Isolation Of Plasma Membrane Vesicles Research Articles

Isolation of Plasma Membrane Vesicles from Mouse Placenta at Term and Measurement of System A and System β Amino Acid Transporter Activity

Placental amino acid transport is essential for optimal fetal growth and development, with a reduced fetal provision of amino acids being implicated as a potential cause of fetal growth restriction (FGR). Understanding placental insufficiency related FGR has been aided by the development of mouse models that have features of the human disease. However, to take maximal advantage of these, methods are required to study placental function in the mouse. Here, we report a method to isolate plasma membrane vesicles from mouse placenta near-term and have used these to investigate two amino acid transporters, systems A and β, the activities of which are reduced in human placental microvillous plasma membrane (MVM) vesicles from FGR pregnancies. Plasma membrane vesicles were isolated at embryonic day 18 by a protocol involving homogenisation, MgCl2 precipitation and centrifugation. Vesicles were enriched 11.3±0.5-fold in alkaline phosphatase activity as compared to initial homogenate, with minimal intracellular organelle contamination as judged by marker analyses. Cytochemistry revealed alkaline phosphatase was localised between trophoblast layers I and II, with intense reaction product deposited on the maternal-facing plasma membrane of layer II, suggesting that vesicles were derived from this trophoblast membrane. System A and system β activity in mouse placental vesicles, measured as Na+-dependent uptake of 14C-methylaminoisobutyric acid (MeAIB) and 3H-taurine respectively confirmed localisation of these transporters to the maternal-facing plasma membrane of layer II. Comparison to human placental MVM showed that system A activity was comparable at initial rate between species whilst system β activity was significantly lower in mouse. This mirrored the lower expression of TAUT observed in mouse placental vesicles. We conclude that syncytiotrophoblast layer II-derived plasma membrane vesicles can be isolated and used to examine transporter function.

Measurement of Hepatobiliary Transport

A major hepatic function involves the removal of xenobiotics from portal blood and their subsequent storage, metabolism, and excretion into bile, or delivery of various products back into the bloodstream. Transport across the liver cell basolateral (or sinusoidal) plasma membrane is a key step in the overall metabolism of many compounds, whereas transport across the apical (or canalicular) plasma membrane into bile is often the principal pathway for their elimination. Although significant progress has been made in the molecular identification of the transport proteins that mediate these processes, much remains to be learned about regulation and physiological integration. The critical limiting factor in studying biliary excretion is that the bile canaliculus is very small (~1 µm in diameter) and is generally inaccessible to sampling by conventional approaches. The bile canalicular membrane is a specialized part of the hepatocellular plasma membrane, such that bile is separated from blood plasma only by the tight junctions that encircle each hepatocyte. Because hepatocyte polarity is rapidly lost during cell isolation, most cell culture models provide only limited information on mechanisms of biliary excretion. Thus, biliary secretion has been studied using four major experimental models: canalicular plasma membrane vesicles, cultured hepatocyte couplets, perfused liver, and in vivo (bile duct-cannulated animals). This unit describes basic methods for collecting bile from anesthetized mice and rats, for carrying out isolated perfused rat liver studies, and for the simultaneous isolation of plasma membrane vesicles derived from the basolateral and canalicular domains of rat liver.

18] Transport and drug binding kinetics in membrane vesicle preparation

This chapter describes the transport and drug binding kinetics in membrane vesicle preparation. It discusses the isolation of plasma membrane vesicles from PC-12 rat pheochromocytoma cells, which express the norepinephrine (NE) transporter. The chapter also highlights how plasma membrane vesicles can be used to study kinetics of substrate transport and drug binding to the transporter. Neurotransmitter transporters are the primary targets of a series of psychoactive compounds. Neurotransmitter transporters are also targets for useful therapeutic drugs. Substances, such as tiagabine, which bind to the γ-aminobutyric acid (GABA) transporter and inhibit GABA reuptake are used as antiepileptic drugs. Binding studies using membrane vesicles isolated from tissues or cells expressing one of these transporters have contributed to understanding of their interaction with transporters. Plasma membrane vesicles isolated from a tissue or from a clonal cell population expressing large amounts of a specific transporter have provided important insights into the driving forces and chemical interconversion associated with transport of molecules across cell membranes.

Phorbol ester-sensitive phospholipase D is mainly localized in the endoplasmic reticulum of BHK cells.

