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R T Leonard

Publications and source records attributed to R T Leonard.

33 records · Page 2Linked to original sources
PubMed1979

Ion Transport in Isolated Protoplasts from Tobacco Suspension Cells: III. Membrane Potential.

The membrane electrical potential difference was measured in cultured cells and isolated protoplasts of tobacco (Nicotiana glutinosa L.) by inserting a microelectrode into cells held fast by a suction micropipette. The potential difference (+/- standard deviation) for unplasmolyzed tobacco cells was -52 +/- 12 millivolts, for cells in 0.3 molar mannitol, -50 +/- 11 millivolts; and for cells plasmolyzed in 0.7 molar mannitol, -49 +/- 12 millivolts all inside negative. The potential difference for isolated protoplasts in 0.7 molar mannitol was -49 +/- 16 millivolts, inside negative. In both cultured cells and protoplasts, the addition of 0.1 millimolar KCN caused a depolarization of the membrane potential. It was concluded that plasmolysis and enzymic release of the protoplast had no significant effect on the membrane potential of cultured tobacco cells.

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PubMed1978

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 x 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 mug/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.

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PubMed1978

Purification of plasma membranes from roots of barley: specificity of the phosphotungstic Acid-chromic Acid stain.

Plasma membrane vesicles from roots of barley (Hordeum vulgare L., var. Arivat) had an equilibrium density in sucrose of about 1.16 grams per cubic centimeter, but could not be purified satisfactorily with the procedure developed for roots of other plant species. The reported procedure involving differential centrifugation to remove mitochondria (peak density of 1.18 grams per cubic centimeter) and subsequent density gradient centrifugation to purify plasma membrane vesicles was modified to include a narrower differential centrifugation fraction (13,000 to 40,000g instead of 13,000 to 80,000g) and a narrower density range in the sucrose gradient (1.15 to 1.18 grams per cubic centimeter instead of 1.15 to 1.20 grams per cubic centimeter). The fraction obtained by the modified procedure was between 60 and 70% pure as determined by staining with the phosphotungstic acid-chromic acid procedure, which was judged to be reliable for identifying plasma membrane vesicles in subcellular fractions from barley roots. The plasma membrane fraction was enriched in K(+)-stimulated ATPase activity at pH 6.5. The presence of nonspecific ATP-hydrolyzing activity in the plasma membrane fraction made it difficult to determine if the ATPase had properties in common with those reported for cation absorption in barley roots.

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PubMed1976

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 x 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.A major peak of ATPase activity at pH 6.5 was observed at low density in Ficoll after nonequilibrium centrifugation in a combination Ficoll-sucrose gradient. Twenty to forty percent of the vesicles in this ATPase fraction stained positively for plasma membranes, and with equilibrium centrifugation the major portion of the ATPase activity shifted to densities in sucrose which were characteristic of plasma membranes. All major vesicular ATPase activities observed in Ficoll or sucrose contained substantial amounts of plasma membranes. For unknown reasons, mitochondria and plasma membranes equilibrated over a broader density range and at lower peak densities in sucrose as a result of equilibrium centrifugation through Ficoll.

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PubMed1976

Association of latent cellulase activity with plasma membranes from kidney bean abscission zones.

Membranes isolated from abscission zones of Phaseolus vulgaris L., cv. Red Kidney, contained cellulase activity. This particulate activity was enhanced 10- to 20-fold by treatment with Triton X-100. Sucrose density gradient analyses of cell fractions showed that the membranes with which cellulase was associated had a peak equilibrium density of 1.16 to 1.17 g/cm(3) which coincided with that of ion-activated ATPase, a marker for plasma membranes. The membrane fraction having the highest cellulase activity also contained a high proportion of plasma membranes as shown by electron microscopy of sucrose density gradient fractions after staining by periodic acid-chromic acid-phosphotungstic acid. It was concluded that the particulate cellulase was associated with the plasma membrane.

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PubMed1976

Cation-stimulated Adenosine Triphosphatase Activity and Cation Transport in Corn Roots.

