Plasmalemma Fraction Research Articles

Activity of Plasma Membrane H+-ATPase in Coleoptile Cells during Development of Maize Seedlings

Hydrolytic activities of the H+-ATPase were compared for plasma membrane fractions isolated from coleoptile cells of 3-, 4-, and 5-day-old etiolated maize seedlings. The membrane preparations obtained by differential centrifugation were additionally purified in the gradient of sucrose density and in the polyethylene glycol-dextran system. The highest level of ATP-hydrolyzing activity was observed in the plasmalemma fraction obtained from 4-day-old seedlings. The pattern of age-dependent changes in H+-ATPase activity of the plasma membranes was clearly different from the monotonic deceleration of coleoptile cell elongation in the period examined. It is supposed that changes in ATPase activity reflect different regulatory roles of this principal ion-transporting enzyme of the plasma membrane at the stage of cell elongation and at a later developmental stage when the coleoptile has completed its physiological function.

Salt loading induces redistribution of the plasmalemmal Na/K-ATPase in proximal tubule cells

We have reported that digitalis-like substances (cardiotonic steroids), including marinobufagenin (MBG), induce endocytosis of the plasmalemmal Na/K-ATPase in LLC-PK1 cells. The current report addresses the potential relevance of plasmalemmal Na/K-ATPase redistribution to in vivo salt handling. Male Sprague-Dawley rats were given 1 week of a high salt (4.0% NaCl) or normal salt (0.4% NaCl) diet. Urinary sodium excretion, as well as MBG excretion, was monitored, and proximal tubules were isolated using a Percoll gradient method. Tubular (86)Rb uptake, Na/K-ATPase enzymatic activity, and Na/K-ATPase alpha1 subunit density were determined. The high salt diet increased urinary sodium (17.8 +/- 1.8 vs. 2.5 +/- 0.3 mEq/day, P < 0.01) and MBG excretion (104 +/- 12 vs. 26 +/- 4 pmol/day), and decreased proximal tubular (86)Rb uptake (0.44 +/- 0.07 vs. 1.00 +/- 0.10, P < 0.01) and Na/K-ATPase enzymatic activity (5.1 +/- 1.1 vs. 9.9 +/- 1.6 micromol/mg pr/hr, P < 0.01) relative to the normal diet. Proximal tubular Na/K-ATPase alpha1 protein density was decreased in the plasmalemma fraction but increased in both early and late endosomes following the high salt diet. In rats fed a high salt diet, anti-MBG antibody caused a 60% reduction in urinary sodium excretion, substantial increases in proximal tubule (86)Rb uptake, and Na/K-ATPase enzymatic activity, as well as significant decreases in the early and late endosomal Na/K-ATPase alpha1 protein content. These data suggest that redistribution of the proximal tubule Na/K-ATPase in response to endogenous cardiotonic steroids plays an important role in renal adaptation to salt loading.

Possible Regulatory Effect of Lipid Peroxidation on the H+-ATPase Activity of the Plasmalemma under Stress Conditions

We studied the effects of high temperature and paraquat on the rate of lipid peroxidation and activity of the H+-ATPase in the plasmalemma fraction isolated from pea leaves. We demonstrated a heat-induced increase in both indices. When lipid-peroxidation was inhibited by pretreatment with butylated hydroxytoluene, a synthetic antioxidant, the H+-ATPase activity increased to a lesser extent than after heat shock without pretreatment. Treatment of plants with paraquat, a prooxidant causing an oxidative stress, resulted in a dramatic increase in lipid peroxidation and H+-ATPase activity. We suggested that the enhanced lipid peroxidation could be one of the causes for the H+-ATPase activation in the plasmalemma under stress conditions.

Modified development in transgenic tobacco plants expressing a rolA::GUS translational fusion and subcellular localization of the fusion protein.

