Characterization of right-side-out membrane vesicles rich in (Na,K)-ATPase and isolated from dog kidney outer medulla

When purified on a sucrose gradient, basolateral membranes from dog kidney outer medulla are found to be very rich in (Na,K)-ATPase; about 50% of the membrane protein is comprised of this enzyme. (Na,K)-ATPase activity is activated 3- to 5-fold by detergent treatment, and this has been previously attributed to the impermeable vesicular nature of the membranes. Porcine trypsin inactivates only that fraction of (Na,K)-ATPase activity seen without detergent, consistent with a right-side-out orientation of membrane vesicles; the trypsin sensitivity and detergent activation of [3H]ouabain binding in the presence of Na+ + Mg2+ + ATP or Mg2+ + Pi are also consistent with this hypothesis. Using nearly isosmotic Hypaque density gradient centrifugation a population of impermeable right-side-out membrane vesicles (H1) is separated from a leaky population (H2). (Na,K)-ATPase activity in the H1 population is 20-fold activated by detergent and insensitive to porcine trypsin. The vesicle volume is 2.4 microliters/mg, and monovalent cations passively equilibrate with the intravesicular volume on a time scale of 5-30 min. Very rapid ouabain sensitive 22Na efflux from the vesicles is observed when ATP is photolytically released from intravesicular caged ATP.     

Myelin Composition and Activities of CNPase and Na+, K+-ATPase in Hypomyelinated "pt" Mutant Rabbit

A disorder of CNS myelination was found in paralytic tremor ("pt") rabbits. The condition is inherited in a sex-linked recessive mode. Ultrastructurally, an obvious myelin deficiency with aberration of myelin sheath formation is observed. The yield of myelin isolation was reduced to 20-30% of control. Myelin isolated from 4-week-old "pt" rabbits contained reduced amounts of galactosphingolipids and of several myelin protein markers. Moreover, myelin basic protein, analyzed by two-dimensional gel electrophoresis, showed a deficit in its more basic components. All these facts suggest a delay in myelin maturation. Ganglioside content was increased as well as Na+,K+-ATPase specific activity. 2',3'-Cyclic nucleotide phosphodiesterase (CNPase) specific activity was the same in "pt" as in control myelin but differed by having greater sensitivity to detergent activation.     

Orientation of vesicles isolated from baso-lataral membranes of renal cortex

Baso-lateral membranes were isolated from the canine and porcine kidney cortex by several different methods currently in use. Sidedness of the isolated membrane vesicles was determined by procedures using 1. ouabain-sensitive (Na+K+)ATPase assays in the presence and in the absence of sodium dodecylsulfate or digitoxigenin plus monensin, 2. (Na+, K+, Mg2+)ATPase assays with valinomycin, 3. sialidase accessibility, and 4. binding of hydrophilic and lipophilic cardiac glycosides. The (Na+K+)ATPase activity in the membrane preparation was increased 10-fold of that found in the crude homogenate. Isolated membrane vesicles, prepared by different techniques, were all found to be overwhelmingly of right-side-out orientation;namely, right-side-out = 51-68%, inside-out = 4-13%, and unsealed vesicles = 26-42%. Results of sidedness determinations by different methods showed a good agreement. Thus, predominantly right-side-out oriented vesicles are formed during conventional isolation procedures for membranes of the kidney cortex.     

Effect of lysophosphatidylcholine on the NADH-ferricyanide reductase activity associated with the plasma membranes of corn roots

Plasma membranes from corn roots (Zea mays L.) were isolated by aqueous two-phase partitioning. A fraction enriched in a vanadate-sensitive ATPase showed characteristics of a plasma membrane ATPase. The sidedness of these vesicles was 89% right-side-out, as evaluated by the ATPase latency. A NADH-ferricyanide reductase was associated with these plasma membrane vesicles. The rate of ferricyanide reduction was 1.3 μmol · min⁻¹·mg⁻¹ protein and was strongly enhanced by the addition of lysophosphatidylcholine (LPC). The effect of this detergent on membrane solubilization and reductase activity was particularly studied. This type of detergent treatment revealed two pH optima (7.0 and 5.0) for the reductase activity, which exhibited biphasic kinetics in the absence or presence of the detergent. These data suggest that two or more reductases could be involved. In addition, membrane vesicle solubilization and determination of ATPase and reductase latency were simultanously studied. From these experiments, it is postulated that the reductase, which exhibits an optimum pH at 7.0 and is slightly stimulated by LPC, could be located on the external side of the plasmalemma. In contrast, the reductase at pH 5.0 strongly stimulated by the detergent treatment, is probably located on the internal side of the membrane, such as the catalytic site of ATPase. Finally, a possible direct action of LPC on the enzymes, is discussed.     

