Isolation and properties of ion-stimulated ATPase activity associated with cauliflower plasma membranes

The association of K⁺-stimulated, Mg²⁺-dependent ATPase activity with plasma membranes from higher plants has been used as a marker for the isolation and purification of a plasma membrane-enriched fraction from cauliflower (Brassica oleraceae L.) buds. Plasma membranes were isolated by differential centrifugation followed by density gradient centrifugation of the microsomal fraction. The degree of purity of plasma membranes was determined by increased sensitivity of Mg²⁺-ATPase activity to stimulation by K⁺ and by assay of approximate marker enzymes. In the purified plasma membrane fraction, Mg²⁺-ATPase activity was stimulated up to 700% by addition of K⁺. Other monovalent cations also markedly stimulated the enzyme, but only in the presence of the divalent cation Mg²⁺. Ca²⁺ was inhibitory to enzyme activity. ATPase was the preferred substrate for hydrolysis, there being little hydrolysis in the presence of ADP, GTP, or p-nitrophenylphosphate. Monovalent cation-stimulated activity was optimum at alkaline pH. Enzyme activity was inhibited nearly 100% by AgNO₃ and about 40% by diethylstilbestrol.     

Labeling and isolation of plasma membranes from corn leaf protoplasts

A plasma membrane-enriched fraction has been isolated from corn leaf mesophyll protoplasts and its identity confirmed with the aid of an external label, diazotized [¹²⁵I]iodosulfanilic acid. Gentle cell disruption enabled internal organelles to be maintained intact and thus facilitated separation from the plasma membrane. The plasma membrane-enriched fraction was devoid of chloroplast or mitochondrial markers, whereas markers for the endoplasmic reticulum and golgi indicated minimal contamination. The highly enriched plasma membrane fraction contained a Mg²⁺-dependent, K⁺-stimulated ATPase with a pH optimum near neutrality. The position of the membranes on sucrose density gradients indicates that the plasma membranes have characteristics similar to other plasma membrane fractions.     

Isolation of plasma membrane fractions from green wheat leaves by free-flow electrophoresis or two-phase partition alone or in combination

Plasma membrane vesicles were isolated from shoots of light-grown wheat seedlings by preparative free-flow electrophoresis, aqueous polymer two-phase partition or both. Plasma membrane vesicles were identified from staining of thin sections prepared for electron microscopy with phosphotungstic acid at low pH. The orientation of the plasma membrane vesicles was determined from latency and trypsin sensitivity of K⁺ Mg²⁺ATPase and of glucan synthase II, and concanavalin A-peroxidase binding and membrane asymmetry visualized by electron microscopy. The K⁺Mg²⁺ATPase and of glucan synthase II activities of plasma membrane fractions isolated by two-phase partition were latent and trypsin resistant. The vesicles bound concanavalin A-peroxidase strongly and exhibited a cytoplasmic side-in morphology. These fractions of cytoplasmic side-in vesicles were less than 10% contaminated by cytoplasmic side-out vesicles. By free-flow electrophoresis, two populations of vesicles which stained with phosphotungstic acid at low pH, designated D and E, were obtained. The vesicle population with the lower electrophoretic mobility, fraction E, contained plasma membrane vesicles with properties similar to those of the plasma membrane vesicles obtained after two-phase partition. The phosphotungstic-reactive vesicles with greater electrophoretic mobility, fraction D, were concanavalin A unreactive with the cytoplasmic membrane leaflet outwards. Less than 50% of the K⁺Mg²⁺-ATPase activity of this fraction was latent and trypsin sensitive. The vesicles of fraction D appeared to be preferentially cytoplasmic side-out. The electrophoretic mobilities of cytoplasmic side-out (non-latent glucan synthase II activity) and cytoplasmic side-in (latent glncan synthase II activity) plasma membrane vesicles isolated from a frozen and thawed wheat plasma membrane fraction, corresponded with the mobilities of fraction D and E, respectively, again showing that the plasma membrane vesicles with the lesser electrophoretic mobility were cytoplasmic side-in. The cytoplasmic side-in and cytoplasmic side-out vesicles therefore showed opposite eletrophoretic mobilities compared with a previous free-flow electrophoretic separation of soybean plasma membranes. The majorities of the plasma membrane vesicles of both fractions D and E entered the upper phase upon two-phase partition with the phase composition used for purification of wheat plasma membranes. Thus, neither electrophoretic mobility nor phase partitioning characteristics can be used as the only criteria for assignment of vesicle orientation.     