The rolA gene is transferred naturally by Agrobacterium rhizogenes to the genome of host plants, where it induces dramatic changes in development of transformed plants, including dwarfism and leaf wrinkling. The predicted translation product of the rolA gene is a small (11.4 kDa), basic (pI = 11.2) protein, which has no clearly significant similarity to sequences in the data bases. We have introduced into the tobacco genome a gene encoding a rolA::GUS fusion protein. Expression of this gene led to synthesis of an RNA and a protein of expected size, and the transformed plants exhibited the dwarfism and leaf wrinkling typical of rolA plants, but to a lesser degree than plants transformed with the wild-type rolA gene. The distribution of beta-glucuronidase (GUS) activity was compared in subcellular fractions of leaf extracts from plants expressing either the rolA::gus gene or a control gus construct. As expected, in the control plants, GUS activity was essentially cytosolic. In contrast, in plants expressing the rolA::gus gene the highest specific activity was associated with the plasmalemma fraction.

Purification of Plasma Membrane and Protein Composition Changes during Ripening and Senescence of Cherry Tomato

Summary Highly plasmalemma-enriched fractions were prepared from the pericarp of cherry tomato (Lycopersicon esculentum Mill. var. cerasiforme Alef) by aqueous polymer two-phase partitioning. The purified vesicles were suitable for electrophoretic characterization of the membrane proteins at five different stages of fruit ripening and senescence. The validity of the polyethylene glycol — dextran partition system has been proved by the distribution between the two phases of marker enzyme activities assayed under optimized conditions for this new fruit material. Although the properties of the vanadate-inhibited K+, Mg2+-ATPase as a plasmalemma marker were not as specific as for other plant species, a several-fold enrichment of its activity was found in the upper phase. This result was essentially confirmed by a similar high enrichment ratio of glucan synthase II activity, representing a more reliable test. Complementary, non-significant contamination of the upper phase was demonstrated using the different endomembrane markers. Assessment of fraction purity also involved electron microscopy, from which an absolute criterion for the plasmalemma origin of the vesicles is obtained. More than 30 polypeptides were clearly resolved by sodium dodecylsulfate-polyacrylamide gel electrophoresis in the plasmalemma fraction at the mature-green stage of the fruit, with the most prominent bands in the 49–57 or 65–72 kDa regions and some further prominent bands corresponding to 18.5,27, 29 and 100 kDa. The changes in protein profile did not indicate the occurrence of any disorganization process during fruit life, even in the last senescence phase, and only a few major constituents decreased (18.5, 27kDa). However, some marked changes in the expression of membrane-associated proteins occurred during the early ripening period, such as the sudden appearance of a 24 kDa major constituent and an increase in the amount of the 41 kDa band. The importance of these putative participants in the ripening and senescence processes is discussed.

Some properties of the H-ATPase activity present in root plasmalemma of Avena sativa L. Two different enzymes or one enzyme with two ATP sites?

The effects of Mg 2+, K + and ATP on a H-ATPase activity from a native plasmalemma fraction of oat roots were explored at 20°C and pH 6.5. In the presence of 3 mM ATP and no K +, H-ATPase activity vs. [Mg 2+] approached a monotonic activation but it became biphasic, with a decline above 3 mM Mg 2+, in the presence of 20 mM K +. Mg 2+ inhibition occurred also in K-free solutions when [ATP] was lowered to 0.05 mM. Also, an apparent monotonic H-ATPase activation by [K +] at 3.0 mM ATP was transformed in biphasic (inhibition by high [K +]) when [ATP] was reduced to 0.05 mM. The best fits of the ATP stimulation curves of hydrolysis satisfied the sum of two Michaelian functions where that with higher affinity had lower V max. Taking into consideration all conditions of activity assay, the high-affinity component (1) had a K m about 11–16 μM and a V max around 0.14–0.28 μmol P i/mg per min whereas that with lower affinity (2) had a K m of 220–540 μM and a V max of 0.5–1.0 μmol P i/mg per min. K m2 was markedly affected by the [K +] and [Mg 2+]; at optimal concentrations of these cations (1 mM Mg 2+ and 10 mM K +) it had a value of 235±24 μM which was increased to 540±35 μM at 20 mM [Mg 2+] and 60 mM [K +]. In addition, V mx1 was reduced to about a half when the concentrations of Mg 2+ and K + were increased to inhibitory levels. These results could be explained by the existence of two different enzymes or one enzyme with two ATP sites. In the second case, we could not tell at this stage if both are catalytic or one is regulatory.