Subcellular distribution of 2′,3′-cyclic nucleotide-3′-phosphodiesterase in cat peripheral nerve

Subcellular distribution of 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) in desheathed, saline perfused cat sciatic nerve is reported. CNPase specific activity was enriched in the total particulate (P₂) fraction and was low in the soluble (S₂) fraction. “Light-myelin” floating above the 0.60 M sucrose phase had the highest CNPase activity, 2.5-fold over the crude homogenate (CH). By contrast, enzyme activity in “heavy myelin” floating above the 0.85 M sucrose interface was equal to that of the CH and accounted for only 12% of total activity. CNPase activity in the membranes floating above the 0.25 and 0.60 and 0.85 M sucrose phases comprised nearly 70% of the total CNPase activity. The “light myelin” fraction floating above the 0.60 M sucrose accounted for approx. 51% of the total CNPase activity. SDS-PAGE of membranes individually harvested from above the 0.25 and 0.60 and 0.85 M sucrose phases revealed the presence of myelin-specific proteins (P₀, P₁; and P₂). Electron microscope examination demonstrated the presence of myelin in each membrane fraction. The results of this study show that the majority of CNPase activity is associated with “light myelin” in cat peripheral nerve.     

A new method for the rapid isolation of basolateral plasma membrane vesicles from rat liver. Characterization, validation, and bile acid transport studies

Basolateral plasma membrane vesicles were prepared from rat liver by a new technique using self-generating Percoll gradients. The method is rapid (total spin time of 2.5 h) and protein yields were high (0.64 mg/g of liver). Transmission electron microscopy studies and measurements of marker enzyme activities indicated that the preparation was highly enriched in basolateral membranes and substantially free of contamination by canalicular membranes or subcellular organelles. High total recoveries for protein yield and marker enzyme activities during the fractionation procedure indicated that enzymatic activity was neither lost (inactivation) nor increased (activation). Thus, the pattern of marker enzyme activities found in the membrane preparation truly reflected substantial enrichment in membranes from the basolateral surface. Analysis of freeze-fracture electron micrographs suggested that approximately 75% of the vesicles were oriented "right-side-out." In order to assess the functional properties of the vesicles, the uptake of [3H]taurocholate was studied. In the presence of a Na+ gradient, taurocholate uptake was markedly stimulated and the bile acid was transiently accumulated at a concentration 1.5- to 2-fold higher than that at equilibrium ("overshoot"). In the absence of a gradient but in the presence of equimolar Na+ inside and outside of the vesicle, taurocholate uptake was faster than in the absence of Na+. These findings support a direct co-transport mechanism for the uptake of taurocholate and Na+. Kinetic studies demonstrated that Na+-dependent taurocholate uptake was saturable with a Km of 36.5 microM and a Vmax of 5.36 nmol mg-1 protein min-1. The high yield, enzymatic profile and retention of transport properties suggest that this membrane preparation is well suited for studies of basolateral transport.     

Biochemical Changes in Central Nervous System Membranes in Experimental Allergic Encephalomyelitis

Biochemical and morphological studies of myelin subfractions were undertaken on Lewis rats during the early stage of the development of experimental allergic encephalomyelitis (EAE). Myelin subfractions, obtained by sucrose density gradient centrifugation at 10 days post-induction, were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and assayed for 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity. Aliquots were processed for electron microscopic analysis. When comparing the myelin subfractions of EAE-affected animals with those of controls, differences were observed only in the light fractions, i.e., a decrease in the specific activity of CNPase and in the percentage of basic proteins relative to the total proteins of the fraction. This decrease was also evident in the basic protein/proteolipid protein ratio which is frequently used in the literature. In addition, electron microscopic observations demonstrated strong differences in the morphology of the same fraction. These findings suggest that the light fraction is the most sensitive in the early stages of the disease and must play a key role in demyelinating processes.     

