Functional and Regulatory Properties of H+ Pumps at the Tonoplast and Plasma Membranes of Zea mays Coleoptiles

1984 ◽  
Vol 39 (9-10) ◽  
pp. 927-937 ◽  
Author(s):  
A. Hager ◽  
W. Biber
Keyword(s):  
Zea Mays ◽  
Atpase Activity ◽  
Plasma Membranes ◽  
Atp Hydrolysis ◽  
Osmotic Shock ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Pronounced Increase

Abstract A microsomal membrane fraction (6000 x g supernatant of a cell homogenate), isolated from coleoptiles of Zea mays, was separated by isopycnic sucrose density gradient centrifugation in the presence of EDTA and without a prior pelleting step to avoid irreversible sticking of different membrane species. The membrane fractions were characterized by assaying commonly used marker enzymes, and the levels of activity investigated of ATP hydrolysis, ATP-dependent H+ transport, and co- and countertransport of ions, such as Cl- , fumarate2-, K+ and Li+. The following results were obtained: (1) ATP hydrolysis is performed by different enzymes associated with different membranes: - vacuolar acid phosphatase (AP; inhibited by molybdate); - Golgi phosphatase, revealing IDPase, pNPase and ATPase activity (not inhibited by molybdate); - ATPase activity of residual submitochondrial particles (sensitive to azide); - a H+-translocating ATPase at tonoplast membranes (Km(ATP) = 0.29 mᴍ; pH 7.5; stimulated by uncouplers and completely inhibited by NO-3); - the H+-translocating ATPase of the plasmalemma (Km(ATP) = 0.39 mᴍ at pH 6.5, inhibited by vanadate, but not by NO-3). The latter activity is evident only after an osmotic shock, indicating that PL vesicles primarily exist as inside-in-vesicles. (2) ATP-fueled H+ pumps are localized at tonoplast (TO) and plasmalemma (PL) vesicles, they differ to some extent in their properties: (a) The PLH+ pump has a very narrow pH optimum and exhibits highest levels of activity at pH 6.5 with a pronounced increase of activity between pH 7.5 and 6.5 (properties, obviously important in vivo for the regulation of active H+-extrusion by certain growth substances, which affect the cytoplasmic pH (Hager and Moser, Planta 1984, in press); in contrast, TO H+ pumps show a considerably wider pH optimum with highest levels of activity around pH 7.5. (b) In variance to the PL H+ pump the activity of TO pumps (Km(ATP) = 0.24 mᴍ) is regulated via the oxidation state of essential thiol groups. Their oxidation to the S -S - form (e.g. by blue light in the presence of a flavin) causes an inactivation, whereas a re-reduction by GSH or cystein restores the activity [51]. (c) The ATP-fueled H+ transport into TO vesicles depends on an anion co-transport; most effective is Cl- , but there is also a stimulation by organic ions, C4 and C5 dicarboxylates, such as malate, succinate, fumarate, 2-oxoglutarate and aspartate; NO-3 is inhibitory. (d) H+-transport into sealed PL vesicles is also anion dependent. In this case, however, NO-3 is as effective as Cl-. (3) The TO membranes contain a H+/K+ exchange mechanism responsible for a secondary active K+ uptake into the vacuole. This mechanism could be the reason for a lower (ATP dependent) acidification of TO vesicles in the presence of K+ compared with Li+. - Similar effects are observed with plasmamembrane vesicles, but in this case there is still the question whether a H+/K+-exchange, a K+ channel, or both are acting.

scholarly journals Immunocytochemical localization and biochemical characterization of a novel plasma membrane-associated, neutral pH optimum α-l-fucosidase from rat testis and epididymal spermatozoa

1996 ◽  
Vol 318 (3) ◽  
pp. 821-831 ◽  
Author(s):  
Manuel AVILÉS ◽  
Irene ABASCAL ◽  
José Angel MARTÍNEZ-MENÁRGUEZ ◽  
María Teresa CASTELLS ◽  
Sheri R. SKALABAN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Gradient Centrifugation ◽  
Light And Electron Microscopy ◽  
Enzymic Activity ◽  
Epididymal Sperm ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Neutral Ph

