomes. Fluorescence-dequenching measurements with octadecylrhodamine B chloride (R18)-labeled membranes showed that the yeast membrane lipids are diluted by the liposome lipids after the freeze-thaw-sonication procedure. At lipid-to-protein ratios greater than 75:1, vesicles with single transporters were formed. Reconstituted specific activity was increased at least twofold if the liposomes contained 50 mol% cholesterol. A further increase in specific activity, from 3- to 10-fold, was achieved by fractionation of the membranes on a Renografin gradient before reconstitution. Examination of the fractions from the Renografin gradient by sodium dodecyl sulfate-gel electrophoresis showed a parallel enrichment of glucose transport activity and a number of proteins including one with an apparent Mr of ca. 60,000, which might be the glucose transporter. Finally, preliminary kinetic analysis of glucose transport activity in vesicles reconstituted at a high lipid-to-protein ratio gave a Vmax of ca. 2.8 mumol/mg of protein per min at 23 degrees C and a Km of ca. 8 mM. The latter value corresponds to the kinase-independent, low-affinity component of glucose transport observed in wild-type cells.     

Cl- transport in apical plasma membrane vesicles isolated from bovine tracheal epithelium

Gradually altered synthetic entities were employed as molecular probes, and arachidonic acid, ADP, human alpha-thrombin and the Ca2+ ionophore A23187 as aggregation-inducing agents, in a comprehensive study on the response profile of human blood platelets with an emphasis on the effects of exogenous and increased intracellular Ca2+. Corroborating further previous conclusions, some representative carbamoylpiperidine derivatives, at concentrations effecting substantial inhibition of ADP-induced aggregation, failed to retain that effect when 5.0 mM Ca2+ was introduced into the otherwise identical test medium; reference compounds chlorpromazine and propranolol registered corresponding inhibitory patterns. At increased concentrations the compounds' inhibitory potency was regenerated even in the presence of 5 mM Ca2+. In fact, in sufficiently high concentrations, the compounds were even capable of inhibiting aggregation elicited by 15 microM of the ionophore A23187; so did chlorpromazine and propranolol. Another set of congeners revealed the striking sensitivity of ionophore A23187-induced human blood platelet aggregation to the surface active potencies of inhibitor molecules. The loss in inhibitory potency was directly related to the lesser hydrophobic character of the molecule.     

Proton potential-dependent polyamine transport by vacuolar membrane vesicles of Saccharomyces cerevisiae.

Vacuolar membrane vesicles of Saccharomyces cerevisiae accumulated spermine and spermidine in the presence of ATP, not in the presence of ADP. Spermine and spermidine transport at pH 7.4 showed saturation kinetics with Km values of 0.2 mM and 0.7 mM, respectively. Spermine uptake was competitively inhibited by spermidine and putrescine, but was not affected by seven amino acids, substrates of active transport systems of vacuolar membrane. Spermine transport was inhibited by the H(+)-ATPase-specific inhibitors bafilomycin A1 and N,N'-dicyclohexylcarbodiimide, but not by vanadate. It was also sensitive to Cu2+ or Zn2+ ions, inhibitors of vacuolar H(+)-ATPase. Both 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile (SF6847) and nigericin blocked completely the spermine uptake, but valinomycin did not. [14C]Spermine accumulated in the vesicles was exchangeable with unlabeled spermine and spermidine. However, it was released by a protonophore only in the presence of a counterion such as Ca2+. These results indicate that a polyamine-specific transport system depending on a proton potential functions in the vacuolar membrane of this organism.     

Amino acid transport in plasma membrane vesicles from isolated rat liver parenchymal cells

Plasma membrane vesicles were prepared from isolated rat liver parenchymal cells. The transport of several amino acids was studied and found to be identical to that in membrane vesicles from whole liver tissue.     

Sodium-dependentl-serine transport in plasma membrane vesicles isolated from Ehrlich cells by two-phase compartmentation

Summary Plasma membrane vesicles were prepared from Ehrlich cells using two-phase system compartmentation. The highly pure plasma membrane vesicles obtained presented a negligible mitochondrial contamination and were suitable for studies of amino acid transport.l-Serine transport showed a clear ionic specificity, maximum incorporation being observed when an inwardly directed NaSCN gradient was used. Na⁺-dependentl-serine transport was dependent on assay temperature and membrane potential, and it seemed to be carried out by two different transport systems. An essential sulfhydryl group seemed to be involved in the transport process.     