The intactness and orientation of acetylcholine receptor-rich membrane from Torpedo californica electric tissue

By a mild and highly reproducible fractionation of Torpedo californica electric tissue, we prepared membrane which was 30 times enriched in nicotinic acetylcholine receptor (AChR). This preparation was neither alkali-stripped nor reconstituted and consequently contained nu (43-kDa protein), which is associated with the cytoplasmic aspect of the receptor. We tested this membrane for the presence of sealed vesicles and determined the orientation of these vesicles by combining three methods. Two of these methods were based on the accessibilities, in the presence and absence of detergent, of the extracellular acetylcholine binding site to alpha-bungarotoxin and of the intracellular nu to trypsin. These two methods are specific for AChR-containing membrane. The third method was morphometry of electron micrographs, by which we estimated the proportion of sequestered membrane. These methods taken together indicated that approximately 45% of the AChR-containing membrane was in the form of leaky vesicles or sheets, 33% was sealed right-side-out vesicles, 11% was sealed inside-out vesicles, and 11% was sequestered within multilamellar or multivesicular vesicles. The complexity of this membrane needs to be taken into account in sidedness studies of the AChR.     

Localization of 2′,3′-cyclic nucleotide 3′-phosphodiesterase in human erythrocyte membranes

The location of 2′,3′-cyclic nucleotide 3′-phosphodiesterase in human erythrocyte membranes was determined. This was accomplished by comparing the enzyme's accessibility with that of glyceraldehyde-3-phosphate dehydrogenase (cytoplasmic surface marker) and acetylcholinesterase (external marker) in sealed and unsealed ghosts and normal and inverted membrane vesicles. The results showed that 2′,3′-cyclic nucleotide 3′-phosphodiesterase, like glyceraldehyde-3-phosphate dehydrogenase, meets several criteria for an inner (cytoplasmic) membrane location: (1) the enzyme was accessible to substrate in unsealed ghosts and inside-out vesicles but not in sealed or right-side-out vesicles, (2) latent activity in sealed ghosts could be exposed with detergent (Triton X-100), (3) activity in unsealed ghosts was gradually sequestered during resealing and could be re-exposed with detergent, and (4) the enzyme was susceptible to trypsin proteolysis only in unsealed ghosts. These results demonstrate that the active site of 2′,3′-cyclic nucleotide 3′-phosphodiesterase faces the cytoplasm of erythrocytes and that the enzyme may not span the lipid bilayer of the membrane. The localization of the phosphodiesterase on the inner membrane surface of erythrocytes suggests that the similar enzyme of myelin may be embedded within the major dense line of the compact lamellae.     

Active Ca2+ transport in plasma membranes of branchial epithelium of the North-American eel, Anguilla rostrata LeSueur

A branchial epithelial membrane fraction, more than 20-fold enriched in Na+/K+-ATPase activity when compared with the crude homogenate of the tissue, was obtained from adult freshwater American eels. In a membrane vesicle preparation that consisted of 33% inside-out, 23% right-side-out and 44% leaky vesicles, the accumulation of 45Ca2+ was stimulated by ATP, but not by ADP. Accumulation of 45Ca2+ was prevented when vesicles were pretreated with detergent or the Ca2+ ionophore A23187; Ca2+ efflux was observed when the ionophore was added to actively 45Ca2+-loading vesicles. Oxalate did not affect Ca2+ accumulation in these vesicles. Kinetic analysis of the Ca2+-transport process by an Eadie-Hofstee plot revealed that the process is homogeneous; its kinetic parameters are a K0.5 for Ca2+ of 0.053 microM and a Vmax of 2.25 nmol Ca2+/min.mg protein (at 37 degrees C). The calmodulin dependency of this Ca2+ transporting process was shown by the inhibitory action of calmodulin antagonists and by the stimulatory effect of calmodulin repletion after EGTA treatment of the membranes. We conclude that an ATP-energized Ca2+ pump is present in the plasma membranes of branchial epithelium, that resembles the Ca2+ pumps of e.g. mammalian intestinal or renal plasma membranes, and propose its involvement in branchial Ca2+-uptake from the water.     