Changes in parameters of the plasmalemma ATPase during cold acclimation of apple (Mains domestica) tree bark tissues

A plasmalemma fraction was isolated from homogenized apple tree (Mains domestica Borkh ‘Golden Delicious’) hark tissues using aqueous phase partitioning and ultra‐centrifugation. Results of marker enzyme assays indicated that a membrane preparation highly enriched in plasma membranes was obtained. ATPase activity in this preparation possessed a high specificity for ATP as substrate, was inhibited by vanadate, diethylstilbcsterol and dicyclohexylcarbocHimide, and was insensitive to inhibitors of mitochondria! and tonoplasl ATPases. Specific activity of the plasma‐lemma ATPase increased during cold acclimation prior to the attainment of vegetative maturity. Kinetic parameters (Km, Vln) determined from assays performed at different temperatures (10, 30°C) indicated a differential effect of cold acclimation on enzyme activity. Vm increased during cold acclimation, whereas Km increased when determined at 30°C but declined at 10°C. Acclimation treatments during April and May resulted in alteration of ATPase kinetics in the absence of any increase in bark frost hardiness. Changes in ATPase kinetics may be related more to enhanced low temperature metabolism than to frost hardiness per se.

Properties of potassium uptake by seedling roots of grape cultivars

Uptake rates of (86Rb)K+ by seedling roots of six cultivars were measured and compared with K+ content of the root, K+ leakage, H+ efflux, and K+-ATPase activity of a partially purified plasmalemma fraction.

Protein Kinase Activities in Tonoplast and Plasmalemma Membranes from Corn Roots

Protein kinase and phosphatase activities were studied in plasmalemma and tonoplast membrane fractions from corn (Zea mays L.) roots in order to test the hypothesis that the tonoplast H(+)-ATPase is regulated by intrinsic protein phosphorylation (G Zocchi, SA Rogers, JB Hanson 1983 Plant Sci Lett 31: 215-221), and to facilitate future purification of kinase activities from these membranes. Kinase activity in the plasmalemma was about three-fold higher than in the tonoplast, and displayed Michaelis Menten-type behavior with a K(m) value for MgATP(2-) of about 50 micromolar. Both activities were optimal at 3 millimolar free Mg(2+) and had pH optima at 6.6 and 7.0 for the plasmalemma and tonoplast, respectively. Kinase activities in both fractions were stimulated by 1 micromolar free Ca(2+), but calmodulin had no stimulatory effect, and chlorpromazine was inhibitory only at high concentrations. The pattern of phosphopeptides on SDS polyacrylamide gel electrophoresis was similar in both fractions except for one band of 50 kilodaltons that was present only in the tonoplast. A partially purified H(+)-ATPase fraction was prepared from tonoplast membranes, incubated under conditions optimal for protein phosphorylation. The three polypeptides (of 67, 57, and 36 kilodaltons), enriched in this fraction, did not become phosphorylated, suggesting that this protein is not regulated by endogenous protein phosphorylation. Protein phosphatase activity was detected only in the plasmalemma fraction. These results indicate that a regulatory cycle of protein phosphorylation and dephosphorylation may operate in the plasmalemma. The activity in the tonoplast appears to originate from plasmalemma contamination.

Evidence that a 25 kilodalton protein associated with sugar beet root plasmalemmas may be involved in the enhanced uptake of iron by iron stressed plants

Abstract Electrophoresis of a root plasmalemma fraction from three sugar beet cultivars with different iron uptake capacities revealed a 25 kilodalton (kd) protein which is produced in enhanced amounts in response to Fe deficiency in a manner corresponding to the Fe uptake capacities of the three cultivars. An assay was developed to measure the plasmalemma‐bound ferric iron reductase. Using this assay, we were able to detect differences in specific activity of the reductase among the three cultivars but not increases in activity in response to iron stress. The results suggest that a 25 kD plasma lemma‐bound protein could be involved in the dramatically enhanced uptake of Fe which occurs when Fe is resupplied to Fe deficient sugar beets.