ISOLATION AND PROPERTIES OF THE PLASMA MEMBRANE OF KB CELLS

Plasma membranes from KB cells were isolated by the method of latex bead ingestion and were compared with those obtained by the ZnCl₂ method. Optimal conditions for bead uptake and the isolation procedure employing discontinuous sucrose gradient centrifugation are described. All steps of preparative procedure were monitored by electron microscopy and specific enzyme activities. The plasma membrane fraction obtained by both methods is characterized by the presence of the Na⁺ + K⁺-activated ATPase and 5'-nucleotidase, and contains NADPH-cytochrome c reductase and cytochrome b₅. The latter two enzymes are also present in lower concentrations in the microsomal fraction. Unlike microsomes which are devoid of the Na⁺ + K⁺-activated ATPase and which contain only traces of 5'-nucleotidase activity, the plasma membrane fraction contains only trace amounts of the rotenone-insensitive NADH-cytochrome c reductase but no cytochrome P-450, both of which are mainly microsomal components. Morphologically the plasma membrane fraction isolated by the latex bead method is composed of vesicles of 0.1–0.3 µm in diameter. On the basis of the biochemical and morphological criteria presented, it is concluded that the plasma membrane fraction isolated by the above methods are of high degree of purity.     

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.     

Cyclic AMP and calcium modulated ATPase activity in the salivary glands of the lone star tick Amblyomma americanum (L.)

Na⁺,K⁺-activated ATPase activity in tick salivary glands increases during the rapid stage of tick feeding paralleling similar increases in dopamine and cAMP-stimulated fluid secretion. High concentrations of cyclic AMP increase Na⁺,K⁺-ATPase activity in a plasma membrane-enriched fraction from the salivary glands of rapidly feeding ticks. Cyclic AMP-dependent protein kinase inhibitor protein blocks activation of Na⁺,K⁺-ATPase activity at low but not high concentrations of cAMP indicating that both activator and inhibitor modulator phosphoproteins of Na⁺,K⁺-ATPase activity exist in the plasma membrane-enriched fraction.ATPase activity in the plasma membrane-enriched fraction is not measurable in the absence of Mg²⁺, Ca²⁺ and Na⁺. Ca-stimulated nucleotidase activity is highest with ATP serving as the preferred substrate in a series including CTP, UTP, GTP and ADP. Calcium, Mg²⁺ stimulated ATPase activity is activated further by calmodulin and partially inhibited by low concentration of vanadate, trifluoperazine and oligomycin. Results suggest that the plasma membrane-enriched fraction of tick salivary glands contains both Ca²⁺-ATPase activity and oligomycin-sensitive Ca²⁺, Mg²⁺-ATPase activities, the latter likely from a small amount of mitochondria in the partially purified organelle fraction.     

Characterization of a k-stimulated adenosine triphosphatase associated with the plasma membrane of red beet

A membrane fraction enriched with a magnesium-dependent, monovalent cation-stimulated ATPase was isolated from red beet (Beta vulgaris L.) storage roots by a combination of differential centrifugation, extraction with KI, and sucrose density gradient centrifugation. This fraction was distinct from endoplasmic reticulum, Golgi, mitochondrial, and possibly tonoplast membranes as determined from an analysis of marker enzymes. The ATPase activity associated with this fraction was further characterized and found to have a pH optimum of 6.5 in the presence of both Mg²⁺ and K⁺. The activity was substrate specific for ATP and had a temperature optimum near 40°C. Kinetics with Mg:ATP followed a simple Michaelis-Menten relationship. However the kinetics of K⁺-stimulation were complex and suggestive of negative cooperativity. When monovalent cations were present at 2.5 millimolarity, ATPase was stimulated in the sequence K⁺ > Rb⁺ > Na⁺ > Li⁺ but when the concentration was raised to 50 millimolarity, the sequence changed to K⁺ ≥ Na⁺ ≥ Rb⁺ > Li. The activity was not synergistically stimulated by combinations of Na⁺ and K⁺. The enzyme was insensitive to NaN₃, oligomycin, ouabain, and sodium molybdate but sensitive to N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, and sodium vanadate. Based on the similarity between the properties of this ATPase activity and those from other well characterized plant tissues, it has been concluded that this membrane fraction is enriched with plasma membrane vesicles.     