ATPase activity of the plasma membrane fraction from primary roots of corn (Zea mays L. WF9 x M14) was activated by Mg(2+) and further stimulated by monovalent cations (K(+) > Rb(+) > Cs(+) > Na(+) > Li(+)). K(+)-stimulated activity required Mg(2+) and was substrate-specific. Maximum ATPase activity in the presence of Mg(2+) and K(+) was at pH 6.5 and 40 C. Calcium and lanthanum (<0.5 mm) were inhibitors of ATPase, but only in the presence of Mg(2+). Oligomycin was not an inhibitor of the plasma membrane ATPase, whereas N,N'-dicyclohexylcarbodiimide was. Activity showed a simple Michaelis-Menten saturation with increasing ATP.Mg. The major effect of K(+) in stimulating ATPase activity was on maximum velocity. The kinetic data of K(+) stimulation were complex, but similar to the kinetics of short term K(+) influx in corn roots. Both K(+)-ATPase and K(+) influx kinetics met all criteria for negative cooperativity. The results provided further support for the concept that cation transport in plants is energized by ATP, and mediated by a cation-ATPase on the plasma membrane.

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PubMed1975

Effect of lanthanum on ion absorption in corn roots.

Short term (10 min) influx of (86)Rb-labeled potassium into corn (Zea mays L. WF9 x M14) root segments was inhibited by La (NO(3))(3) or LaCl(3). Half maximal inhibition of K(+) influx from 0.25 mm KCl was obtained with 0.025 mm La(3+). Kinetic analysis indicated the inhibition to be of a competitive nature. With absorption periods exceeding one hour, La(3+) no longer inhibited, but rather stimulated K(+) influx rates. La(3+) was not an inhibitor of (36)Cl or (32)P absorption. Separated cortex and stele absorbed labeled potassium (and phosphate) at comparable rates, and La(3+) inhibited potassium influx in both tissues. The effects of La(3+) on ion absorption were similar to those of Ca(2+), suggesting that the two polyvalent cations act at the same site. Based on this and the observation that La(3+) does not seem to penetrate the plasma membrane, it was concluded that La(3+) and Ca(2+) affect changes in ion transport without entering cells.

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PubMed1973

Membrane-bound Adenosine Triphosphatase Activities of Oat Roots.

Homogenates of oat (Avena sativa cv. Goodfield) roots contained at least five membrane-associated adenosine triphosphatase (ATPase) activities. The membrane-bound ATPases were separated on sucrose gradients and distinguished by membrane density, pH optima, sensitivity to monovalent salts, and substrate specificity.A membrane fraction sedimenting at low centrifugal force (13,000g) contained two ATPase activities at pH 9.0. One membrane ATPase was coincident with cytochrome c oxidase activity and had a density of 1.18 grams per cubic centimeter. This membrane system was identified as mitochondria. The other pH 9.0 ATPase in this fraction occurred at a density of 1.16 grams per cubic centimeter. The identity of this membrane is unknown.Three additional ATPases were in a membrane fraction sedimenting at high centrifugal forces (13,000-80,000g). One membrane ATPase coincided with NADH-cytochrome c reductase activity, had a density of about 1.09 grams per cubic centimeter, and was equally active at pH 6.0 and 9.0. A second membrane ATPase of the 13,000 to 80,000g fraction had a density of 1.13 grams per cubic centimeter and was more active at pH 9.0 than at pH 6.0. A third membrane ATPase had greater activity at pH 6.0 than at pH 9.0, and the membrane had an apparent density of 1.17 grams per cubic centimeter on the sucrose gradient. This ATPase was especially sensitive to KCI. The identity of the membranes which contain ATPases is discussed in relation to the distribution of other enzymes on the gradient.

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PubMed1973

Characterization of Plasma Membrane-associated Adenosine Triphosphase Activity of Oat Roots.