Active phosphate ion transport in plasma membrane vesicles isolated from mouse fibroblasts.

Inorganic phosphate accumulated 8-fold in plasma membrane vesicles derived from simian virus 40-transformed 3T3 mouse fibroblasts when a NaCl gradient (external greater than internal) was artificially imposed across the membrane. Preincubation with Na+ or addition of monensin markedly reduced phosphate accumulation. Na+-stimulated phosphate transport was not affected by addition of either dicarboxylic acids, antimycin A, or ouabain and persisted after addition of proton ionophores. The coupling of phosphate transport to Na+ gradients was pH-dependent, with maximal stimulation by Na+ below pH 7. These findings suggest that monovalent phosphate anion moves across the plasma membrane in co-transport with sodium ion.     

Cyclic AMP controls the plasma membrane H+-ATPase activity from Saccharomyces cerevisiae

The thermosensitive G1-arrested cdc35-10 mutant from Saccharomyces cerevisiae, defective in adenylate cyclase activity, was shifted to restrictive temperature. After 1 h incubation at this temperature, the plasma membrane H+-ATPase activity of cdc35-10 was reduced to 50%, whereas that in mitochondria doubled. Similar data were obtained with cdc25, another thermosensitive G1-arrested mutant modified in the cAMP pathway. In contrast, the ATPase activities of the G1-arrested mutant cdc19, defective in pyruvate kinase, were not affected after 2 h incubation at restrictive temperature. In the double mutants cdc35-10 cas1 and cdc25 cas1, addition of extracellular cAMP prevented the modifications of ATPase activities observed in the single mutants cdc35-10 and cdc25. These data indicate that cAMP acts as a positive effector on the H+-ATPase activity of plasma membranes and as a negative effector on that of mitochondria.     

Glucose transport in a kinaseless Saccharomyces cerevisiae mutant.

Wild-type Saccharomyces cerevisiae organisms contain three kinases which catalyze the phosphorylation of glucose: two hexokinase isozymes (PI and PII) and one glucokinase. Glucose transport measurements for triple-kinaseless mutants, which lack all three of these kinases, confirm that the kinases are involved in the low apparent Km transport process observed in metabolizing cells. Thus kinase-positive cells containing one or more of the three kinases exhibit biphasic transport kinetics with a low apparent Km (1 to 2 mM) and high apparent Km (40 to 50 mM) component. Triple-kinaseless cells, however, exhibit only the high apparent Km component of kinase-positive cells (60 mM). Kinetic analysis of glucose transport in the triple-kinaseless cells shows that glucose is transported by a facilitated diffusion process which exhibits trans-stimulated equilibrium exchange and influx counterflow.     

Characterization of solute transport in plasma membrane vesicles isolated from cotyledons ofRicinus communis L.

Evidence is presented for the proton-coupled transport of sucrose and glutamine in purified plasma membrane vesicles isolated from cotyledons ofRicinus communis. Imposition of a pH gradient (internal alkaline) across the plasma membrane resulted in a rapid uptake of sucrose and glutamine which was inhibited in the presence of carbonyl cyanide-m-chlorophenyl hydrazone. Imposition of a pH gradient plus an internal negative membrane potential stimulated uptake further. Glucose and fructose uptakes were negligible under these conditions. Sucrose uptake into the vesicles demonstrated saturation kinetics with a K_m of 0.87 mol·m^-3, indicating carrier-mediated transport. In support of this, uptake was very sensitive to the protein-modifying reagentp-chloromercuribenzenesulphonic acid. N-Ethylmaleimide, another sulphydryl reagent, was only slightly inhibitory. However, both reagents strongly inhibited sucrose uptake into intact cotyledons; the possible reasons for the difference between the intact and isolated systems are assessed. The value of this system for the study of sucrose and amino acid carriers is discussed.     

