Sucrose-dependent H+ transport in plasma-membrane vesicles isolated from sugarbeet leaves (Beta vulgaris L.)

The mechanism of sucrose transport across the plasma membrane (PM) was investigated in membrane vesicles isolated from sugarbeet (Beta vulgaris L.) leaves. In the presence of a membrane potential () generated as a K⁺-diffusion potential, negative inside, sucrose induced a rapid and transient alkalization of the medium. Alkalization was inhibited by carbonyl cyanide m-chlorophenylhydrazone, was specific for the sucrose sugar and was dependent on the sucrose concentration with a K_m of approx. 1 mM. Sucrose-induced alkalization and sucrose transport were inhibited by the sulfhydryl-reactive reagent, p-chloromercuribenzene sulfonic acid, and by the histidine-reactive reagent, diethyl pyrocarbonate. Parallel analysis of sucrose uptake and alkalization indicated that the stoichiometry of sucrose uptake to proton consumed was 11. These results provide clear evidence that the saturable mechanism of sucrose transport across the PM in plants is a coupled H⁺-sucrose symport.Abbreviations and Symbols CCCP carbonyl cyanide m-chlorophenylhydrazone - DEPC diethyl pyrocarbonate - PCMBS p-chloromercuribenzene sulfonic acid - pH pH gradient - membrane potential difference - PM plasma membrane The financial support for a portion of thus study was provided by the Deutsche Forschungsgemeinschaft. We thank Kimberly A. Mitchell for her excellent technical assistance and dedicate this report to the memory of Mr. William A. Dungey.     

Adult rat brain synaptic vesicles I. Isolation and characterization

Synaptic vesicles were isolated from adult rat brain in a form which seemed to be 90–95% pure by chemical and enzymatic assay. The only significant contaminant was the synaptosomal plasma membrane. Contamination with Golgi apparatus and lysosomes appeared limited although some uncertainty remains on this point. The vesicles are sufficiently pure for valid analytical studies to be performed, but the possibility of internal heterogeneity of the preparations must be taken into account.     

Rabbit distal colon epithelium: I. Isolation and characterization of basolateral plasma membrane vesicles from surface and crypt cells

Summary A method has been developed for the simultaneous isolation of basolateral plasma membrane vesicles from surface and crypt cells of rabbit distal colon epithelium by sequential use of differential sedimentation, isopycnic centrifugation and Ficoll 400 barrier centrifugation. The protein yield was high (total 0.81 mg/g mucosa) and surface and crypt cell-derived basolateral membrane fractions have been purified 34- and 9-fold with respect to the homogenate. The pattern of marker enzyme enrichments revealed only minor contamination by subcellular organelles. Latency of ouabain-sensitive (Na⁺, K⁺)-ATPase activity prior and after trypsin treatment of membranes indicated a vesicle configuration of sealed right side-out: sealed inside-out: leaky of approximately 211. The presence of sealed vesicles was also evident from the osmotic sensitivity of thed-[1-¹⁴C] mannitol equilibrium space determined with either fraction. Although considerably different in protein profile, surface and crypt basolateral membranes were similar in cholesterol to phospholipid molar ratio and membrane fluidity as determined by steady-state fluorescence polarization.Stopped-flow light scattering experiments revealed a rather low water permeability of the membranes with a permeability coefficient of 6 m/sec at 35°C, which is one order of magnitude lower than reported for small intestinal plasma membranes. Both membrane fractions have been shown to effectively generate outward uphill potassium ion gradients, a process that is energized by ATP and inhibited by the membrane-permeant cardiacglycoside digitoxin. These characteristics are consistent with the activity of a (Na⁺, K⁺) pump operating in inside-out vesicles.     

Amino acid transport in brush-border-membrane vesicles isolated from human small intestine.