1. Immunocytochemical and biochemical techniques have been used to localize and characterize a novel plasma membrane-associated, neutral-pH-optimum α-l-fucosidase from rat spermatozoa. Light and electron microscopy specifically localized the fucosidase on the plasma membrane of the convex region of the principal segment of testicular and cauda epididymal sperm heads. Immunoreactivity for α-l-fucosidase was also detected in the Golgi apparatus of spermatocytes and spermatids but no immunoreactivity was observed in the acrosome. 2. Fractionation of epididymal sperm homogenates indicated that over 90% of the α-l-fucosidase activity was associated with the 48000 g pellet. This pellet-associated activity could be solubilized with 0.5 M NaCl but not with 0.5% Triton X-100, suggesting that fucosidase is peripherally associated with membranes. Sucrose-density-gradient centrifugation of sperm homogenates indicated that fucosidase was enriched in the plasma membrane-enriched fraction. Analysis of α-l-fucosidase on intact epididymal sperm indicated that the enzyme was active, displayed linear kinetics and had a pH–activity curve (with an optimum near 7) which was comparable to that of fucosidase from epididymal sperm extracts. These results further suggest that fucosidase is associated with plasma membranes, and that its active site is accessible to fucoconjugates. Evidence that most of the fucosidase is associated with the exterior of the plasma membrane came from studies in which intact sperm had fucosidase activity comparable to that of sperm sonicates, and from studies in which approx. 90% of the fucosidase activity on intact sperm could be released from the sperm by gentle shaking with 0.5 M NaCl. Isoelectric focusing indicated that the NaCl-solubilized epididymal sperm fucosidase appears to have one major and one minor isoform with pIs near 7.2 and 5.2, respectively. SDS/PAGE and Western blotting indicated that the NaCl-solubilized extract of epididymal sperm contains two protein bands of 54 and 50 kDa which were highly immunoreactive with the IgG fraction of anti-fucosidase antibodies. Although the function of the novel sperm fucosidase is not known, its specific localization to the plasma membrane of the region of the rat sperm head involved in sperm–egg binding and its high enzymic activity at neutral pH on intact sperm suggest that this enzyme may have a role in sperm–egg interactions.

Tonoplast and plasma membrane ATPases from maize lines of high or low vigour

1992 ◽  
Vol 2 (2) ◽  
pp. 105-111 ◽  
Author(s):  
S. Sánchez-Nieto ◽  
R. Rodríguez-Sotres ◽  
P. González-Romo ◽  
I. Bernal-Lugo ◽  
M. Gavilanes-Ruíz
Keyword(s):  
Zea Mays ◽  
Plasma Membrane ◽  
Acid Phosphatase ◽  
Atpase Activity ◽  
Kinetic Analysis ◽  
Atp Hydrolysis ◽  
Membrane Atpases ◽  
Substrate Concentrations ◽  
Tonoplast Atpase ◽  
Specific Inhibitors

AbstractThe effectiveness of ATPase in germinated seed may play an important role in the vigour of germination. The activities of tonoplast and plasma membrane ATPases in two maize (Zea mays L.) lines with different vigour of germination were determined. ATP hydrolysis was measured in microsomal fractions from coleoptiles along with the responses to specific inhibitors for the plasma membrane, tonoplast and mitochondrial ATPases as well as for acid phosphatase. Nitrate-sensitive ATPase activity was 1.5–3.0 times lower in the low-vigour line than in the high-vigour line. Kinetic analysis of ATP hydrolysis at different substrate concentrations revealed the existence of two enzymes in the microsomal fractions of the two lines. The Vmax of enzyme 1 in the low-vigour line was a third of that in the high-vigour line. This enzyme was identified as the nitrate-sensitive or tonoplast ATPase on the basis of measurements of ATP hydrolysis in the presence of specific inhibitors at high (8.12mm) and low (0.77mm) ATP concentrations.

The centriolar complex isolated from starfish spermatozoa

1981 ◽  
Vol 49 (1) ◽  
pp. 33-49 ◽  
Author(s):  
R. Kuriyama ◽  
H. Kanatani
Keyword(s):  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Microtubule Assembly ◽  
High Concentration ◽  
Microtubule Organization ◽  
Sucrose Density Gradient Centrifugation ◽  
The Difference ◽  
Distal Centriole