An immunologic probe for a defined region of the myelin proteolipid

Antiserum has been prepared against an isolated polypeptide fragment, designated BPS4, which comprises residues 181-211 of the bovine myelin proteolipid. The antiserum recognizes the intact bovine proteolipid protein but not several other polypeptide fragments within the molecule, nor the myelin basic protein, thus demonstrating specificity of the antiserum. In a competitive enzyme-linked immunosorbent assay, both the major proteolipid and the DM 20 bands observed on sodium dodecyl sulfate-polyacrylamide gels reacted equally well with the antiserum, indicating that the BPS4 segment is present in both molecular species. The rat myelin proteolipid protein cross-reacted with antiserum against the intact bovine protein but showed minimal cross-reactivity with the antiserum against the bovine BPS4 fragment. This was demonstrated in parallel experiments using three types of preparations, namely, sodium dodecyl sulfate-solubilized myelin, delipidated myelin, and isolated proteolipid apoprotein. The difference between the bovine and rat proteins, which presumably reflects amino acid sequence differences, is thus detectable by the antiserum against the polypeptide fragment but not by the antiserum against the intact protein. Isolated bovine myelin membranes did not bind the antiserum in the absence of detergent or without delipidation. On the other hand, in vesicles reconstituted with the intact bovine apoprotein, the BPS4 segment was oriented on the exterior face of the liposome where it was capable of binding antibody and was susceptible to Pronase digestion.     

Localization of enzymes involved in polyphosphoinositids metabolism on the cytoplasmic surface of the human erythrocyte membrane.

1. Impermeable inside-out and right-side-out vesicles were prepared from membranes of human erythrocytes. During preparation of each kind of impermeable vesicle, permeable vesicles were also obtained. 2. Incubation of vesicles with [gamma-32P]ATP at 37 degrees C for periods of up to 1 hr did not change the topography or the permeability of the vesicles. 3. Vesicles incorporated labeled phosphate from [gamma-32P]ATP into both diphosphoinositide and triphosphoinositide, but impermeable inside-out vesicles incorporated significantly more nuclide than did right-side-out vesicles. 4. Permeable vesicles derived during the preparation of inside-out vesicles were as active as impermeable inside-out vesicles in the incorporation of labeled phosphate into the polyphosphoinositides. However, permeable vesicles derived during the preparation of right-side out vesicles were not as active. 5. Impermeable right-side-out vesicles, treated with 0.01 percent saponin, incorporated labeled phosphate into the polyphosphoinositides at a level comparable to that of impermeable inside-out vesicles. 6. These data show that the enzymes involved in metabolism of diphosphoinositide and triphosphoinositide are located on the cytoplasmic surface of the erythrocyte membrane.     

Oriented reconstitution of red cell membrane proteins and assessment of their transmembrane disposition by immunoquenching of fluorescence

The two major membrane glycoproteins of human red cells, glycophorin and band 3, the anion exchange protein, were isolated from cells exofacially labeled with fluorescein and reconstituted into vesicles with defined transmembrane disposition. Uniform orientation of polypeptides was accomplished by two procedures: Vesicles with single protein units were obtained by a one-step dilution of a protein/detergent suspension with a vast excess of phospholipid. Vesicles with uniform orientation of protein were selected by affinity chromatography on derivatized Sepharoses (organomercurial, wheat germ agglutinin, aminoethyl or diethylaminoethyl). Vesicles with multiple protein units with uniform orientation were generated by vectorial immobilization of detergent solubilized proteins on the above affinity matrices and in situ formation of proteoliposomes by detergent substitution for phospholipid. The proteoliposomes were released from the column by addition of excess free ligand. The orientation of band 3 and glycophorin in the reconstituted vesicles was first assessed by immunofluorescence quenching, using anti-fluorescein antibodies, to quantitatively quench fluorescein residues exposed on the outer surface of vesicles. Further assessment was achieved by chromatographing the vesicles through various affinity and ionic matrices. Vesicle populations of higher than 90% homogeneity in protein orientation (right-side-out or inside-out) were obtained with both procedures. The above methods provide a convenient experimental tool for the oriented reconstitution of proteins and the evaluation of their transmembrane disposition.     