Orientation and integrity of plasma membrane vesicles obtained from carrot protoplasts

Two fractions enriched in plasma membrane derived from suspension-cultured carrot (Daucus carota L.) cells were examined to determine if they differed from each other either in physical nature or in orientation. Parameters studied included the protein composition of purified membranes derived from trypsinized and nontrypsinized protoplasts as well as from trypsinized purified plasma membranes, the effect of inhibitors and membrane perturbants on ATPase activity, the binding of [acetyl-¹⁴C]concanavalin A to purified membrane fractions, and the competitive removal of [acetyl-¹⁴C]concanavalin A from purified membranes derived from [acetyl-¹⁴C]concanavalin A-labeled protoplasts. One fraction (at density of 1.102 grams per cubic centimeter on Renografin gradients) appears to be a mixed population of `tightly' sealed vesicles with the majority being rightside-out vesicles of plasma membrane, and the other fraction (density 1.128 grams per cubic centimeter) apparently is a population of predominantly `leaky' vesicles and/or nonvesicular fragments of plasma membrane, a large portion of which appear to be `leaky' inside-out vesicles. In addition, it is shown that plasma membrane-enriched fractions can be distinguished from cellular endomembranes on the basis of protein and glycoprotein composition.     

ATP-dependent Ca2+ transport in wheat root plasma membrane vesicles

Plasma membrane preparations of high purity were obtained from roots of dark-grown wheat (Triticum aestivum L. cv. Drabant) by aqueous polymer two-phase partitioning. These preparations mainly contained sealed, right-side-out vesicles (ca 90% exposing the original outside out). By subjecting the preparations to 4 freeze/thaw cycles the proportion of sealed, inside-out (cytoplasmic side out) vesicles increased to ca 30%. Inside-out and right-side-out plasma membrane vesicles were then separated by partitioning the freeze/thawed plasma membranes in another aqueous polymer two-phase system. In this way, highly purified, sealed, inside-out (>60% inside-out) vesicles were isolated and subsequently used for characterization of the Ca²⁺ transport system in the wheat plasma membrane. The capacity for ⁴⁵Ca²⁺ accumulation, nonlatent ATPase activity and proton pumping (the latter two markers for inside-out plasma membrane vesicles) were all enriched in the inside-out vesicle fraction as compared to the right-side-out fraction. This confirms that the ATP-binding site of the ⁴⁵Ca²⁺ transport system, similar to the H⁺-ATPase, is located on the inner cytoplasmic surface of the plant plasma membrane. The ⁴⁵Ca²⁺ uptake was MgATP-dependent with an apparent K_m for ATP of 0.1 mM and a high affinity for Ca²⁺ [K_m(Ca²⁺/EGTA) = 3 μM]. The pH optimum was at 7.4–7.8. ATP was the preferred nucleotide substrate with ITP and GTP giving activities of 30–40% of the ⁴⁵Ca²⁺ uptake seen with ATP. The ⁴⁵Ca²⁺ uptake was stimulated by monovalent cations; K^? and Na⁺ being equally efficient. Vanadate inhibited the ⁴⁵Ca²⁺ accumulation with half-maximal inhibitions at 72, 57 and 2 μM for basal, total (with KCI) and net K⁺-stimulated uptake, respectively. The system was also highly sensitive to erythrosin B with half-maximal inhibition at 25 nM and total inhibition at 1μM. Our results demonstrate the presence of a primary Ca²⁺ transport ATPase in the plasma membrane of wheat roots. The enzyme is likely to be involved in mediating active efflux (ATP-binding sites on the cytoplasmic side) to the plant cell exterior to maintain resting levels of cytoplasmic free Ca²⁺ within the cell.     