Calcium transport driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae

Vacuolar membrane vesicles of Saccharomyces cerevisiae accumulate Ca2+ ion in the presence of ATP, not in the presence of ADP or adenyl-5'-yl imidodiphosphate. Calcium transport showed saturation kinetics with a Km value of 0.1 mM and optimal pH of 6.4. Ca2+ ion incorporated in the vesicles was exchangeable and released completely by a protonophore uncoupler, 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile (SF6847), or calcium-specific ionophore, A23187. The transport required Mg2+ ion but was inhibited by Cu2+ or Zn2+ ions, inhibitors of H+-ATPase of the vacuolar membrane. The transport activity was sensitive to the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide, but not to oligomycin or sodium vanadate. SF6847 or nigericin blocked Ca2+ uptake completely, but valinomycin stimulated it 1.35-fold. These results indicate that an electrochemical potential difference of protons is a driving force for this Ca2+ transport. The ATP-dependent formation of the deltapH in the vesicles and its partial dissipation by CaCl2 were demonstrated by fluorescence quenching of quinacrine. This Ca2+ uptake by vacuolar membrane vesicles is suggested to be catalyzed by a Ca2+/H+ antiport system.     

Kinetic characterization of plasma membrane atpase from Saccharomyces cerevisiae

1. Plasma membrane preparations have been isolated from spheroplasts of Saccharomyces cerevisiae, strain R XII, via lysis and subsequent differential centrifugation. These preparations are almost devoid of mitochondrial contamination. 2. The plasma membrane ATPase is fairly stable when refrigerated, but loses activity at 8 degrees C and above. Below pH 5.6 the ATPase is irreversibly inactivated. The enzyme also splits GTP and ITP, although to a lesser extent. 3. Mg2+-ions are essential as part of the reactive substrate, MgATP, and furthermore they activate the ATPase. Optimal conditions depend on substrate concentration. When the concentration of free Mg2+ ions exceeds about 0.1 mM, competitive inhibition occurs. 4. In the range of pH 5.6-9.2 two functional groups dissociate. One, with pKb = 8.1 +/- 0.1 participated in substrate binding and another one with pKb' = 8.1 +/- 0.1 is involved in substrate splitting. 5. The experiments with group-specific inhibitors suggest that an alpha-amino group and a sulfhydryl residue are involved in substrate binding and conversion. Furthermore, imidazole, tryptophan and carboxyl residues may be important for the catalytic process.     

Tyrosine transport by membrane vesicles isolated from rat brain

Tyrosine uptake by membrane vesicles derived from rat brain has been investigated. The uptake is dependent on an Na⁺ gradient ([$\text{Na}{}^{\mathrm{+}}\text{]}\text{outside}\text{> [}\text{Na}{}^{\mathrm{+}}\text{]}\text{inside}$). 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 process is stimulated by a membrane potential (negative inside) as demonstrated by the effect of the ionophores valinomycin and carbonyl cyanide $\text{m-}\text{chlorophenylhydrazone}$ and anions with different permeabilities. Kinetic data show that tyrosine is accumulated by two systems with different affinities. Tyrosine uptake is inhibited by the presence of phenylalanine and tryptophan.     

Calcium transport and calcium-ATPase activity in human lymphocyte plasma membrane vesicles

We have studied Ca transport and the Ca-activated Mg-ATPase in plasma membrane vesicles prepared from normal human lymphocytes. Membrane vesicles that were exposed to oxalate as a Ca-trapping agent accumulated Ca in the presence of Mg2+ and ATP. ADP, AMP, GTP, UTP, ITP, TTP, or CTP did not substitute for ATP in energizing uptake. The Vmax for Ca uptake was 2.4 pmol of Ca/micrograms of protein/min, and the Km values for Ca and ATP were 1.0 and 80 microM, respectively. One microM A23187, added initially, completely inhibited net Ca uptake and, if added later, caused the release of Ca accumulated previously. Cyanide, oligomycin, ouabain, or varying Na+ or K+ concentrations had no effect on Ca uptake. A Ca-activated ATPase was present in the same membrane vesicles, which had a Vmax of 25 pmol of Pi/micrograms of protein/min at a free Ca concentration of 4-5 microM. This Ca-ATPase had Km values for Ca and ATP of 0.6 and 90 microM, respectively. These kinetic parameters were similar to those observed for uptake of Ca by the vesicles. The Ca-ATPase activity was insensitive to azide, oligomycin, ouabain, or varying Na+ or K+ concentrations. No Ca-activated hydrolysis of GTP or UTP was observed. Both Ca transport and the Ca-ATPase activity of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-treated lymphocyte plasma membranes were stimulated 2-fold by a cytoplasmic component (calmodulin) that was purified 500-fold from lymphocyte cytoplasm. Thus, human lymphocyte plasma membranes have both a Ca transport activity and a Ca-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivities to calmodulin.     

