Beta-alanine transport in synaptic plasma membrane vesicles from rat brain. Efflux, exchange and stoichiometry

The efflux and exchange of beta-alanine were studied in synaptic plasma membrane vesicles from rat brain. The mechanism of beta-alanine translocation has been probed by comparing the ion dependence of net efflux to that of exchange. Dilution-induced efflux requires the simultaneous presence of internal sodium and chloride ions while influx is dependent on the presence of these two ions on the outside [Zafra, F., Aragón, M. C., Valdivieso, F. and Giménez, C. (1984) Neurochem Res. 9, 695-707]. These data show that the release of beta-alanine occurs via the carrier system and that it is cotransported with sodium and chloride ions. beta-Alanine efflux from the membrane vesicles is stimulated by external beta-alanine. This exchange does not require external sodium and chloride but it is dependent on the external concentration of beta-alanine. Half-maximal stimulation is obtained at a beta-alanine concentration similar to the Km for beta-alanine influx. Results of the direct measurements of the coupling of sodium and chloride to the transport of beta-alanine by using a kinetic approach allow us to propose a stoichiometry for the translocation cycle catalyzed by the beta-alanine transporter of three sodium ions and one chloride ion per beta-alanine zwitterion. To account for all the observed effects of external ions, beta-alanine concentrations and membrane potential on beta-alanine influx and efflux, a kinetic model of the Na+/Cl-/beta-alanine cotransport system is discussed.     

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.     

Passive calcium influx by plasma membrane vesicles isolated from rat liver

Passive Ca2+ influx independent of ATP addition to the incubation medium, took place in plasma membrane vesicles isolated from rat liver. The rate of Ca2+ influx was found to depend on the concentration of added Ca2+, and on the incubation temperature, and was inhibited by La3+, Hg2+ and by p-chloromercuribenzoate. Influx was not blocked by calcium channel blockers, or affected by a range of uncouplers. Addition of the Ca2+ ionophore A23187 to vesicles that had taken up the ion induced a rapid efflux of Ca2+ especially when EGTA also was added to the incubation medium. A number of divalent cations inhibited Ca2+ influx. The vesicles could be frozen and stored overnight with little loss in activity. The kinetics of Ca2+ influx could be related to that which occurs in the unstimulated perfused rat liver. The data suggest that the plasma membrane vesicle preparation may be useful for further studies on the basal liver cell Ca2+ influx system in vitro.     

L-alanine transport by systems A and ASC in plasma membrane vesicles isolated from Ehrlich cells.

Plasma membrane vesicles were prepared from Ehrlich cells using two-phase system compartmentation. These vesicles accumulated L-alanine mainly by means of Na(+)-dependent transport systems A and ASC. The kinetic of both transport systems could be elucidated by specific inhibition of system A with methyl-aminoisobutyric acid.     

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.     

Amino acid uptake in plasma membrane vesicles isolated from proliferating tumor cells and tissues

Summary The transport of L-alanine, a natural substrate of system A, across plasma membrane vesicle preparations has been studied in the early stages of rat DENA-PH hepato-carcinogenesis and in a very undifferentiated rat ascites hepatoma cell line (Yoshida AH-130) in the exponential and stationary phase of growth.Kinetic analyses indicated an increase of the V_max value in DENA-PH-treated rats 30 h after partial hepatectomy as well as in exponential growing Yoshida ascites cells. In DENA-PH-treated rats the Km value was drastically reduced 7 and 60 days after surgery, when enzyme-altered hyperplastic and preneoplastic lesions were present in rat liver. Drastically reduced Km values were also found in Yoshida ascites cells.The results suggest that an altered alanine transporter might take place in liver plasma membranes from carcinogen-treated rats. This appears to occur also in an established tumor cell line, grown in vivo.Abbreviations AAF 2-acetylaminofluorene - DENA diethylnitrosamine - PH partial hepatectomy - PMSF phenylmethanesulfonyl fluoride     

Depolarization-induced release of glycine and beta-alanine from plasma membrane vesicles derived from rat brain synaptosomes

Glycine and beta-alanine actively loaded into brain synaptic plasma membrane vesicles were released into the external medium by using the classical depolarization agents high K+ and veratridine. This release occurs via a Ca2+-independent process. Measurements of membrane depolarization using tetraphenylphosphonium uptake show a close correlation between changes in the membrane potential and stimulation of the efflux process. Results shown herein and previously reported by our group (Aragón, M.C. and Giménez, C. (1986) Biochim. Biophys. Acta 855, 257-264; Agulló, L., Jiménez, B., Aragón, M.C. and Giménez, C. (1986) Eur. J. Biochem. 159, 611-617), suggest that the glycine and beta-alanine transport systems in synaptic plasma membranes are susceptible of modulation by changes in ionic fluxes and hence in the membrane potential, similar to those occurring during depolarization and repolarization.     