Plasma Membrane-associated Adenosine Triphosphatase Activity of Isolated Cortex and Stele from Corn Roots

The plasma membrane fractions from separated cortex and stele of primary roots of corn (Zea mays L. WF9 × M14) contained cation ATPase activity at similar levels but with somewhat different properties. ATPase activity from cortex was optimum at pH 6.5, showed a simple Michaelis-Menten saturation with increasing ATP·Mg, and showed complex kinetic data for K⁺ stimulation similar in character to the kinetic data for K⁺-ATPase and K⁺ influx in primary roots. The results for cortex indicate that homogenates of primary roots are dominated by membranes from cortical cells.

ATPase activity from stele was optimum at pH 6.5 and showed another maximum at pH 9. At pH 6.5, activity from stele had properties similar to that from cortex except that the kinetics of K⁺ stimulation closely approached that expected for a Michaelis-Menten enzyme. At pH 9, the enzyme activity from stele was inhibited by 5 μg/ml oligomycin, suggesting that a significant portion of the activity was of mitochondrial origin. Sucrose density gradient analysis indicated some contamination of mitochondrial membranes in the plasma membrane fraction from stele. The results for stele are consistent with the view that stelar parenchyma cells are not deficient in ion pumps.

     

Involvement of plasma membrane potential in the tolerance mechanism of plant roots to aluminium toxicity

H⁺-ATPase activity of a plasma membrane-enriched fraction decreased after the treatment of barley (Hordeum vulgare) seedlings with Al for 5 days. A remarkably high level of Al was found in the membrane fraction of Al-treated roots. A long-term effect of Al was identified as the repression of the H⁺-ATPase of plasma membranes isolated from the roots of barley and wheat (Triticum aestivum) cultivars, Atlas 66 (Al-tolerant) and Scout 66 (Al-sensitive). To monitor short-term effects of Al, the electrical membrane potentials across plasma membranes of both wheat cultivars were compared indirectly by measuring the efflux of K⁺ for 40 min under various conditions. The rate of efflux of K⁺ in Scout was twice that in Atlas at low pH values such as 4.2. Vanadate, an inhibitor of the H⁺-ATPase of the plasma membrane, increased the efflux of K⁺. Al repressed this efflux at low pH, probably through an effect on K⁺ channels, and repression was more pronounced in Scout. Al strongly repressed the efflux of K⁺ irrespective of the presence of vanadate. Ca²⁺ also had a repressive effect on the efflux of K⁺ at low pH. The effect of Ca²⁺, greater in Scout, might be related to the regulation of the net influx of H⁺, since the effect was negated by vanadate. The results suggest that extracellular low pH may cause an increase in the influx of H⁺, which in turn is counteracted by the efflux of K⁺ and H⁺. These results suggest that the ability to maintain the integrity of the plasma membrane and the ability to recover the electrical balance at the plasma membrane through a net influx of H⁺ and the efflux of K⁺ seem to participate in the mechanism of tolerance to Al stress under acidic conditions.     

Preparation of a human lung purified plasma membrane fraction: Confirmation by enzyme markers,electron microscopy,and histamine H1 receptor binding

Summary A simple and rapid method of isolating plasma membranes from human peripheral lung tissue is described. The method involves homogenization of tissue in 0.25m sucrose-buffered medium followed by differential and sucrose density gradient centrifugation. Enzymatic and morphological characterization of the plasma membrane fraction revealed minimal contamination by nonplasma membrane fragments. The isolated plasma membranes showed an 18-fold purification of 5-nucleotidase activity compared to the original homogenate. Electronmicroscopic studies of the plasma membrane fraction revealed the presence of small membrane vesicles having a trilaminar membrane structure. To further examine the purity of the plasma membrane preparation, the binding of the H₁ receptor antagonist,³H pyrilamine, to the plasma membrane-enriched fraction was compared to the binding to crude membrane preparations. Both the plasma membrane-enriched fraction and the crude membrane preparation had similar K_d's for the histamine antagonist, but the plasma membrane-enriched fraction had a threefold greater binding capacity, reflecting the relative enrichment of plasma membranes of the preparation. Thus, a method has been developed for the isolation of plasma membranes from human peripheral lung which should provide material for a variety of biochemical and pharmacological studies.     