Lipid-mediated glycosylation of endogenous proteins in isolated plasma membrane of Saccharomyces cerevisiae.

A highly purified plasma membrane fraction from Saccharomyces cerevisiae was obtained by centrifugation on discontinuous sucrose and Urografin gradients. This plasma membrane fraction was capable of glycosylating endogenous proteins. It is shown that glycolipids play an intermediate role in these glycosylation reactions; with uridine 5'-diphosphate-N-acetylglucosamine as sugar donor the intermediate lipids possessed stability towards alkali and chromatographic mobilities similar to polyprenyl diphosphate N-acetylglucosamine and polyprenyl diphosphate di-N-acetylchitobiose.     

L-glutamate transport in renal plasma membrane vesicles

Summary This review describes the uptake of L-glutamate by well-characterized preparations of renal brush border (luminal) and baso-lateral membrane vesicles derived from the plasma membrane of the polar proximal tubular cell. L-glutamate is taken up against its concentration gradient, from both sides, by co-transport systems in which the movement of the amino acid into the cell is coupled to the influx of Na⁺ and efflux of K⁺ down their respective electrochemical gradients. The presence of these ion gradient-energized systems, specific for L-glutamate, may account for the exceedingly high intracellular concentration of this metabolically important amino acid in the renal tubule.     

ATP-dependent calcium transport in plasma membrane vesicles from neutrophil leukocytes

Plasma membrane vesicles were prepared from guinea pig peritoneal exudate neutrophils, using nitrogen cavitation to rupture the plasma membrane and differential centrifugation to separate the vesicles. The vesicles were enriched 13.2-fold in (Na+, K+)-ATPase activity and had a cholesterol:protein ratio of 0.15, characteristic of plasma membranes. Contamination of the vesicle preparation with DNA or marker enzyme activities for intracellular organelles was very low. Studies designed to determine vesicle sidedness and integrity indicated that 33% were sealed, inside-out; 41% were sealed, right side-out, and 26% were leaky. The vesicles accumulated 45Ca2+ in a linear fashion for 45 min. The uptake was dependent on the presence of oxalate and MgATP in the incubating medium. Uptake showed a Ka for free Ca2+ of 164 nM and a Vmax of 17.2 nmol/mg . min (based on total protein). GTP, ITP, CTP, UTP, ADP, or AMP supported uptake at rates less than or equal to 11% of ATP. Ca2+ uptake was maximal at pH 7-7.5. Calcium stimulated the hydrolysis of ATP by the vesicles with a Ka for free Ca2+ of 440 nM and Vmax of 17.5 nmol/mg . min (based on total protein). When the Ca2+ uptake rate was based upon those vesicles expected to transport Ca2+ (33% sealed, inside-out vesicles) and Ca2+-stimulated ATPase activity was based upon those vesicles expected to express that activity (26% leaky + 33% sealed, inside-out vesicles), the molar stoichiometry of Ca2+ transported:ATP hydrolyzed was 2.12 +/- 0.12. Calmodulin did not increase either Vmax or Ka for free Ca2+ of the uptake system in the vesicles, even when they were treated previously with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The high affinity of this system for Ca2+, specificity for ATP, physiological pH optimum, and stoichiometry of Ca2+ transported:ATP hydrolyzed suggest that it represents an important mechanism by which neutrophils maintain low levels of cytoplasmic free Ca2+.