Plasma membranes from Candida tropicalis grown on glucose or hexadecane. I. Isolation, identification and purification.

Plasma membranes from Candida tropicalis grown on glucose or hexadecane were isolated using a method based on the difference in surface charge of mitochondria and plasma membranes. After mechanical disruption of the cells, a fraction consisting of mitochondrial and plasma membrane vesicles was obtained by differential centrifugation. Subsequently the mitochondria were separated from the plasma membrane vesicles by aggregation of the mitochondria at a pH corresponding to their isoelectric point. Additional purification of the isolated plasma membrane vesicles was achieved by osmolysis. Surface charge densities of mitochondria and plasma membranes were determined and showed substrate-dependent differences. The isolated plasma membranes were morphologically characterized by electron microscopy and, as a marker enzyme, the activity of Mg2+-dependent ATPase was determine. By checking for three mitochondrial marker enzymes the plasma membrane fractions were estimated to be 94% pure with regard to mitochondrial contamination.     

Receptors for gonadotropins and prostaglandins in lysosomes of bovine corpora lutea.

The total mitochondrial fraction of bovine corpus luteum specifically bound [³H]prostaglandin (PG) E₁, [³H] PGF_2α, and ¹²⁵I-labeled human lutropin (hLH) despite very little 5′-nucleotidase activity, a marker for plasma membranes. Since the total mitochondrial fraction isolated by conventional centrifugation techniques contains both mitochondria and lysosomes, it was subfractionated into mitochondria and lysosomes to ascertain the relative contribution of these fractions to the binding. Subfractionation resulted in an enrichment of cytochrome c oxidase (a marker for mitochondria) in mitochondria and of acid phosphatase (a marker for lysosomes) in lysosomes. The lysosomes exhibited little or no contamination with Golgi vesicles, rough endoplasmic reticulum, or peroxisomes as assessed by their appropriate marker enzymes. Subfractionation also re ulted in [³H] PGE₁, [³H] PGF_2α, and ¹²⁵I-labeled hLH binding enrichment with respect to homogenate in lysosomes but not in mitochondria. The lysosomal binding enrichment and recovery were, however, lower than in plasma membranes. The ratios of marker enzyme to binding, an index of organelle contamination, revealed that plasma membrane and lysosomal receptors were intrinsic to these organelles. Freezing and thawing had markedly increased lysosomal binding but had no effect on plasma membrane binding. Exposure to 0.05% Triton X-100 resulted in a greater loss of plasma membrane compared to lysosomal binding. In summary, the above results suggest that lysosomes, but not mitochondria, in addition to plasma membranes, intrinsically contain receptors for PGs and gonadotropins. Furthermore, lysosomes overall contain a greater number of PGs and gonadotropin receptors compared to plasma membranes and these receptors are associated with the membrane but not the contents of lysosomes.     

Separation of dense, polysaccharide-containing vesicles from secreting, cultured oat cells. Characterization of a putative secretory vesicle fracton

Suspension cultured oat (Avena sativa L. cv. Garry) cells, which secrete polysaccharides into the medium, were used as starting material for analyses of Golgi-derived vesicle membranes and plasma membranes isolated during cell fractionation. Vesicles collected by a procedure employing ultrafiltration followed by ultracentrifugation into a sucrose step gradient exhibited an equilibrium density of 1.27 g cm^?3 when run on continuous sucrose gradients, a feature which is most likely attributable to the high concentration of enclosed polysaccharides. Brief sonication lowered the density of these vesicles to about 1.15 g cm^?3, as judged from the coincidence of the protein peak and the marker enzymes for Golgi [Triton-stimulated UDPase, cold-storage IDPase (EC 3.6.1.6)] and plasma membrane [vanadate-inhibited K⁺, Mg²⁺-ATPase (EC 3.6.1.3)]. Sonication of these vesicles also greatly diminished the amount of detectable polysaccharide observed in a colorimetric assay for sugars. Fractionation of a plasma membrane-enriched preparation from these cells on continuous sucrose gradients showed the major protein peak and the peak activity for the plasma membrane marker at 1.17 g cm^?3, however, there was also significant overlap with a mitochondrial [cytochrome c oxidase (EC 1.9.3.1)] peak at 1.18 g cm^?3, Smaller peaks of the Golgi markers were seen at 1.14 g cm^?3. Analyses of marker enzymes for ER and mitochondria (EC 1.6.99.3) showed little contamination of the membranes of presumptive secretory vesicles from these sources, and the lack of significant vanadate-insensitive ATPase activity in the density range from 1.13–1.18 g cm^?3 in either fractionation scheme suggests that these membranes do not include material from the tonoplast. The coincidence of markers for Golgi and plasma membrane with from the tonoplast. The coincidence of markers for Golgi and plasma membrane with the membranes of sonicated, dense vesicles, at a density slightly lower than that of plasma membranes prepared from the same cells, supports the possibility that membranes en route to the plasma membrane are incompletely differentiated.     