Characterization of the plasmalemma ATPase activity from roots of Plantago major ssp. pleiosperma, purified by the two‐phase partitioning method

A purified plasmalemma preparation from roots of Plantago major L. ssp. pleiosperma (Pilger) was obtained by the two‐phase partitioning method, using 6.5% (w/w) of Dextran T‐500 and polyethylene glycol 3350, respectively. The distribution of murker enzymes proved the purity of the plasmalemma fraction. The ATPase activity was characterized by determining its sensitivity to anions, cations and inhibitors. The Mg2+‐dependent ATPase activity peaked at pH 7.25, K+‐stimulation at pH 6.75, and the Cl− ‐stimulation both at pH 6.75 and 7.5 (all in the presence of 3 mM MgSO4). The plasmalemma preparations hydrolyzed preferentially ATP (in the presence of Mg2+), although they were less specific for ATP at pH 7.5 than at pH 6.75. The Cl− ‐ stimulated ATPase is probably associated with and located on the plasmalemma. The question if the Cl− ‐stimulated activity is due to an ATPase distinct from the classical K+‐stimulated ATPase is considered.

Plasmalemma from the roots of cucumber: Isolation by two‐phase partitioning and characterization

Plasmalemma was isolated from the roots of 2‐week‐old cucumber plants (Cucumis sativus L. cv. Rhensk druv) by utilizing an aqueous polymer two‐phase system with 6.5%:6.5% (w/w) Dextran T500 and polyethylene glycol (PEG) 3350 at pH 7.8. The plasmalemma fraction comprised ca 6% of the membrane proteins contained in the microsomal fraction. The specific activity of the plasma membrane marker enzyme (K+, Mg2+‐ATPase) was 14‐ to 17‐times higher in the upper (PEG‐rich) than in the lower (Dextran‐rich) phase, and the reverse was true for marker enzymes (cytochrome c oxidase, EC 1.9.3.1, and antimycin A‐resistant NADPH cytochrome c reductase) of intracellular membranes. The ATPase was highly stimulated by the addition of detergent (Triton X‐100), so that the isolated plasmalemma vesicles appear tightly sealed and in a right‐side‐out orientation. Further characterization of the ATPase activities showed a pH optimum at 6.0 in the presence of Mg2+. This optimum was shifted to pH 5.8 after addition of K+. K+ stimulated the ATPase activity below pH 6 and inhibited above pH 6. The ATPase activity was specific for ATP and sensitive to N,N‐dicyclohexylcarbodiimide and sodium vanadate, with K+ enhancing the vanadate inhibition. The enzyme was insensitive to sodium molybdate, NO−3, azide and oligomycin. No Ca2+‐ATPase was detected, and even as little as 0.05 mM Ca2+ inhibited the Mg2+‐ATPase activity.

Epidermal growth factor receptor kinase translocation and activation in vivo.

The rat liver epidermal growth factor (EGF) receptor was assessed for EGF-dependent autophosphorylation as well as phosphorylation of a defined exogenous substrate in purified plasmalemma and Golgiendosome fractions isolated from rat liver homogenates. While EGF-dependent kinase activity was readily detected in plasmalemma the corresponding activity in Golgi-endosome fractions required detergent. Consequent to the systemic injection of EGF in vivo, the majority (approximately 60%) of receptor as evaluated by 125I-EGF binding was rapidly lost (T 1/2 approximately 8 min) from the plasmalemma and correspondingly accumulated in the Golgi-endosome fraction in a dose-dependent manner. Electron microscope radioautography of 125I-EGF uptake into Golgi-endosome fractions identified internalization into lipoprotein-filled vesicles of heterogenous size and shape but not into stacked saccules of the Golgi apparatus. Evaluation of receptor kinase activity in plasmalemma fractions isolated at various times after EGF injection in vivo showed more rapid loss of EGF-dependent autophosphorylation activity (T 1/2 approximately 10 s) than of receptor content (T 1/2 approximately 8 min). In contrast to the EGF receptor kinase of the plasmalemma fraction, kinase activity accumulating in endosomes was activated, i.e. maximally stimulated, in the absence of EGF or Triton X-100 in vitro. Furthermore, following the peak time of accumulation of EGF receptor kinase in endosomes (5-15 min) EGF-dependent autophosphorylation activity and EGF receptor content were lost more slowly (T 1/2 approximately 27 and 87 min for the loss of autophosphorylation activity and receptor content, respectively). The rapidity of translocation of activated EGF receptor into endosomes (30 s) and the dose response to low levels (1 microgram) of EGF injected are consistent with a physiological role for internalized EGF receptor kinase activity.