Isolation and characterization of plasma and smooth membranes of the marine diatom Nitzschia alba

A procedure is described for isolating plasma, smooth and other cellular membranes from hypotonically lysed protoplasts of the marine diatom, Nitzschia alba. From starting material of approximately 10 g wet weight (10¹⁰ cells), about 168 mg (organic weight) of a membrane-enriched fraction, exclusive of mitochondria, is obtained by differential centrifugation. From this, six membrane fractions are separated on a discontinuous sucrose gradient by isopycnic centrifugation.The plasma membranes, from the density region 1.23-1.29 g/cc, consist of small vesicles and sheets. They are purified approximately 20-fold, based on the increase in specific activity of a (Na⁺-K⁺-Mg²⁺)-ATPase, an enzyme found predominantly in these membranes. They also contain the highest specific and total activity of a (Mg²⁺)-ATPase and, in addition, are distinguished chemically by their high sterol specific content and high molar ratio of sterol/phospholipid (0.792-0.854). The carbohydrate/ protein ratio (0.070-0.072) is appreciably lower than that of the smooth membranes.The smooth membranes separate into two distinct fractions, a light and heavy component, which occur at the top of the sucrose gradient in densities of 1.13 and 1.18 g/cc, respectively. Both fractions are composed of relatively large membrane vesicles and membrane sheets and are distinguished from other membrane fractions by an exceptionally high carbohydrate/protein ratio (0.194-0.294).The light component shows the highest specific content of lipid, phospholipid, neutral lipid, carbohydrate, sialic acid, and RNA, and the highest specific activity of NADPH cytochrome c reductase, 5′-nucleotidase and phosphodiesterase compared to the other five fractions. It shows the lowest Na⁺ plus K⁺ stimulation of the (Mg²⁺)-ATPase. This fraction is probably enriched in endoplasmic reticulum.The heavy component contains some Golgi-like vesicles, sacs and tubules. It is characterized by the highest total content of chemical constituents analyzed, with the exception of RNA, and by the highest specific activity of thiamine pyrophosphatase, uridine diphosphatase, acid and alkaline phosphatase, and glucose-6-phosphatase, suggesting that this component is enriched in Golgi membranes approximately 13-fold.A most striking feature of these diatom membranes is the presence in all fractions of (Mg²⁺)-ATPase activity which is stimulated 5- to 10-fold by the presence of equimolar Na²⁺ plus K⁺. The data clearly differentiate these membrane fractions from each other as well as from membranes prepared from animal cells.     

Ca-Mg-ATPase activity in brain coated microvesicles purified on immunosorbents

Coated microvesicle fractions isolated from ox forebrain cortex by the ultracentrifugation procedure of Pearse (1) and by the modified, less time consuming method of Keen et al (2) had comparable Ca²⁺+Mg²⁺ dependent ATPase activities (about 9 μmol/h per mg protein). The Na⁺+K⁺+Mg²⁺ dependent ATPase activity was 3.2 μmol/h per mg (±1.0, S.D., n=3) when microvesicles were prepared according to (1) and 1.5 μmol/h per mg (±1.0, S.D., n=3) when prepared according to (2).Oligomycin, ruthenium red, and trifluoperazine, inhibitors of Ca²⁺ transport in mitochondria and erythrocyte membranes had no effect on Ca²⁺+Mg²⁺ dependent ATPase from any of the preparations.As demonstrated both by ATPase assays and electron microscopy, coated microvesicles could be bound to immunosorbents prepared with poly-specific antibodies against a coated microvesicle fraction obtained by the method of Pearse (1). The binding could be inhibited by dissolved coat protein using partially purified clathrin. The fraction of coated vesicles eluted from the immunosorbent was purified relative to the starting material as judged by electron microscopy.The Ca²⁺+Mg²⁺ ATPase activity and calmodulin content was copurified with the coated microvesicles and the specific activity of Na⁺+K⁺+Mg²⁺ ATPase was decreased.Na⁺+K⁺+Mg²⁺ dependent ATPase activity in the coated microvesicle fraction could be ascribed to membranes with the appearance of microsomes. These membranes were also bound to the immunosorbents, but the binding was not influenced by clathrin. The capacity of the immunosorbents for these membranes was less than for the coated microvesicles, resulting in a decrease of Na⁺+K⁺+Mg²⁺ dependent ATPase activity in the eluted coated microvescile fraction.It was concluded that Ca²⁺+Mg²⁺ ATPase activity is not a contamination from plasma membrane vesicles or mitochondrial membranes but seems to be an integral part of the coated vesicle membrane.     