In vitro reconstitution of exocytosis from plasma membrane and isolated secretory vesicles

We describe the reconstitution of exocytotic function through recombination of purified cortical secretory vesicles (CVs) and plasma membrane from sea urchin eggs. CVs were dislodged from a cell surface complex preparation by gentle homogenization in an isotonic dissociation buffer, and purified by differential centrifugation. CV-free plasma membrane fragments were obtained by mechanically dislodging CVs from cortical lawn (CL) preparations with a jet of CL isolation buffer. This procedure produced a "plasma membrane lawn" preparation, consisting of plasma membrane fragments attached via their vitelline layer (an extracellular glycocalyx) to a polylysine-coated microscope slide. When freshly prepared CVs were incubated with plasma membrane lawns, CVs reassociated with the cytoplasmic face of the plasma membrane, forming an exocytotically competent, reconstituted cortical lawn (RL). Exocytosis in RLs was monitored by phase-contrast microscopy, and quantitated with a sensitive microphotometric assay. Half-maximal exocytosis in RLs occurred at 18.5 microM free Ca2+; half-maximal exocytosis in control lawns occurred at 5.7 microM free Ca2+. Greater than 90% of the purified CVs that were not attached to a plasma membrane lawn remained intact when bathed in a buffer containing millimolar Ca2+. This result excluded the possibility that Ca2+-triggered CV lysis was responsible for our observations, and confirmed that the association of CVs with the plasma membrane was required for exocytosis in RLs. Evidence that the Ca2+-stimulated release of CV contents in CLs and RLs is the in vitro equivalent of exocytosis was obtained with an immunofluorescence-based vectorial transport assay, using an antiserum directed against a CV content protein: stimulation of RLs or partially CV-depleted CLs with Ca2+ resulted in fusion of the CV and plasma membranes, and the vectorial transport of CV contents from the cytoplasmic to the extracytoplasmic face of the egg plasma membrane.     

Epidermal growth factor stimulates diacylglycerol kinase in isolated plasma membrane vesicles from A431 cells

We have examined the effect of epidermal growth factor(EGF) on three kinds of kinases activities, phosphatidylinositol(PI) kinase, phosphatidylinositol 4-phosphate[PI(4)P] kinase and diacylglycerol(DG) kinase that make important roles in the regulation of inositol phospholipids metabolism. When isolated plasma membrane vesicles from A431 cells were incubated at 30 degrees C with [gamma-32P]ATP and exogenously added DG, EGF enhanced the activity of DG kinase approximately 2-fold. This stimulation is found to be dose-dependent with a half maximal activation at 1 nM. In this case, EGF increased Vmax without changing Km Value for ATP or DG. Although this activation was observed in the absence of detergent, it was more evident when membrane vesicles were treated with 1 mM deoxycholate. Interestingly, the effect of EGF was only detected in magnesium containing medium. The use of manganese instead of magnesium diminished the stimulatory effect in either condition, presence or absence of deoxycholate. On the other hand, the stimulation of PI kinase or PI(4)P kinase activity was not caused by EGF. These results suggest that DG kinase activation by EGF makes important roles in cellular responses leading to cell growth.     

Glucose transport activity in isolated plasma membrane vesicles from Saccharomyces cerevisiae

Purified plasma membranes prepared from yeast cells by mechanical agitation with glass beads exhibit no detectable sugar transport activity. However, the addition of phospholipid (asolectin) liposomes to the purified plasma membranes followed by freezing, thawing, and brief sonication produces membrane vesicles which exhibit D-glucose-specific transport activity. The characteristics of zero trans, equilibrium exchange, and influx counterflow exhibited by the membrane vesicles are similar to those of intact cells.     