Uptake of L-alanine and L-phenylalanine by purified bursh-border-membrane vesicles isolated from human small intestine was investigated by using a rapid-filtration technique. L-Alanine entered the same osmotically reactive space as D-glucose, indicating that transport into the vesicle rather than binding to the membranes was being observed. The uptake rate for L-alanine was higher in the presence of a Na+ gradient than in the presence of a K+ gradient. In the presence of a Na+ gradient, the lipophilic anion SCN- caused an increase in L-alanine transport, whereas the nearly impermeant SO42- anion decreased the uptake of L-alanine compared with its uptake in the presence of Cl-. The uptake of L-phenylalanine into the brush-border-membrane vesicle was also stimulated by Na+. The results indicate co-transport of Na+ and neutral amino acids inthe human intestinal brush-border membrane.     

Isolation and characterization of gas vesicles from Microcyclus aquaticus

Intact gas vesicles of Microcyclus aquaticus S1 were isolated by using centrifugally accelerated flotation of vesicles and molecular sieve chromatography. Isolated gas vesicles were cylindrical organelles with biconical ends and measured 250×100 nm. The gas vesicle membrane was composed almost entirely of protein; neither lipid nor carbohydrate was detected, although one mole of phosphate per mole of protein was found. Amino acid analysis indicated that the protein contained 54.6% hydrophobic amino acid residues, lacked sulfur-containing amino acids, and had a low aromatic amino acid content. The protein subunit composition of the vesicles was determined by gel electrophoresis in (i) 0.1% sodium dodecyl sulfate at pH 9.0 and (ii) 5 M urea at pH 2.0. The membrane appeared to consist of one protein subunit of MW 50 000 daltons. Charge isomers of this subunit were not detected on urea gels. Antiserum prepared against purified gas vesicles of M. aquaticus S1 cross-reacted with the gas vesicles of all other gas vacuolate strains of M. aquaticus, as well as those of Prosthecomicrobium pneumaticum, Nostoc muscorum, and Anabaena flos-aquae, indicating that the gas vesicles of these widely divergent organisms have some antigenic determinants in common.Abbreviations SDS sodium dodecyl sulfate - MW molecular weight - Tris tris(hydroxymethyl)aminomethane - EDTA disodium ethylenediaminetetraacetic acid - BSA bovine serum albumin - TCA trichloroacetic acid - P _c pressure necessary to collapse gas vesicles     

Isolation and characterization of coated vesicles from filamentous fungi

Coated vesicles have been shown to exist in Neurospora crassa (Ascomycetes) and Uromyces phaseoli (Basidiomycetes) growing germlings. Separation of coated vesicles in both fungi was obtained when the high-speed (100,000g) pellet was fractioned on a Sephacryl S-1000 gel filtration column, according to the procedure of Mueller and Branton. Electron micrographs of negatively stained coated vesicles from fractions of gel filtration show the same striking lattice coated vesicles similar to vertebrate coated vesicles. We observe two major size classes of coated vesicles in both fungi: the larger class (100-180 nm) is similar in size to vertebrate coated vesicles; the smaller class (50-80 nm) is mostly found in both fungi. When examined by SDS-PAGE, the Sephacryl column fractions containing the maximum concentration of electron microscopically visible coated vesicles coincide with the bands of the protein coat reported as clathrin. The protein composition on SDS-PAGE of the coated vesicles indicates a major polypeptide species of 180 kDa and minor 30 to 36 kDa species. Polypeptides of 100 kDa and 64 kDa are also found in the fractions containing coated vesicles.     

Glycodeoxycholate transport in brush border membrane vesicles isolated from rat jejunum and ileum.

The transport of the bile salt, glycodeoxycholate, was studied in vesicles derived from rat jejunal and ileal brush border membranes using a rapid filtration technique. The uptake was osmotically sensitive, linearly related to membrane protein and resembled D-glucose transport. In ileal, but not jejunal, vesicles glycodeoxycholate uptake showed a transient vesicle/medium ratio greater than 1 in the presence of an initial sodium gradient. The differences between glycodeoxycholate uptake in the presence and absence of a Na+ gradient yielded a saturable transport component. Kinetic analysis revealed a Km value similar to that described previously in everted whole intestinal segments and epithelial cells isolated from the ileum. These findings support the existence of a transport system in the brush border membrane that: (1) reflects kinetics and characteristics of bile salt transport in intact intestinal preparations, and (2) catalyzes the co-transport of Na+ and bile salt across the ileal membrane in a manner analogous to D-glucose transport.     