Binding of the Calcium Antagonist [3H] Nitrendipine to Human Myometrial Plasmalemma

Membrane fragments were prepared from nonpregnant human myometrium and fractionated by differential and sucrose gradient centrifugation. Reversible high-affinity binding sites of the dihydropyridine calcium antagonist nitrendipine were identified in highest density in that membrane fraction most enriched in markers for plasmalemma. In determinations done on several preparations, the equilibrium dissociation constant for nitrendipine at 37 degrees C was found to be 0.3 to 0.8 nM and was the same for each fraction. The maximum binding capacity was found to be 400-500 fmol/mg protein in the plasmalemma fraction.

Aflatoxin-induced alterations in Glycine max, cv. 'Essex' root cell membrane protein content.

Aflatoxins (AFTs) are hepatocarcinogens, mutagens, teratogens and toxins. Isolates of AFTs-producing strains of Aspergillus flavus can grow upon both autoclaved soybeans and to a lesser extent field-grown beans. Besides inhibiting the elongation of excised, in vitro cultured soybean roots, the AFTs can impair the roots' ability to remove [14C]-leucine from a culture medium and to incorporate the amino acid into acid-insoluble, cytoplasmic protein. In this connection, exogenous AFTs once taken-up and compartmentalized by the excised roots can reduce the acid-insoluble protein content of what appears to be a non-enriched plasmalemma fraction. Here, we report a combined biochemical and microscopical attempt to determine whether the protein reduction occurs throughout the excised, in vitro-cultured root and whether the plasmalemma proteins which are affected by AFTs can be both solubilized and characterized. Histochemistry of Carnoy-fixed roots revealed a reduction in Ninhydrin-Schiff-stainability throughout the AFTs-treated root. Transmission electron microscopy of 80 000 X g pellets derived from homogenates of both non-treated (NT) and treated (T) roots provided further evidence to our previously reported marker enzyme analyses for the occurrence of the plasmalemma in the 80 000 X g pellet. Treatment of the latter with the Laemmli SDS procedure released greater than 85% of the protein associated with the pellets obtained from homogenates of either NT or T roots. Gel permeation chromatography on Biogel-100 of released proteins from 80 000 X g pellets of both NT and T roots yielded both void volume and retarded peaks. Both the amplitude and the quantities of protein recovered within the void volume peak were less within the void volume of the t than the NT root. Polyacrylamide tube gel electrophoresis of G-100 void volume peaks of chromatographed, SDS-released proteins from the pellets revealed quantitative but not qualitative differences in Coomassie Blue-gel staining patterns between T and NT roots. These results suggest that the SDS methods which we employed could solubilize the 80 000 X g pellet proteins but that combined gel permeation chromatography and tube gel electrophoresis were not sensitive enough to reveal whether AFTs could alter the types of proteins associated with the 80 000 X g pellet (purported plasmalemma).

The role of calcium ions in cytological effects of hypogravity

Electron-cytochemical and biochemical methods made it possible to reveal certain differences in ATPase activity stimulation by calcium ions in root apex cells of pea seedlings and moss protonema Funaria hygrometrica grown under stationary and slow clinostatic (2 rev/min) conditions. It was showed that under clinostatic conditions in comparison with the control variant the ATPase activity decreases in plasmalemma. The protein content in the plasmalemma fraction was also twice as low under these conditions. The root apex cells of the pea seedlings grown under spaceflight conditions were found to contain high concentrations of membrane-bound calcium. The data obtained are discussed in relation to problems of possible mechanisms of disturbance in calcium balance and the system of active calcium ion transport through plasmalemma under hypogravity.