ATPase activity of plasma membranes isolated from oat (Avena sativa L. cv. Goodfield) roots was activated by divalent cations (Mg(2+) = Mn(2+) > Zn(2+) > Fe(2+) > Ca(2+)) and further stimulated by KCl and a variety of monovalent salts, both inorganic and organic. The enzyme exhibited greater specificity for cations than anions. The presence of Mg(2+) was necessary for KCl stimulation. Ca(2+) was ineffective in replacing Mg(2+) for activation of plasma membrane ATPase, but it did activate other membrane-bound ATPases. The pH optima for Mg(2+) activation and KCl stimulation of the plasma membrane ATPase were 7.5 and 6.5, respectively.The plasma membrane ATPase showed little synergistic effects of K(+) and Na(+), and it was only slightly sensitive to ouabain. Oligomycin did not inhibit the ATPase, while N,N'-dicyclohexylcarbodiimide was a potent inhibitor of the enzyme.The apparent Km for Mg(2+) activation (0.84 mm) of the ATPase was about twice that of the apparent Km for ATP (0.38 mm). The effect of KCl in stimulating the enzyme was not on the apparent Km values for ATP and Mg(2+) but rather on maximum velocity. The kinetics of KCl stimulation of the plasma membrane ATPase were similar to the kinetics of (42)K(+) influx into oat roots and neither followed the Michaelis-Menten equation but rather were best described by a single activity curve with continually changing kinetic parameters. These results support the concept that cation transport at the plasma membrane of root cells is coupled to a cation-activated ATPase which is functional from low (0.01 mm) to high (50.0 mm) concentrations of KCl.

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PubMed1972

Purification of an ion-stimulated adenosine triphosphatase from plant roots: association with plasma membranes.

A membrane-bound adenosine triphosphatase (EC 3.6.1.3) that requires Mg(++) and that is stimulated by monovalent ions has been purified 7- to 8-fold from homogenates of oat (Avena sativa L. Cult. Goodfield) roots by discontinuous sucrose-gradient centrifugation. The enzyme was substrate specific; adenosine triphosphate was hydrolyzed 25 times more rapidly than other nucleoside triphosphates. The membrane fraction containing adenosine triphosphatase was enriched in plasma membranes, which were identified by the presence of a glucan synthetase (EC 2.4.1.12), a high sterol to phospholipid ratio, and by a stain consisting of periodic acid, chromic acid, and phosphotungstic acid that is specific for plant plasma membranes. Oat-root plasma membranes and the associated adenosine triphosphatase were purified on either a 6-layer discontinuous sucrose gradient or on a simplified gradient consisting of only two sucrose layers.These results represent the first demonstration that plant plasma membranes contain an adenosine triphosphatase that is activated by monovalent ions, and this finding further implicates the enzyme in the absorption of inorganic ions by plant roots.

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PubMed1972

Induction and development of increased ion absorption in corn root tissue.

Washing excised or intact primary roots of corn (Zea mays L., WF9 x M14) in aerated distilled water or dilute salt solutions for 2 hours induced doubling of the rate of accumulation of various nutrient ions and solutes. This response to washing depended upon aerobic metabolism, but involved no increase in aerobic respiration. Excision of root tissue was not required as the effect could be obtained with intact root systems. Increased phosphate absorption followed after a lag period of 30 to 40 minutes and continued for 6 hours before leveling off at about 3.5 times the initial rate. Chloramphenicol was not inhibitory to the development of increased absorption, while inhibitors of RNA and protein synthesis were. Auxins and kinetin were also inhibitory, but so was the antiauxin, 2,4,6-trichlorophenoxyacetic acid.Kinetic analyses of phosphate and potassium ((86)Rb) absorption showed no change in apparent Michaelis constant. Electron microscopic examination and analysis for membrane protein and lipid-phosphate content after washing showed no proliferation of membrane or other changes in cell structure and composition. It appears that washing augments or activates existing membrane transport mechanisms by processes involving protein synthesis.

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PubMed1972

Increased Membrane-bound Adenosine Triphosphatase Activity Accompanying Development of Enhanced Solute Uptake in Washed Corn Root Tissue.

Washing of excised corn (Zea mays L., variety WF9xM14) root tissue is accompanied by an increase in (Mg(2+) + K(+))-stimulated adenosine triphosphatase. This is the adenosine triphosphatase described by Fisher, Hansen, and Hodges as positively correlated with ion accumulation rates. The increase in activity is confined to the microsomal fraction. A close parallel exists between increases in adenosine triphosphatase and phosphate absorption, and they respond similarly to inhibitors of RNA and protein synthesis. However, the amplitude of change is much smaller in adenosine triphosphatase. Possible reasons for this discrepancy are discussed.

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