Rat liver canalicular membrane vesicles. Isolation and topological characterization

Canalicular plasma membranes were isolated from rat liver homogenates using nitrogen cavitation and calcium precipitation methods. Compared with homogenates, the membranes were enriched 55- to 56-fold in gamma-glutamyltransferase, aminopeptidase M, and alkaline phosphatase activities and showed very low enrichment in markers of other membranes. By electron microscopy, the membrane preparation contained neither junctional complexes nor contaminating organelles and consisted exclusively of vesicles. The presence of vesicles was also evident from the osmotic sensitivity of D-[6-3H]glucose uptake into the membrane preparation. Antisera obtained from rabbits immunized with highly purified rat kidney gamma-glutamyltransferase inhibited the transferase activity of intact or Triton X-100-solubilized membranes by 45-55%. Treatment of vesicles with anti-gamma-glutamyltransferase antisera and anti-rabbit IgG antisera increased the apparent density of the membranes during sucrose density gradient centrifugation. gamma-Glutamyltransferase and aminopeptidase M activities were selectively removed from the vesicles by limited proteolysis with papain without changing the intravesicular space or alkaline phosphatase activity of the membranes. Specific binding of anti-gamma-glutamyltransferase antibody to the outer surface of isolated hepatocytes was observed as measured by the antisera and 125I-labeled protein A; binding followed saturation kinetics with respect to antibody concentration. These data indicate that the isolated canalicular membrane vesicles are exclusively oriented right-side-out and that gamma-glutamyltransferase and aminopeptidase M are located on the luminal side of rat liver canalicular plasma membranes.     

Isolation of basolateral and brush-border membranes from the rabbit kidney cortex: Vesicle integrity and membrane sidedness of the basolateral fraction

A rapid and reproducible method has been developed for the simultaneous isolation of basolateral and brush-border membranes from the rabbit renal cortex. The basolateral membrane preparation was enriched 25-fold in (Na⁺ + K⁺)-ATPase and the brush-border membrane fraction was enriched 12-fold in alkaline phosphatase, whereas the amount of cross-contamination was low. Contamination of these preparations by mitochondria and lysosomes was minimal as indicated by the low specific activities of enzyme markers, i.e., succinate dehydrogenase and acid phosphatase. The basolateral fraction consisted of 35–50% sealed vesicles, as demonstrated by detergent (sodium dodecyl sulfate) activation of (Na⁺ + K⁺)-ATPase activity and [³H]ouabain binding. The sidedness of the basolateral membranes was estimated from the latency of ouabain-sensitive (Na⁺ + K⁺)-ATPase activity assayed in the presence of gramicidin, which renders the vesicles permeable to Na⁺ and K⁺. These studies suggest that nearly 90% of the vesicles are in a right-side-out orientation.     

Subcellular localization of the BtpA protein in the cyanobacterium Synechocystis sp. PCC 6803.

Photosystem I is a large pigment-protein complex embedded in the thylakoid membranes of chloroplasts and cyanobacteria. In the cyanobacterium Synechocystis sp. PCC 6803, the btpA gene encodes a 30-kDa polypeptide. Mutations in this gene significantly affect accumulation of the reaction center proteins of photosystem I in Synechocystis 6803 [Bartsevich, V. V. & Pakrasi, H. B. (1997) J. Biol. Chem. 272, 6372-6378]. We describe here the intracellular localization of the BtpA protein. Immunolocalization in Synechocystis 6803 cells demonstrated that the BtpA protein is tightly associated with the thylakoid membranes. Phase fractionation in the detergent Triton X-114 indicated that BtpA is a peripheral membrane protein. To determine which surface of the thylakoid membrane BtpA is exposed to, we used a two-phase polymer partitioning technique to develop a novel method to isolate inside-out and right-side-out thylakoid vesicles from Synechocystis 6803. Treatments of such vesicles with different salts and protease showed that the BtpA protein is an extrinsic membrane protein which is exposed to the cytoplasmic face of the thylakoid membrane.     