Isolation of highly purified fractions of plasma membrane and tonoplast from the same homogenate of soybean hypocotyls by free-flow electrophoresis

A procedure is described whereby highly purified fractions of plasma membrane and tonoplast were isolated from hypocotyls of dark-grown soybean (Glycine max L. var Wayne) by the technique of preparative free-flow electrophoresis. Fractions migrating the slowest toward the anode were enriched in thick (10 nanometers) membranes identified as plasma membranes based on ability to bind N-1-naphthylphthalamic acid (NPA), glucan synthetase-II, and K⁺-stimulated, vanadate-inhibited Mg²⁺ ATPase, reaction with phosphotungstic acid at low pH on electron microscope sections, and morphological evaluations. Fractions migrating farthest toward the anode (farthest from the point of sample injection) were enriched in membrane vesicles with thick (7-9 nanometers) membranes that did not stain with phosphotungstic acid at low pH, contained a nitrate-inhibited, Cl-stimulated ATPase and had the in situ morphological characteristics of tonoplast including the presence of flocculent contents. These vesicles neither bound NPA nor contained levels of glucan synthetase II above background. Other membranous cell components such as dictyosomes (fucosyltransferase, latent nucleosidediphosphate phosphatase), endoplasmic reticulum vesicles (NADH- and NADPH- cytochrome c reductase), mitochondria (succinate-2(p-indophenyl)-3-p-nitrophenyl)-5-phenyl tetrazolium-reductase and cytochrome oxidase) and plastids (carotenoids and monogalactosyl diglyceride synthetase) were identified on the basis of appropriate marker constituents and, except for plastid thylakoids, had thin (<7 nanometers) membranes. They were located in the fractions intermediate between plasma membrane and tonoplast after free-flow electrophoretic separation and did not contaminate either the plasma membrane or the tonoplast fraction as determined from marker activities. From electron microscope morphometry (using both membrane measurements and staining with phosphotungstic acid at low pH) and analysis of marker enzymes, both plasma membrane and tonoplast fractions were estimated to be about 90% pure. Neither fraction appeared to be contaminated by the other by more than 3%.     

Marker enzymes,phospholipids and acyl group composition of a somal plasma membrane fraction isolated from rat cerebral cortex: a comparison with microsomes and synaptic plasma membranes

Subjecting brain homogenates to differential speed and sucrose density gradient centrifugation resulted in the isolation of a membrane fraction from the post-mitochondrial supernatant with properties and marker enzyme profiles typical of plasma membranes. This membrane fraction is compared with the microsomes and the synaptic plasma membranes isolated from synaptosomes. Like the synaptic plasma membranes, membranes obtained from the post-mitochondrial supernatant were enriched five-fold in 5′-nucleotidase activity. However, the latter membranes were lower in (Na⁺, K⁺)-ATPase activity and higher in NADPH-cytochrome C reductase activity as compared to the synaptic plasma membranes. The post-mitochondrial plasma membranes were also different from the microsomes in their respective marker enzyme activities. Electron microscopic examination indicated largely membranous vesicles for both plasma membrane fractions with little contamination by myelin, mitochondra and intact synaptosomes. The phospholipid and acyl group profiles of the two plasma membrane fractions were surprisingly similar, but they were different from the characteristic profiles of myelin and mitochondria. It is concluded that plasma membranes isolated from the post-mitochondrial supernatant fraction are derived largely from neuronal and glial soma and are thus designated the somal plasma membrane fraction.     

Effects of centrifugal force and centrifugation time on the sedimentation of plant organelles

The effect of centrifugal force and length of centrifugation time on the sedimentation of plant organelles was determined for corn (Zea mays L.) root homogenates. A centrifugal force of 6000g for at least 20 minutes was necessary to pellet 90% of the mitochondrial marker (cytochrome c oxidase). This initial centrifugation step is optimal for separating mitochondria from microsomes, since cross-contamination of endoplasmic reticulum and plasma membrane vesicles with mitochondria is minimized. Centrifugal forces of 8000g or 10,000g for 20 minutes and 13,000g for 15 minutes pellet 90% of the mitochondrial marker; however, these centrifugation conditions also sediment more plasma membrane and endoplasmic reticulum.     