Pulsed-laser creation and characterization of giant plasma membrane vesicles from cells

Femtosecond-pulsed laser irradiation was found to initiate giant plasma membrane vesicle (GPMV) formation on individual cells. Laser-induced GPMV formation resulted from intracellular cavitation and did not require the addition of chemical stressors to the cellular environment. The viscosity, structure, and contents of laser-induced GPMVs were measured with fluorescence microscopy and single-particle tracking. These GPMVs exhibit the following properties: (1) GPMVs grow fastest immediately after laser irradiation; (2) GPMVs contain barriers to free diffusion of incorporated fluorescent beads; (3) materials from both the cytoplasm and surrounding media flow into the growing GPMVs; (4) the GPMVs are surrounded by phospholipids, including phosphatidylserine; (5) F-actin is incorporated into the vesicles; and (6) caspase activity is not essential for GPMV formation. The effective viscosity of 65 nm polystyrene nanoparticles within GPMVs ranged from 32 to 434 cP. The nanoparticle diffusion was commonly affected by relatively large, macromolecular structures within the bleb.     

Biochemical characterization and membrane fluidity of membranous vesicles isolated from boar seminal plasma

Mammalian seminal plasma contains membranous vesicles (MV), which differ in composition and origin. Among these particles, human prostasomes and equine prostasome-like MV have been the most studied. The aim of the present work is to characterize the biochemical composition and membrane fluidity of MV isolated from boar seminal plasma. The MV from boar seminal plasma were isolated by ultracentrifugation and further purification by gel filtration on Sephadex G-200. The MV were examined by electron microscopy (EM), amount of cholesterol, total phospholipid, protein content, and phospholipid composition were analyzed. Membrane fluidity of MV and spermatozoa were estimated from the electron spin resonance (ESR) spectra of the 5-doxilstearic acid incorporated into the vesicle membranes by the order parameter (S). The S parameter gives a measure of degree of structural order in the membrane and is defined as the ratio of the spectral anisotropy in the membranes to the maximum anisotropy obtained in a rigidly oriented system. The S parameter takes into consideration that S = 1 for a rapid spin-label motion of about only one axis and S = 0 for a rapid isotropic motion. Intermediate S values between S = 0 and S = 1 represents the consequence of decreased membrane fluidity. The EM revealed the presence of bilaminar and multilaminar electron-dense vesicles. Cholesterol to phospholipid molar ratio from the isolated MV was 1.8. Phospholipid composition showed a predominance of sphingomyelin. The S parameter for porcine MV and for boar spermatozoa was 0.73 +/- 0.02 and 0.644 +/- 0.008, respectively, with the S for MV being greater (p < 0.001) than the S for spermatozoa. The high order for S found for boar MV was in agreement with the greater cholesterol/phospholipids ratio and the lesser ratio for phosphatidylcholine/sphingomyelin. Results obtained in the present work indicate that MV isolated from boar semen share many biochemical and morphological characteristics with equine prostasome-like MV and human prostasomes. The characteristics of the porcine MV of the seminal plasma, however, differed from those of boar sperm plasma membranes.     

Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.     

Symport of proton and sucrose in plasma membrane vesicles isolated from spinach leaves

The mechanism of sucrose transport was investigated in plasma membrane (PM) vesicles isolated from spinach (Spinacia oleracea L.) leaves. PM vesicles were isolated by aqueous two-phase partitioning and were equilibrated in pH 7.8 buffer containing K⁺. The vesicles rapidly accumulated sucrose in the presence of a transmembrane pH gradient (ΔpH) with external pH set at 5.8. The uptake rate was slow at pH 7.8. The K⁺-selective ionophore, valinomycin, stimulated uptake in the presence of a ΔpH, and the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), greatly inhibited ΔpH-dependent sucrose uptake. Addition of sucrose to the vesicles resulted in immediate alkalization of the medium. Alkalization was stimulated by valinomycin, was abolished by CCCP, and was sucrose-specific. These results demonstrate the presence of a tightly coupled H⁺/sucrose symporter in PM vesicles isolated from spinach leaves.     

Stoichiometry of sodium- and chloride-coupled glycine transport in synaptic plasma membrane vesicles derived from rat brain

The stoichiometric properties of the glycine transporter were studied in synaptic plasma membrane vesicles from rat brain. The present results, together with previous data from our laboratory, allow us to suggest a stoichiometry of 2 Na+ and 1 Cl- per glycine zwitterion for the translocation cycle catalyzed by the glycine carrier. We propose a kinetic model with an ordered mechanism for the binding/debinding of solutes.     

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.     

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.