Comparison of the properties of active Ca2+ transport by the islet-cell endoplasmic reticulum and plasma membrane

The properties of active or ATP-dependent calcium transport by islet-cell endoplasmic reticulum and plasma membrane-enriched subcellular fractions were directly compared. These studies indicate that the active calcium transport systems of the two membranes are fundamentally distinct. In contrast to calcium uptake by the endoplasmic reticulum-enriched fraction, calcium uptake by islet-cell plasma membrane-enriched vesicles exhibited a different pH optimum, was not sustained by oxalate, and showed an approximate 30-fold greater affinity for ionized calcium. A similar difference in affinity for calcium was exhibited by the Ca²⁺-stimulated ATPase activities which are associated with these islet-cell subcellular fractions. Consistent with the effects of calmodulin on calcium transport, calmodulin stimulated Ca²⁺-ATPase in the plasma membranes, but did not increase calcium-stimulated ATPase activity in the endoplasmic reticulum membranes. The physiological significance of the differences observed in calcium transport by the endoplasmic reticulum and plasma membrane fractions relative to the regulation of insulin secretion by the islets of Langerhans is discussed.     

Photosynthate partitioning in split-root citrus seedlings with mycorrhizal and nonmycorrhizal root systems

The plasma membrane ATP-phosphohydrolase (ATPase) from red beet (Beta vulgaris L.) storage tissue was solubilized with the zwitterionic detergent Zwittergent 3-14 from a plasma membrane-enriched fraction which was extracted with the anionic detergent, sodium deoxycholate. For both the extraction of extraneous proteins by deoxycholate and the solubilization of active plasma membrane ATPase by Zwittergent 3-14, the optimal concentration of detergent was 0.1% (weight per volume) with a detergent to protein ratio of 1.0 (milligram per milligram). The properties of the solubilized ATPase were found to be similar to the membrane-bound enzyme with respect to pH optimum, substrate specificity, inhibitor sensitivity, and kinetics of K⁺ stimulation. The solubilized ATPase preparation formed a rapidly turning over phosphoenzyme, the breakdown velocity of which was increased in the presence of 50 millimolar KCl. Solubilization with 0.1% Zwittergent 3-14 following extraction with 0.1% deoxycholate resulted in an increase in both ATPase activity and steady state phosphoenzyme level; however, a direct correspondence between the increase in ATPase activity and phosphorylation level did not exist. It is proposed that this discrepancy may be the result of a detergent-mediated modification of kinetic rate constants in the mechanism of the enzyme.     

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%.     

Phosphorylation of the adenosine triphosphatase in a deoxycholate-treated plasma membrane fraction from corn roots

The ATP phosphohydrolase (ATPase) activity of a corn (Zea mays L., WF9 × Mo17) root plasma membrane fraction was enriched almost 2-fold by selective extraction with 0.1% (w/v) deoxycholate. The detergent treatment solubilized about 30% of the total membrane protein and some ATP hydrolyzing activity that was not K⁺-stimulated, but the major portion of the ATPase activity could be pelleted with membranes. The properties of the ATPase associated with the detergent-extracted plasma membrane fraction were similar to those for the ATPase of the untreated plasma membrane fraction with respect to substrate specificity, pH optimum, kinetics with MgATP, ion stimulation, and inhibitor sensitivity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed only minor differences in protein composition resulting from the detergent treatment.