Plasma membranes from oats prepared by partition in an aqueous polymer two-phase system : on the use of light-induced cytochrome B reduction as a marker for the plasma membrane

Presumptive plasma membrane fractions have been prepared from oat (Avena sativa L. cv. Brighton) roots and shoots, respectively, by partition of microsomal fractions in a dextran-polyethylene glycol two-phase system. The plasma membranes had a high affinity for the polyethylene glycol-rich upper phase, whereas membranes from mitochondria and other organelles partitioned in the dextran-rich lower phase or at the interface. Thus, relatively pure plasma membranes were obtained by only two partition steps, and within 3 hours from homogenization of the material.

The plasma membranes from both organs were enriched in K⁺-stimulated Mg²⁺-dependent ATPase and glucan synthetase II, two tentative markers for the plant plasma membrane. Silicotungstic acid, an indicative stain for the plasma membrane, stained the vesicles recovered from the upper phase, but failed to stain the membranes partitioning in the lower phase or at the interface.

The plasma membranes were also enriched in a light-reducible b-cytochrome. This b-cytochrome can be measured by its light-induced absorbance change and may serve as a marker for the plant plasma membrane.

     

Isolation of plasma membranes from corn roots by sucrose density gradient centrifugation: an anomalous effect of ficoll

An investigation was conducted into the isolation of plasma membrane vesicles from primary roots of corn (Zea mays L., WF9 × 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.     

ASSEMBLY OF LIPIDS INTO MEMBRANES IN ACANTHAMOEBA PALESTINENSIS : II. The Origin and Fate of Glycerol3H-Labeled Phospholipids of Cellular Membranes

The membranes of Acanthamoeba palestinensis were studied by examination in fixed cells, and then by following the movements of glycerol-³H-labeled phospholipids by cell fractionation. Two previously undescribed structures were observed: collapsed cytoplasmic vesicles of cup shape, and plaques in food vacuole and plasma membrane similar in size to the collapsed vesicles. It appeared that the plaques formed by insertion of collapsed vesicles into membranes and/or that collapsed vesicles formed by pinching off of plaques. Fractions were isolated, enriched with nuclei, rough endoplasmic reticulum (RER), plasma membrane, Golgi-like membranes, and collapsed vesicles. The changes in specific activity of glycerol-³H-labeled phospholipids in these membranes during incorporation, turnover, and after pulse-labeling indicated an ordered sequence of appearances of newly synthesized phospholipids, first in nuclei and RER, then successively in Golgi membranes, collapsed vesicles, and finally, plasma membrane. In previous work we had found no large nonmembranous phospholipid pool in A. palestinensis. These observations are consistent with the hypothesis that membrane phospholipids are synthesized, perhaps as integral parts of membranes, in RER and nuclei. Subsequently, some of the newly synthesized phospholipids are transported to the Golgi complex to become integrated into the membranes of collapsed vesicles, which are precursors of the plasma membrane. Collapsed vesicles from the plasma membrane by inserting into it as plaques. When portions of the plasmalemma from food vacuoles, collapsed vesicles pinch off from their membranes and are recycled back to the cell surface.     

Sugar transport and potassium permeability in yeast plasma membrane vesicles

Plasma membrane vesicles were isolated from homogenised yeast cells by filtration, differential centrifugation and aggregation of the mitochondrial vesicles at pH 4. As judged by biochemical, cell electrophoretic and electron microscopic criteria a pure plasma membrane vesicle preparation was obtained.The surface charge density of the plasma membrane vesicles is similar to that of intact yeast cells with an isoelectric point below pH 3. The mitochondrial vesicles have a higher negative surface charge density in the alkaline pH range. Their isoelectric point is near pH 4.5, where aggregation is maximal.The yield of vesicles sealed to K⁺ was maximal at pH 4 and accounted for about one third of the total vesicle volume.The plasma membrane vesicles demonstrate osmotic behaviour, they shrink in NaCl solutions when loosing K⁺.As in intact yeast cells the entry and exit of sugars like glucose or galactose in plasma membrane vesicles is inhibited by UO₂²⁺.Counter transport in plasma membrane vesicles with glucose and mannose and iso-counter transport with glucose suggests that a mobile carrier for sugar transport exists in the plasma membrane.After galactose pathway induction in the yeast cells and subsequent preparation of plasma membrane vesicles the uptake of galactose into the vesicles increased by almost 100% over the control value without galactose induction. This increase is explained by the formation of a specific galactose carrier in the plasma membrane.     