Centrioles from spermatozoa of the starfish, Asterina pectinifera, were isolated and partially purified by solubilization of chromatin followed by sucrose density-gradient centrifugation. The ultrastructure of the isolated centriolar complex was investigated in whole mount preparations by electron microscopy. The complex unit was composed of a pair of centrioles and a pericentriolar structure, which associated with the distal end of the distal centriole by 9 spoke-like satellites extending radially to a marginal ring. Each satellite bifurcated at a dense node forming 2 fan-like shapes with a periodic striated pattern. The tubular structure of the centrioles easily disintegrated, leaving the pericentriolar structure or axonemal microtubules intact. The distal centriole in a spermatozoon served as an initiating site for flagellar microtubule assembly; that is, a number of “9 + 2′ axonemal tubules were observed adhering just beneath the distal end of the basal body. In experiments in vitro, polymerization of microtubule proteins purified from porcine brain was initiated by the structure at the ends of both proximal and distal centrioles, but not from the satellites or the marginal ring. Also, few if any microtubules were formed from the sides of each centriole, even in the presence of a high concentration of exogenous tubulin. On the other hand, centrioles of spermatozoa, when they were in mature ooplasm, could initiate the formation of sperm asters by microtubules. Therefore, centrioles in spermatozoa seem to be able to initiate microtubules in a 2 ways. A possible explanation of the difference between the 2 types of microtubule organization in vivo, i.e. in the sperm cell itself and in the ooplasm, it discussed.

3 ß-Hydroxysteroid Oxidoreductase in Suspension Cultures of Digitalis lanata EH RH

1990 ◽  
Vol 45 (9-10) ◽  
pp. 963-972 ◽  
Author(s):  
Hildegard Maria Warneck ◽  
Hanns Ulrich Seitz
Keyword(s):  
Incubation Temperature ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Cell Compartment ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Digitalis Lanata ◽  
Substrate Kinetics ◽  
Soluble Part

Abstract A 3 β-hydroxysteroid oxidoreductase was isolated and characterized in the microsomes of Digitalis lanata cell cultures. The enzyme catalyzes the conversion of 5α-pregnane-3,20-dione to 5a-pregnan-3 β-ol-20-one and requires NAD(P)H2. The enzyme was found to have a pH optimum of 80. The reaction had an optimum incubation temperature of 25 °C with linear reduction for the first 4 h, reaching maximum enzyme activity after 7 h. Substrate kinetics for 5a-pregnane-3,20-dione and NADPH2 resulted in apparent Km-values of 18.5-20 (µM for 5a-pregnane-3,20-dione and 50-120 µM for the co-substrate NADPH2. In order to localize 3β-hydroxysteroid oxidoreductase differential centrifugation as well as linear sucrose density gradient centrifugation were performed. The results obtained lead to the conclusion that 3β-hydroxysteroid oxidoreductase is not associated with a single cell compartment, but consists of a major soluble part and a markedly smaller part of endoplasmic reticulum-associated activity

scholarly journals Separation and Paired Proteome Profiling of Plant Chloroplast and Cytoplasmic Ribosomes

Plants ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 892
Author(s):  
Alexandre Augusto Pereira Firmino ◽  
Michal Gorka ◽  
Alexander Graf ◽  
Aleksandra Skirycz ◽  
Federico Martinez-Seidel ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Gradient Centrifugation ◽  
Leaf Tissue ◽  
Proteome Analysis ◽  
Sucrose Density Gradient ◽  
Cross Linking ◽  
Sucrose Density Gradient Centrifugation ◽  
Cytoplasmic Ribosome ◽  
Plant Chloroplast

Conventional preparation methods of plant ribosomes fail to resolve non-translating chloroplast or cytoplasmic ribosome subunits from translating fractions. We established preparation of these ribosome complexes from Arabidopsis thaliana leaf, root, and seed tissues by optimized sucrose density gradient centrifugation of protease protected plant extracts. The method co-purified non-translating 30S and 40S ribosome subunits separated non-translating 50S from 60S subunits, and resolved assembled monosomes from low oligomeric polysomes. Combining ribosome fractionation with microfluidic rRNA analysis and proteomics, we characterized the rRNA and ribosomal protein (RP) composition. The identity of cytoplasmic and chloroplast ribosome complexes and the presence of ribosome biogenesis factors in the 60S-80S sedimentation interval were verified. In vivo cross-linking of leaf tissue stabilized ribosome biogenesis complexes, but induced polysome run-off. Omitting cross-linking, the established paired fractionation and proteome analysis monitored relative abundances of plant chloroplast and cytoplasmic ribosome fractions and enabled analysis of RP composition and ribosome associated proteins including transiently associated biogenesis factors.