Aflatoxin‐induced alteration in soybean membrane protein

AbstractIsolates of aflatoxin‐producing strains ofAspergillus can grow on Glycine max beans. Furthermore, both mixed aflatoxins and aflatoxin B1 inhibit the elongation of both attached and excised G. max roots. In addition, the toxin inhibits the capacity of the excised roots to take up [14C]leucine. This suggests that the toxin may inhibit the activity of an uptake system responsible for uptake of low‐molecular‐weight (MW) compounds by possibly altering membrane structure and/or function. In this connection, incubation of excised roots in media containing either mixed aflatoxins or aflatoxin B1 results in a diminution of acid‐insoluble protein, but neither sterol nor lipid phosphorus levels, of an 80,000 X g pellet (purported “crude” plasmalemma fraction). To provide preliminary evidence that mixed aflatoxins can decrease the amount of a specific plasmalemma protein (which might regulate the uptake of low‐MW compounds), we incubated roots with and without mixed aflatoxins and then gel‐filtrated integral proteins which were released by detergents from 80,000 X g pellets that had been obtained by differential centrifugation of Miracloth filtrates. The released proteins were gelfiltrated on Sephadex G‐100 columns. Sodium dodecyl sulfate, Triton X‐100 and Tween 20 each solubilized greater than 85% of the pellet's protein. Gel filtration yielded 280 nm absorbing void volume and retarded peaks for substances which were either precipitated by trichloroacetic acid (total protein) or solubilized by detergents (integral protein) from pellets that were derived from aflatoxin‐treated and nontreated roots. The amounts of protein which were recovered within column void volumes following gel filtration of 80,000 X g, acid‐precipitable protein were not significantly different for treated and nontreated roots, suggesting that incubation of roots with aflatoxins does not reduce the pellet's content of an acid‐precipitable, high‐MW (100,000 or more) protein. However, this conclusion is highly tentative as less than 15% of the acid‐insoluble protein which was layered onto the column was recovered. The amplitude of the void volume peaks for detergent‐released proteins from treated roots was consistently less than that for nontreated roots, suggesting that treatment of roots with aflatoxins diminishes the “crude” plasmalemma fraction's content of high‐MW protein(s). This suggestion was supported through calculation of the amounts of protein which were recovered within both the void volume and retarded peaks and comparing these to the total protein levels that were recovered from the columns. The amount of protein which was found within the void volume peak following gel filtration of either Triton X‐100 or Tween 20‐released proteins from pellets that were derived from treated roots was less than that for nontreated roots.

Intracellular distribution and origin of pentacyclic triterpenes in Calendula officinalis leaves

In Calendula officinalis leaves the cyclization of squalene to β-amyrin and its further oxidation to oleanolic acid as well as the biosynthesis of all derivatives of oleanolic acid 3-glucoside and some derivatives of oleanolic acid 3-glucuronoside occur in the microsomal fraction. The final metabolites of oleanolic acid 3-glucoside series i.e. pentaglycosides, are translocated from this fraction, one to the cell wall and plasmalemma fraction and the other to the cytosol. The derivatives of oleanolic acid 3-glucoronoside are synthesized partially in other fractions and accumulate in the different membraneous structures of the cell.

The molecular organization of nerve membranes: I. Isolation and characterization of plasma membranes from the retinal axons of the squid: an axolemma-rich preparation

Squid retinal nerves, because of their high ratio of axolemma to Schwann cell membrane, were used to develop a procedure to isolate plasma membranes. NADH oxidoreductase and (Na + K)-ATPase were studied as enzyme of plasma membranes in various fractions separated by differential centrifugation of hypotonic homogenates of retinal axons. The pellet which sedimented at 100,000 g (F-100) was selected as a crude plasmalemma fraction because of their highest specific activity for both enzymes. Chemical analysis of F-100 revealed a protein: cholesterol: phospholipids ratio of 37:9:29 as percent of membrane dry weight. RNA and DNA were 0.72 and 0.07%, respectively. Assays of mitochondrial and endoplasmic reticulum enzymes indicated that rare contamination of these structures was present in F-100. Negatively stained electron micrographs of F-100 revealed vesicles formed by concentric membranes. Further purification of F-100 was achieved by linear gradient centrifugatiou which separated two types of membranes. ATPase and NADH oxidoreductase were found in a first protein peak at 37% sucrose (PM 1), but mainly ATPase was found in the second peak at 27% sucrose (PM 2). Sections of osmium-fixed membranes studied under the electron microscope showed striking density of plasma membranes, 70–80 Å in thickness. Proteins from F-100, PM 1, and PM 2 were solubilized by various procedures and resolved either by sucrose-gradient centrifugation or acrylamide-gel electrophoresis. PM 1 and PM 2 differed in their number of protein bands, as well as in their uv spectrum and pH titration curve. These data together with the EM and the enzymic profile indicate that two types of plasma membranes chemically and structurally different were purified from the retinal axons of the squid. Their probable cellular origin is discussed.