Separation of visual pigments on columns of controlled-pore glass beads

Plasma membranes have been isolated from 3T3 and SV-3T3 cells grown in culture. Cells were harvested mechanically and disrupted in simple isotonic buffered salt solutions without resorting to hypotonic swelling or chemical membrane “hardeners.” The method of storing collected cells, the cell concentration during disruption, and the method of mechanical disruption were found to be significant variables affecting the yield of plasma membranes. The plasma membranes were separated from mitochondria and other cellular organelles by a single centrifugation through a step sucrose gradient containing a viscosity barrier of Dextran T-500 (modified fromA. S. Sun and B. Poole (1975)Anal. Biochem.68, 260). The isolated plasma membranes were located by assay for the “marker” enzyme, alkaline phosphatase (EC 3.1.3.1). The isolated plasma membrane fraction was free of mitochondrial and essentially free of lysozymal and endoplasmic reticulum contamination, which were assayed by measuring cytochrome c reductase, arylsulfatase, and hydrolysis of α-naphthol acetate, respectively. Of the enzymes tested, the phosphodiesterase activity was found to be the most specific assay for the plasma membrane from culture mouse fibroblast cells. The 5′-nucleotidase (EC 3.1.3.5) activity, the other plasma membrane marker, was extremely low in activity and gave an additional peak of activity when 5′-adenilic acid was used as substrate as compared to the expected single peak obtained with 5′-cytidilic acid as substrate. Overall recovery of isolated plasma membranes was greater than 75% as measured by the final recovery of phosphodiesterase activity.     

Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein: Is PAM involved in the capacitative calcium entry?

A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca²⁺ signalling and maintenance of Ca²⁺ homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca²⁺-ATPase, Na⁺, K⁺-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca²⁺ ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca²⁺ entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca²⁺ entry, and their formation and rebuilding have an important regulatory role in cellular Ca²⁺ homeostasis.     

Isolation and Identification of Plasma Membrane from Light-Grown Winter Rye Seedlings (Secale cereale L. cv Puma)

An effective method for the isolation of plasma membrane from light-grown winter rye seedlings (Secale cereale L. cv Puma) was established using a liquid two-polymer phase separation. The conditions for the specific partition of plasma membrane into the polyethylene glycol-enriched upper phase were examined, including variations in the polymer concentration, buffer system, pH, and NaCl addition in the phase partition system. The most effective phase partition system for the isolation of plasma membrane from winter rye consisted of 5.6/5.6% (w/w) polyethylene glycol 4000/dextran T500 in 0.25 molar sucrose-10 millimolar potassium phosphate-30 millimolar NaCl (pH 7.8), repeated once. When the isolated plasma membrane was centrifuged on a linear sucrose density gradient, a single band was found at the 34% (w/w) sucrose layer (1.141 grams per cubic centimeter) which co-fractionated with the pH 6.5-ATPase.

Identification of plasma membrane was performed by the combination of phosphotungstic acid-chromic acid stain and specific binding of N-1-naphthylphthalamic acid. Based on morphometrical observations after phosphotungstic acid-chromic acid stain, the isolated plasma membrane consisted mostly of vesicles of high purity. The isolated plasma membrane also showed extremely high specificity for N-1-naphthylphthalamic acidbinding, 10-fold higher than other membranes. It was also confirmed that there is a distinct difference in properties between plasma membrane and other membranes. The endomembranes such as from chloroplasts, mitochondria, and endoplasmic reticulum were observed to be highly sensitive to Zn²⁺ ion and lower pH, which resulted in an abrupt aggregation of membranes. On the contrary, plasma membrane was very stable to these treatments and no aggregation was observed. These unique properties of isolated plasma membrane are generally observed in a wide variety of plant species and can be utilized for the assessment of the purity of preparations of isolated plasma membranes and for their identification.