Localization of Atrazine in Corn (Zea mays), Oat (Avena sativa), and Kidney Bean (Phaseolus vulgaris) Leaf Cells

Weed Science ◽  
1983 ◽  
Vol 31 (5) ◽  
pp. 664-671 ◽  
Author(s):  
Robert F. Norris ◽  
Ivy E. Fong
Keyword(s):  
Zea Mays ◽  
Phaseolus Vulgaris ◽  
Avena Sativa ◽  
Gradient Centrifugation ◽  
Leaf Tissue ◽  
Kidney Bean ◽  
Vascular Bundles ◽  
Sucrose Density Gradient Centrifugation ◽  
Test Species ◽  
Subcellular Organelles

Corn (Zea maysL. ‘DeKalb 640′), kidney beans (Phaseolus vulgarisL. ‘Light red’) and oats (Avena sativaL. ‘Kanota’) were grown in solution culture and the roots exposed to radioactively labeled atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine]. The radioactivity from3H-atrazine, or its metabolites, was located in microautoradiographs of corn leaf sections, almost exclusively at the perimeter of the vascular bundles, and was primarily confined to the cell walls and intercellular spaces, with no activity associated with subcellular organelles. Radioactivity from3H-atrazine in oat leaf section microautoradiographs was primarily associated with the chloroplasts, with no evidence of radioactivity specifically associated with other subcellular organelles. Sucrose density gradient centrifugation of homogenized leaf tissue following exposure of the plants to14C-atrazine showed that most of the radioactivity was associated with the light cell fragments and cytoplasm for the test species. There was no accumulation of14C-activity in any of the heavier subcellular organelles from corn leaf cells. A peak of radioactivity was associated with the chloroplast fraction from oat and kidney bean leaves.

Anion-stimulated ATPase activity of brush border from rat small intestine.

1979 ◽  
Vol 236 (1) ◽  
pp. E70 ◽  
Author(s):  
M H Humphreys ◽  
L Y Chou
Keyword(s):  
Alkaline Phosphatase ◽  
Atpase Activity ◽  
Brush Border ◽  
Atp Hydrolysis ◽  
Intestinal Transport ◽  
Mitochondrial Enzymes ◽  
Intestinal Alkaline Phosphatase ◽  
Ph Optimum ◽  
Low Concentrations ◽  
Small Intestinal

Differential centrifugation of rat small intestinal homogenates produced a crude brush border (BB) fraction that was enriched 15-fold for the marker enzymes, alkaline phosphatase and sucrase; contamination with mitochondrial enzymes, monoamine oxidase and succinate dehydrogenase, was minimal. ATP hydrolysis by this BB fraction was stimulated by addition of several anions to the incubation medium: HCO3 and Cl were equally effective in this regard, with NO3, NO2, SO4, and acetate being less stimulatory. SCN and CNO inhibited ATPase activity, whereas the divalent anion SO3 was stimulatory at low concentrations (less than 25 mM) but inhibitory at 100 mM. Maximum anion stimulation was observed at a Mg concentration of 0.5 mM, and pH optimum was 8.5. Kinetic analysis showed that HCO3 increased the Vmax without altering the Km for ATP; the Ka for this effect of HCO3 was 35 mM. This enzyme activity was completely inhibited by 20 mM L-phenylalanine, 10 mM L-cysteine, and 3 mM EDTA, compounds that also inhibited intestinal alkaline phosphatase. These results demonstrate the presence of anion-stimulated ATPase activity in rat small intestinal brush border and suggest that this activity may be related to intestinal alkaline phosphatase. The role of this enzyme in intestinal transport is not known, but could relate to the regulation of intestinal absorption and secretion.

Mass spectrometry is a powerful tool for identification of proteins associated with lipid rafts of Jurkat T-cell line

2004 ◽  
Vol 32 (5) ◽  
pp. 777-779
Author(s):  
P. Pompach ◽  
P. Man ◽  
P. Novák ◽  
V. Havlíček ◽  
A. Fišerová ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Extraction Procedure ◽  
Sucrose Density Gradient ◽  
Cellular Adhesion ◽  
Jurkat Cells ◽  
Membrane Microdomains ◽  
Sucrose Density Gradient Centrifugation ◽  
Biologically Relevant ◽  
Jurkat T Cell

Many proteins involved in signal-transduction pathways are concentrated in membrane microdomains enriched in lipids with distinct physical properties. Since these microdomains are insoluble in non-ionic detergents in cold, proteins associated with them could be efficiently purified by techniques such as sucrose-density gradient centrifugation. The complexity of the resulting protein mixture requires powerful MS technique for its analysis. We have found that successful identification of biologically relevant proteins is critically dependent on the enrichment of the starting material (plasma membranes), and on the extraction procedure. Applying these conditions in combination with microHPLC-ESI (electrospray ionization)-MS/MS, we have identified proteins involved in signalling, cytoskeletal association and cellular adhesion in Jurkat cells that are not stimulated by any antibody incubation.