The plasma membrane fraction from corn roots contained an endogenous protein kinase activity. This was shown by the time course of phosphate incorporation and by the labeling of a number of protein bands on SDS-polyacrylamide gel electrophoresis. The deoxycholate treatment removed measurable protein kinase activity and allowed the demonstration of a rapidly turning over covalent phosphorylated intermediate associated with the detergent-extracted plasma membrane fraction. The phosphorylated intermediate was present as a 100,000 dalton polypeptide and may represent the catalytic subunit of the plasma membrane K⁺-ATPase.

     

Studies on the subcellular distribution of (Na+ + K+)-ATPase,K+-stimulated ATPase and HCO3−-stimulated ATPase activities in malpighian tubules of Locusta migratoria L.

The present study confirms previous reports of the presence of (Na⁺ + K⁺)-ATPase and anion-stimulated ATPase activity in Malpighian tubules of Locusta. In addition, the presence of a K⁺-stimulated, ouabain-insensitive ATPase activity has been identified in microsomal fractions. Differential and sucrose density-gradient centrifugation of homogenates has been used to separate membrane fractions which are rich in mitochondria, apical membranes and basolateral membranes; as indicated by the presence of succinate dehydrogenase and the presence or absence of non-specific alkaline phosphatase activity, respectively. Relatively high specific (Na⁺ + K⁺)-ATPase activity was associated with the basolateral membrane-rich fractions with only low levels of this activity being associated with the apical membrane-rich preparation. K⁺-stimulated ATPase activity was also associated, predominantly, with the basolateral membrane-rich fractions. However, comparison of the distribution of this activity with that of the (Na⁺ + K⁺)-ATPase suggests that the two enzymes did not co-separate. The possibility that the K⁺-stimulated ATPase was not associated with the basolateral plasma membrane is discussed.Anion-stimulated ATPase activity was found in the apical and basolateral membrane-rich fractions and in the fraction contaning mainly mitochondria. Nevertheless, the fact that this bicarbonate-stimulated activity did not co-separate with succinate dehydrogenase activity suggests that it was not exclusively mitochondrial in origin. These results are consistent with physiological studies indicating a basolateral (Na⁺ + K⁺)-ATPase but do not support the K⁺-stimulated ATPase as a candidate for the apical electrogenic pump. The possible role of the bicarbonate-stimulated ATPase activity in ion transport across both the basolateral and apical cell membranes is discussed.     

Separation of two types of electrogenic h-pumping ATPases from oat roots

Microsomal vesicles of oat roots (Avena sativa var Lang) were separated with a linear dextran (0.5-10%, w/w) or sucrose (25-45%, w/w) gradient to determine the types and membrane identity of proton-pumping ATPases associated with plant membranes. ATPase activity stimulated by the H⁺/K⁺ exchange ionophore nigericin exhibited two peaks of activity on a linear dextran gradient. ATPase activities or ATP-generated membrane potential (inside positive), monitored by SCN⁻ distribution, included a vanadate-insensitive and a vanadate-sensitive component. In a previous communication, we reported that ATP-dependent pH gradient formation (acid inside), monitored by quinacrine fluorescence quenching, was also partially inhibited by vanadate (Churchill and Sze 1983 Plant Physiol 71: 610-617). Here we show that the vanadate-insensitive, electrogenic ATPase activity was enriched in the low density vesicles (1-4% dextran or 25-32% sucrose) while the vanadate-sensitive activity was enriched at 4% to 7% dextran or 32% to 37% sucrose. The low-density ATPase was stimulated by Cl⁻ and inhibited by NO⁻₃ or 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS). The distribution of Cl⁻-stimulated ATPase activity in a linear dextran gradient correlated with the distribution of H⁺ pumping into vesicles as monitored by [¹⁴C]methylamine accumulation. The vanadate-inhibited ATPase was mostly insensitive to anions or DIDS and stimulated by K⁺. These results show that microsomal vesicles of plant tissues have at least two types of electrogenic, proton-pumping ATPases. The vanadate-insensitive and Cl⁻-stimulated, H⁺-pumping ATPase may be enriched in vacuolar-type membranes; the H⁺-pumping ATPase that is stimulated by K⁺ and inhibited by vanadate is most likely associated with plasma membrane-type vesicles.