     

Surface properties of right side-out plasma membrane vesicles isolated from barley roots and leaves

Highly purified plasma membrane vesicles were obtained from roots and leaves of 7-day-old light-grown barley (Hordeum vulgare L. cv Kristina) seedlings by partitioning of crude microsomal fractions in a dextran-polyethylene glycol two-phase system. Sodium dodecylsulfate polyacrylamide gel electrophoresis showed the polypeptide composition of plasma membranes from the two organs to be qualitatively similar, but with different relative amounts of some of the polypeptides. Between 80 and 100% of the K⁺,Mg²⁺-ATPase activity was latent indicating that the vesicles were sealed and right side-out. The isoelectric points of the outer surface of root and leaf plasma membranes as determined by cross-partitioning were similar and quite acidic—about pH 3.6. In contrast, the net negative surface charge density at pH 7.0 as measured by 9-aminoacridine fluorescence differed significantly, being −29 mC·m⁻² for the leaf plasma membrane and only −19 mC·m⁻² for the root plasma membrane. As isolated, both types of plasma membrane vesicles had Ca²⁺ and Mg²⁺ bound to the outer surface as shown by the combined use of chelators and 9-aminoacridine fluorescence; however, the leaf plasma membrane had a relatively higher proportion of Ca²⁺ bound (0.57) than did the root plasma membrane (0.45). This difference probably reflects differences in the in vivo conditions as no chelator was present during the isolation procedure. Also Ni²⁺ could bind to the root vesicles as indicated by the effect of Ni²⁺ on 9-aminoacridine fluorescence, and by the binding of ⁶³Ni²⁺ (44 nanomoles bound per milligram protein) at 100 micromolar NiCl₂.     

Yeast mitochondrial outer membrane specifically binds cytoplasmically-synthesized precursors of mitochondrial proteins

The precursor of cytochrome b₂ (a cytoplasmically-synthesized mitochondrial protein) binds to isolated mitochondria or to isolated outer membrane vesicles. Binding does not require an energized inner membrane, is diminished by trypsin treatment of the membranes and is not observed with the partially processed (intermediate) form of the cytochrome b₂ precursor or with non-mitochondrial proteins. Upon energization of the mitochondria, the bound precursor is imported and cleaved to the mature form. Similar results were obtained with the precursor of citrate synthase. This receptor-like binding activity was present in isolated outer, but not inner membrane. It was solubilized from outer membrane with non-ionic detergent and reconstituted into liposomes.     

Specific binding of dexamethasone to plasma membranes from skeletal muscle

A procedure was developed to isolate plasma membranes from rabbit skeletal muscle. K⁺-dependent phosphatase activity was used as marker enzyme for plasma membranes and was determined in the presence of CHAPS (3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate), a zwitterionic detergent. Ca²⁺-ATPase and succinate dehydrogenase activities were used as marker enzymes for sarcoplasmic reticulum and mitochondria, respectively. Electron-microscopy revealed that plasma membranes were in the form of vesicles. Significant proteolysis of membrane proteins was observed during extraction, which was inhibited by EGTA and 20 mM molybdate. SDS-polyacrylamide gel electrophoresis revealed the disappearance of an intense 96 kDa protein band when membranes were purified in the absence of EGTA and molybdate. Specific binding sites for [³H]dexamethasone were identified in plasma membranes after freezing and incubation with CHAPS. Dithiothreitol was essential for steroid binding and ATP increased it. Under standardized assay conditions, binding was complete with 50 min à 37°C. No binding occurred at 0°C, nor if EGTA and molybdate were absent from the extraction medium.     

Mitochondrial membrane permeabilisation by amyloid aggregates and protection by polyphenols

Alzheimer's disease and Parkinson's disease are neurodegenerative disorders characterised by the misfolding of proteins into soluble prefibrillar aggregates. These aggregate complexes disrupt mitochondrial function, initiating a pathophysiological cascade leading to synaptic and neuronal degeneration. In order to explore the interaction of amyloid aggregates with mitochondrial membranes, we made use of two in vitro model systems, namely: (i) lipid vesicles with defined membrane compositions that mimic those of mitochondrial membranes, and (ii) respiring mitochondria isolated from neuronal SH-SY5Y cells. External application of soluble prefibrillar forms, but not monomers, of amyloid-beta (Aβ₄₂ peptide), wild-type α-synuclein (α-syn), mutant α-syn (A30P and A53T) and tau-441 proteins induced a robust permeabilisation of mitochondrial-like vesicles, and triggered cytochrome c release (CCR) from isolated mitochondrial organelles. Importantly, the effect on mitochondria was shown to be dependent upon cardiolipin, an anionic phospholipid unique to mitochondria and a well-known key player in mitochondrial apoptosis. Pharmacological modulators of mitochondrial ion channels failed to inhibit CCR. Thus, we propose a generic mechanism of thrilling mitochondria in which soluble amyloid aggregates have the intrinsic capacity to permeabilise mitochondrial membranes, without the need of any other protein. Finally, six small-molecule compounds and black tea extract were tested for their ability to inhibit permeation of mitochondrial membranes by Aβ₄₂, α-syn and tau aggregate complexes. We found that black tea extract and rosmarinic acid were the most potent mito-protectants, and may thus represent important drug leads to alleviate mitochondrial dysfunction in neurodegenerative diseases.     