scholarly journals The Walker B motif of the second nucleotide-binding domain (NBD2) of CFTR plays a key role in ATPase activity by the NBD1–NBD2 heterodimer

2006 ◽  
Vol 401 (2) ◽  
pp. 581-586 ◽  
Author(s):  
Fiona L. L. Stratford ◽  
Mohabir Ramjeesingh ◽  
Joanne C. Cheung ◽  
Ling-JUN Huan ◽  
Christine E. Bear
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Vital Role ◽  
Atp Binding ◽  
Primary Role ◽  
Binding Domains ◽  
Membrane Spanning ◽  
Atp Binding And Hydrolysis

CFTR (cystic fibrosis transmembrane conductance regulator), a member of the ABC (ATP-binding cassette) superfamily of membrane proteins, possesses two NBDs (nucleotide-binding domains) in addition to two MSDs (membrane spanning domains) and the regulatory ‘R’ domain. The two NBDs of CFTR have been modelled as a heterodimer, stabilized by ATP binding at two sites in the NBD interface. It has been suggested that ATP hydrolysis occurs at only one of these sites as the putative catalytic base is only conserved in NBD2 of CFTR (Glu1371), but not in NBD1 where the corresponding residue is a serine, Ser573. Previously, we showed that fragments of CFTR corresponding to NBD1 and NBD2 can be purified and co-reconstituted to form a heterodimer capable of ATPase activity. In the present study, we show that the two NBD fragments form a complex in vivo, supporting the utility of this model system to evaluate the role of Glu1371 in ATP binding and hydrolysis. The present studies revealed that a mutant NBD2 (E1371Q) retains wild-type nucleotide binding affinity of NBD2. On the other hand, this substitution abolished the ATPase activity formed by the co-purified complex. Interestingly, introduction of a glutamate residue in place of the non-conserved Ser573 in NBD1 did not confer additional ATPase activity by the heterodimer, implicating a vital role for multiple residues in formation of the catalytic site. These findings provide the first biochemical evidence suggesting that the Walker B residue: Glu1371, plays a primary role in the ATPase activity conferred by the NBD1–NBD2 heterodimer.

scholarly journals Cellular gene transfer mediated by influenza virosomes with encapsulated plasmid DNA

2007 ◽  
Vol 405 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Jørgen de Jonge ◽  
Johanna M. Leenhouts ◽  
Marijke Holtrop ◽  
Pieter Schoen ◽  
Peter Scherrer ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Gradient Centrifugation ◽  
Cationic Lipid ◽  
Sucrose Density Gradient ◽  
Target Cells ◽  
Sucrose Density Gradient Centrifugation ◽  
Viral Membrane ◽  
Viral Envelopes

Reconstituted influenza virosomes (virus membrane envelopes) have been used previously to deliver pDNA (plasmid DNA) bound to their external surface to a variety of target cells. Although high transfection efficiencies can be obtained with these complexes in vitro, the virosome-associated DNA is readily accessible to nucleases and could therefore be prone to rapid degradation under in vivo conditions. In the present study, we show a new method for the production of DNA–virosomes resulting in complete protection of the DNA from nucleases. This method relies on the use of the short-chain phospholipid DCPC (dicaproylphosphatidylcholine) for solubilization of the viral membrane. The solubilized viral membrane components are mixed with pDNA and cationic lipid. Reconstitution of the viral envelopes and simultaneous encapsulation of pDNA is achieved by removal of the DCPC from the mixture through dialysis. Analysis by linear sucrose density-gradient centrifugation revealed that protein, phospholipid and pDNA physically associated to particles, which appeared as vesicles with spike proteins inserted in their membranes when analysed by electron microscopy. The DNA–virosomes retained the membrane fusion properties of the native influenza virus. The virosome-associated pDNA was completely protected from degradation by nucleases, providing evidence for the DNA being highly condensed and encapsulated in the lumen of the virosomes. DNA–virosomes, containing reporter gene constructs, transfected a variety of cell lines, with efficiencies approaching 90%. Transfection was completely dependent on the fusogenic properties of the viral spike protein haemagglutinin. Thus, DNA–virosomes prepared by the new procedure are highly efficient vehicles for DNA delivery, offering the advantage of complete DNA protection, which is especially important for future in vivo applications.

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