Sodium-dependent methyl 1-thio-beta-D-galactopyranoside transport in membrane vesicles isolated from Salmonella typhimurium.

Membrane vesicles isolated from Salmonella typhimurium G-30 grown in the presence of melibiose catalyze methyl 1-thio-beta-D-galactopyranoside (TMG) transport in the presence of sodium or lithium, as shown initially with intact cells by Stock and Roseman (Stock, J., and Roseman, S. (1971), Biochem. Biophys. Res. Commun. 44, 132). TMG-dependent sodium uptake is also observed, but only when a potassium diffusion potential (interior negative) is induced across the vesicle membrane. Cation-dependent TMG accumulation varies with the electrochemical gradient of protons generated as a result of D-lactate oxidation, and the vesicles catalyze D-lactate-dependent sodium efflux in a manner which is consistent with the operation of a proton-sodium exchange mechanism. Although the stoichiometry between sodium and TMG appears to be 1:1 when transport is induced by a potassium diffusion potential, evidence is presented which indicates that the relationship may exceed unity under certain conditions. The results are explained in terms of a model in which TMG-sodium (lithium) symport is driven by an electrochemical gradient of protons which functions to maintain a low intravesicular sodium or lithium concentration through proton--sodium (lithium) antiport.     

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.     

Siderochromes from Pseudomonas fluorescens. I. Isolation and characterization

Several iron-binding pigments (siderochromes) produced by Pseudomonas fluorescens have been isolated and partially characterized. They include ferribactin and various forms of pyoverdine, as well as some previously unreported compounds. In particular, the existence of ferribactin has been independently confirmed for the first time. Column and thin layer chromatographic procedures have been developed to fractionate, purify, and identify the siderochromes. We find ferribactin to contain nine amino acids, one residue each of glutamine, tyrosine, and glycine, and two each of serine, lysine, and N-hydroxyornithine, rather than 10 as earlier reported. Pyoverdine is a peptide with the same composition as ferribactin except for the absence of glutamine and the substitution of a fluorescent chromophore for tyrosine. Paper electrophoresis reveals an extra ionizable group in ferric pyoverdine relative to pyoverdine or ferribactin which provides that complex a definite cathodic mobility at pH 3. Optical spectra of the pyoverdine fluorescent component indicate that, in conjunction with the two hydroxamate groups, it is involved in the metal ion coordination, conferring on pyoverdine a dramatically increased affinity for Fe(III) relative to ferribactin.     

Phosphate transport into brush-border membrane vesicles isolated from rat small intestine.

Uptake of Pi into brush-border membrane vesicles isolated from rat small intestine was investigated by a rapid filtration technique. The following results were obtained. 1. At pH 7.4 in the presence of a NaCl gradient across the membrane (sodium concentration in the medium higher than sodium concentration in the vesicles), phosphate was taken up by a saturable transport system, which was competitively inhibited by arsenate. Phosphate entered the same osmotically reactive space as D-glucose, which indicates that transport into the vesicles rather than binding to the membranes was determined. 2. The amount of phosphate taken up initially was increased about fourfold by lowering the pH from 7.4 to 6.0.3. When Na+ was replaced by K+, Rb+ or Cs+, the initial rate of uptake decreased at pH 7.4 but was not altered at pH 6.0.4. Experiments with different anions (SCN-,Cl-, SO42-) and with ionophores (valinomycin, monactin) showed that at pH 7.4 phosphate transport in the presence of a Na+ gradient is almost independent of the electrical potential across the vesicle membrane, whereas at pH 6.0 phosphate transport involves the transfer of negative charge. It is concluded that intestinal brush-border membranes contain a Na+/phosphate co-transport system, which catalyses under physiological conditions an electroneutral entry of Pi and Na+ into the intestinal epithelial cell. In contrast with the kidney, probably univalent phosphate and one Na+ ion instead of bivalent phosphate and two Na+ ions are transported together.