Highly purified plasma membranes from rat hepatocytes following rate-isopycnic zonal centrifugation

Plasma membranes from liver parenchymal cells were isolated by rate-isopycnic zonal centrifugation. A method is described for the Beckman size 15 zonal rotor. It involved preparation from a perfused liver of a parenchymal cell-enriched homogenate in isoosmotic sucrose. The nuclear fraction containing membranes was recovered by centrifugation. The resuspended pellet was applied on the gradient of the zonal rotor. The isolated membranes had the same isopycnic banding density as 37% sucrose (w/w). The specific activity of 5′-nucleotidase, a widely used plasma membrane marker, was 105 μmoles·(mg protein)^?1·h^?1 being enriched by a factor of 50 as compared with parenchymal cell homogenate. The plasma membrane fraction was free of the mitochondrial and lysosomal enzymes, succinate dehydrogenase and acid phosphatase. No DNA and 10 μg RNA per mg plasma membrane protein were found. The purity of the membranes and their morphological appearance were controlled by electron microscopy. The preparation consisting of large membrane sheets showed a considerable purification away from other cellular components. A comparison with similar methods indicates that plasma membranes of a higher degree of purity can be obtained from parenchymal cells.     

Studies on isolated subcellular components of cat pancreas

Summary Pancreas of the cat was fractionated into its subcellular components by centrifugation through an exponential ficoll-sucrose density gradient in a zonal rotor. This enables a preparation of four fractions enriched in plasma membranes, endoplasmic reticulum, mitochondria and zymogen granules, respectively. The first fraction, enriched by 9- to 15-fold in the plasma membrane marker enzymes, hormone-stimulated adenylate cyclase, (Na⁺K⁺)-ATPase, and 5-nucleotidase, is contaminated by membranes derived from endoplasmic reticulum but is virtually free from mitochondrial and zymogen-granule contamination. The second fraction from the zonal gradient shows only moderate enrichment of the above marker enzymes but contains a considerable quantity of plasma membrane marker enzymes and represents mostly rough endoplasmic reticulum. The third fraction contains the bulk of mitochondria and the fourth mainly zymogen granules as assessed by electron microscopy and marker enzymes for both mitochondria and zymogen granules, namely succinic dehydrogenase, trypsin and amylase. Further purification of the plasma membrane fractions by differential and sucrose step-gradient centrifugation yields plasma membrane enriched 40-fold in basal and hormone-stimulated adenylate cyclase and (Na⁺K⁺)-ATPase.     

Proton transport in isolated vacuoles from corn coleoptiles

Vacuoles were isolated from corn coleoptile protoplasts and ATP-dependent proton transport was measured by quinacrine fluorescence quenching or by the uptake of [¹⁴C]methylamine. Intact vacuoles were judged to be free of a surrounding plasma membrane based on fluorescent staining with fluoroscein-diacetate. Essentially all of the detectable ATP-stimulated methylamine uptake and α-mannosidase activities present in intact protoplasts were recovered in isolated vacuoles. In contrast, the activities of marker enzymes for plasma membranes, Golgi, endoplasmic reticulum, and mitochondria were reduced to 5 to 17% in vacuolar preparations. The characteristics of proton pumping by isolated vacuoles were compared to those of light microsomal membranes possibly derived from the tonoplast. ATP-dependent proton pumping by both isolated vacuoles and light microsomal vesicles was stimulated by Cl⁻, and inhibited by NO₃⁻, carbonyl cyanide-m-chlorophenylhydrazone, N,N′-dicyclohexylcarbodiimide, N-ethylmaleimide, 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid, diethylstilbestrol, and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but not by vanadate. Both activities also showed substrate specificity for Mg-ATP. Finally, proton transport activities of vacuolar and microsomal fractions exhibited similar profiles after flotation in linear dextran gradients. We conclude that the microsomal proton pump previously characterized in corn coleoptiles (Mettler et al. 1982 Plant Physiol 70: 1738-1742) is derived from the tonoplast.