Stereospecificity of NADH-ferricyanide reductase is a convenient marker for the endoplasmic reticulum of plant cells

The stereospecificity of NADH-ferricyanide reductase and NADH-cytochrome c reductase in the endoplasmic reticulum (ER) for the α-hydrogen on the nicotinamide ring is presented as a very sensitive and convenient assay to detect ER contamination in preparations of membranes lacking α-specific NADH-acceptor reductase, such as the plasma membrane and the tonoplast. The experimental details of the assay are given and the limitations explored (time-course, amount of protein, possible side reactions, speed, reproducibility, etc.). The NADH-ferricyanide reductase activity of plasma membranes from spinach and sugarbeet leaf was completely β-specific and always showed a latency (increase upon addition of Triton X-100), whereas the α-specificity in the ER was non-latent. This is consistent with the presence of mainly right-side-out vesicles in preparations of plasma membranes with the binding site for NADH and ferricyanide on the inner, cytoplasmic surface. In contrast, right-side-out ER vesicles have the binding site on the outer, cytoplasmic surface. The addition of as little as 1% of the α-specific ER (on an NADH-ferricyanide activity basis) to the spinach leaf plasma membrane could be detected with the stereospecificity assay. Wheat root plasma membrane showed some α-specificity (in addition to β-specificity) which was probably due to ER contamination since the activity was non-latent. The stereospecificity assay is also shown to be useful in monitoring the separation of tonoplast vesicles from ER vesicles by countercurrent distribution of a light microsomal fraction. It follows that the NADH-acceptor reductase activities in preparations of plasma membrane and tonoplast are due to distinct enzymes characteristic for those membranes.     

The membrane-bound domain of the phosphotransferase enzyme IImtl of Escherichia coli constitutes a mannitol translocating unit

The orientation of the mannitol binding site on the Escherichia coli phosphotransferase enzyme IImtl in the unphosphorylated state has been investigated by measuring mannitol binding to cytoplasmic membrane vesicles with a right-side-out and inside-out orientation. Enzyme IImtl is shown to catalyze facilitated diffusion of mannitol at a low rate. At equilibrium, bound mannitol is situated at the periplasmic side of the membrane. The apparent binding constant is 40 nM for the intact membranes. Solubilization of the membranes in detergent decreases the affinity by about a factor of 2. Inside-out membrane vesicles, treated with trypsin to remove the C-terminal cytoplasmic domain of enzyme IImtl, showed identical activities. These experiments indicate that the translocation of mannitol is catalyzed by the membrane-bound N-terminal half of enzyme IImtl which is a structurally stable domain.     

Interaction of myelin basic protein and 2′,3′-cyclic nucleotide phosphodiesterase with mitochondria

The content and distribution of myelin basic protein (MBP) isoforms (17 and 21.5 kDa) as well as 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) were determined in mitochondrial fractions (myelin fraction, synaptic and non-synaptic mitochondria) obtained after separation of brain mitochondria by Percoll density gradient. All the fractions could accumulate calcium, maintain membrane potential, and initiate the opening of the permeability transition pore (mPTP) in response to calcium overloading. Native mitochondria and structural contacts between membranes of myelin and mitochondria were found in the myelin fraction associated with brain mitochondria. Using Western blot, it was shown that addition of myelin fraction associated with brain mitochondria to the suspension of liver mitochondria can lead to binding of CNPase and MBP, present in the fraction with liver mitochondria under the conditions of both closed and opened mPTP. However, induction of mPTP opening in liver mitochondria was prevented in the presence of myelin fraction associated with brain mitochondria (Ca²⁺ release rate was decreased 1.5-fold, calcium retention time was doubled, and swelling amplitude was 2.8-fold reduced). These results indicate possible protective properties of MBP and CNPase.     

The toolbox of vesicle sidedness determination

Vesicles prepared from cellular plasma membranes are widely used in science for different purposes. The outer membrane leaflet differs from the inner membrane leaflet of the vesicle, and during vesicle preparation procedures two types of vesicles will be generated: right-side-out vesicles, of which the outer leaflet is topologically equivalent to the outer monolayer of the cellular plasma membrane, and inside-out vesicles. Because two populations of vesicles exist, sidedness information of the vesicle preparation is indispensable. This note focuses on the ins and outs of sidedness determination of vesicles and compares various methodologies used to establish this ratio.