The localization of phorbol ester-sensitive phospholipase D (PLD) in baby hamster kidney cells has been investigated by determining the subcellular distribution of the phosphatidylbutanol produced when the cells are incubated with phorbol 12-myristate 13-acetate and n-butanol. Results derived by isolation of plasma membrane vesicles from intact cells or by subcellular fractionation on a sucrose density gradient suggest the PLD is specific for phosphatidylcholine and its primary site of action is not the plasma membrane but the endoplasmic reticulum.

Isolation of plasma membrane vesicles from the gill epithelium of the crayfish, Orconectes limosus Rafinesque, and properties of the Na +/H + exchanger

1. 1. Membrane vesicles have been isolated from the gill epithelium of the crayfish by differential centrifugation followed by Percoll density gradient centrifugation. 2. 2. In order to characterize the membrane fractions putative “apical” markers like leucine aminopeptidase (EC 3.4.11.2), nucleoside diphosphatase (EC 3.6.1.6) and alkaline phosphatase were assayed. However, their use may be limited by their high solubility. 3. 3. After the separation procedure, less than 20% of their initial activities were recovered in the membrane fractions. 4. 4. Nevertheless, this fractionation procedure led to a splitting of the used marker enzyme activities into two peaks. 5. 5. Most of the activity of the “apical” marker enzymes was found in the less dense part of the Percoll gradient and the unquestionably basolateral Na +,K +-ATPase in the more dense part. 6. 6. An amiloride-sensitive and electroneutral Na +/H +-exchanger, as assayed by acridine orange fluorescence-quench, appeared to be localized mainly in the “apical” membrane fraction, according to its distribution within the Percoll gradient which was opposite to the Na +,K +-ATPase but mainly in cofractionation with the “apical” markers. 7. 7. The activity of the Na +H +-exchanger revealed saturation kinetics with increasing Na + concentration. 8. 8. The corresponding Lineweaver-Burk plot showed a K m of 17mmol/l Na +. 9. 9. With a Hill coefficient of about 1, cooperative effects concerning Na + seem to be excluded. 10. 10. Amiloride inhibits this exchanger with a K i of about 3.5 × 10 −5mol/l.

Isolation of canine neutrophil plasma membranes.

This paper describes the isolation of plasma membrane vesicles formed by nitrogen cavitation of canine neutrophils. Plasma membranes from disrupted cells were separated from other membranes and organelles by Percoll-density gradient centrifugation. Transmission electron microscopic examination of membrane preparations chromatographed on either Sephacryl S-1000 or Sepharose 4B revealed that two populations of plasma membrane vesicles were formed: large (176 +/- 22nm), and small (119 +/- 11nm). Purified large vesicles were separated from Percoll and contaminating cytosol by Sephacryl S-1000 chromatography. Small vesicles were obtained free of Percoll by recavitating purified large vesicles. Problems encountered due to the presence of a soluble 5'-nucleotidase inhibitor also are discussed. Large and small membrane vesicles were separated into adherent and non-adherent populations by affinity chromatography on either concanavalin A-Sepharose or lentil lectin-Sepharose columns.

Isolation of plasma membrane vesicles from rabbit skeletal muscle and their use in ion transport studies.

A method has been developed for the isolation of sealed plasma membrane vesicles from rabbit white skeletal muscle. The final preparation was highly purified as indicated by enrichment of plasma membrane marker enzymes (i.e. ouabain-sensitive (Na+,K+)-ATPase, adenylate cyclase, and acetylcholinesterase). The absence of sarcoplasmic reticulum and mitochondria as contaminants was indicated by the low specific activity of marker enzymes, i.e. Ca2+-ATPase, succinate-cytochrome c reductase, and monoamine oxidase. Thin section and negative staining electron microscopy confirmed the absence of sarcoplasmic reticulum and mitochondrial contamination. The plasma membrane preparation consisted largely of sealed vesicles as observed by electron microscopy and as also demonstrated by latency of enzymic activities, which were unmasked by preincubation with detergent (sodium dodecyl sulfate). Membrane sidedness was estimated from latency of ouabain-sensitive (Na+,K+)-ATPase activity and acetylcholinesterase activity. The latency studies suggest that most of the vesicles are oriented inside out with respect to the orientation of the sarcolemma membrane in the muscle fiber. The inside-out plasma membrane vesicles actively accumulated sodium ions upon addition of ATP. The sodium ions were concentrated greater than 8-fold inside the vesicles and were released upon addition of the ionophore monensin. The sodium ions were taken up in the presence of K+ or NH4+ but not of choline. Uptake was inhibited by low concentrations of vanadate or digitoxin. The Na+ uptake was concomitant with Rb+ efflux. Therefore, the sodium ion transport and the resulting gradients formed appear to have been generated by the ouabain-sensitive (Na+,K+)-ATPase. Batrachotoxin, which opens Na+ channels in excitable tissues, prevents most of the Na+ uptake, suggesting the presence of toxin-activated Na+ channels in these plasma membrane vesicles.

Isolation of plasma membrane vesicles of human platelets by affinity chromatography

A new approach to prepare plasma membrane vesicles (PMV) of human platelets was attempted, utilizing wheat germ aggulutinin (WGA) affinity chromatography. The isolation was much faster than the centrifugal method of Barber and Jamieson and as low as 20 ml of human citrated whole blood may be used as a starting material. Upon characterization of the PMV fraction by electron microscopy and marker enzyme assays, the quality of the fraction was found comparable with the centrifugal method. The yield by this method was approximately two times higher than by the centrifugal method.

Isolation of plasma membrane vesicles, derived from transverse tubules, by selective homogenization of subcellular fractions of frog skeletal muscle in isotonic media

A new technique for isolating fragmented plasma membranes from skeletal muscle has been developed that is based on gentle mechanical disruption of selected homogenate fractions. (Na + + K +)-stimulated , Mg 2+-dependent ATPase was used as an enzymatic marker for the plasma membrane, Ca 2+-stimulated, Mg 2+-dependent ATPase as a marker for sarcoplasmic reticulum, and succinate dehydrogenase for mitochondria. Cell Cell segments in an amber low-speed ( 800 × g) pellet of a frog muscle homogenate were disrupted by repeated gentle shearing with a Polytron homogenizer. Sarcoplasmic reticulum was released into the low-speed supernatant, whereas most of the plasma membrane marker remained in a white, fluffy layer of the sediment, which contained sarcolemma and myofibrils. Additional gentle shearing of the white low-speed sediment extracted plasma membranes in a form that required centrifugation at 100 000 × g for pelleting. This pellet, the fragmented plasma membrane fraction, had a relatively high specific activity of (Na + + K +)-stimulated ATPase compared with the other fractions, but it had essentially no Ca 2+-stimulated ATPase activity and only a small percentage of the succinate dehydrogenase activity of the homogenate. Experimental evidence suggests that the fragmented plasma membrane fraction is derived from delicate transverse tubules rather than from the thicker, basement membrane-coated sarcolemmal sheath of muscle cells. Electron microscopy showed small vesicles lined by a single thin membrane. Hydroxyproline, a characteristic constituent of collagen and basement membrane, could not be detected in this fraction.

Isolation of Plasma Membranes from Corn Roots by Sucrose Density Gradient Centrifugation

An investigation was conducted into the isolation of plasma membrane vesicles from primary roots of corn (Zea mays L., WF9 x M14) by sucrose density gradient centrifugation. Identification of plasma membranes in cell fractions was by specific staining with the periodic-chromic-phosphotungstic acid procedure. Plasma membrane vesicles were rich in K(+)-stimulated ATPase activity at pH 6.5, and equilibrated in linear gradients of sucrose at a peak density of about 1.165 g/cc. It was necessary to remove mitochondria (equilibrium density of 1.18 g/cc) from the homogenate before density gradient centrifugation to minimize mitochondrial contamination of the plasma membrane fraction. Endoplasmic reticulum (NADH-cytochrome c reductase) and Golgi apparatus (latent IDPase) had equilibrium densities in sucrose of about 1.10 g/cc and 1.12 to 1.15 g/cc, respectively. A correlation (r = 0.975) was observed between K(+)-stimulated ATPase activity at pH 6.5 and the content of plasma membranes in various cell fractions. ATPase activity at pH 9 and cytochrome c oxidase activity were also correlated.A major peak of ATPase activity at pH 6.5 was observed at low density in Ficoll after nonequilibrium centrifugation in a combination Ficoll-sucrose gradient. Twenty to forty percent of the vesicles in this ATPase fraction stained positively for plasma membranes, and with equilibrium centrifugation the major portion of the ATPase activity shifted to densities in sucrose which were characteristic of plasma membranes. All major vesicular ATPase activities observed in Ficoll or sucrose contained substantial amounts of plasma membranes. For unknown reasons, mitochondria and plasma membranes equilibrated over a broader density range and at lower peak densities in sucrose as a result of equilibrium centrifugation through Ficoll.