Characteristics of vanadate-sensitive ATPase activities migrating to different densities on a sucrose gradient in microsomes from Acer pseudoplatanus cells

Patti, Vittoria; Curti, G; Lado, P.

doi:10.1016/0168-9452(92)90215-8

Cited by 3 publications

(2 citation statements)

References 23 publications

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“…A preliminary experiment showed that the specific activity of the PPase in a tonoplast-enriched fracti'On (prepared according to Patti et al, 1992) was higher in adapted tlian in unadapted cells confirming the results reported above (data not shown).…”

Section: Resultssupporting

confidence: 86%

“…Protoplasts were prepared from cells by enzyrnatic digestion of the wall (Patti et al 1992). The pelleted pro-toplasts were suspended in a medium contaitiing 30 mM Tris-MES (pH 7.5), 5 mM EGTA, 3 mM DTT, 1 mM PMSF, 3 mM MgSOj, 0.5% (w/v) BSA and 500 mM mannitol, counted in a Nageotte chamber, and diluted with an appropriate volame of the same medium in order to obtain a suspetision of about 6x10*' protoplasts ml"'.…”

Section: Isolation Of Protoplasts and Preparation Of Microsomal Fractionmentioning

confidence: 99%

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Enhanced K+-stimulated pyrophosphatase activity in NaCl-adapted cells of Acer pseudoplatanus

Zingarelli¹,

Anzani²,

Lado³

1994

Physiol Plant

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P. aod Lado. P, 1994. Enhanced K*-st!mulated pyropbosphatase activity in NaCl-adapted cells of Acer pseudoplaiatms, Cell suspension cultures of Acer pseudoplatanus L. (Bligny cell line) adapted to growth in the presence of NaCi, are a useful tool for investigating mechanistns for cellular salt tolerance. We eotnpared the activities of vana"date-sensitive {plasma membrane) and nitrate-sensitive (tonoplast) ATPases, and tonoplast K+-stimulated PPase in microsomal fractions (8()0O-108 000 g) from unadapted and NaCi-adapted (80 mtW) cells of A. pseudoplaiatms. Since NaCI reduces the growth rate of the culture, the two cell lines were harvested and compared at both the same cellulai' density and at the same growth phase (middle exponential phase or beginning of the stationary phase). The ATPase activity of the plasma membrane (expressed both on the hasis of protoplast number and in relation to protein contenl) was not affected by the adaptation to salinity. The two enzyme activities of the tonoplast (mainly as expressed on a protein basis) were higher in adapted than in unadapted ceHs. However, a preferential increase in PPase activity took place, although the pH dependeoce, ionic requirements, and apparent Kn, of the PPase activity were the same in the two cell lines. The three enzyme activities showed different sensitivities to detergents such as Triton X-100. Brij 58 and lysophospbatidylcholine (LPC). The stimulation of K*-stimulated PPase activity by detergents was higher in adapted than in unadapted cells. This suggests that the salt-induced enhancement of the PPase activity might partially depend on a modification of the iipid component of the tonoplast.

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Section: Resultssupporting

confidence: 86%

Section: Isolation Of Protoplasts and Preparation Of Microsomal Fractionmentioning

confidence: 99%

Enhanced K+-stimulated pyrophosphatase activity in NaCl-adapted cells of Acer pseudoplatanus

Zingarelli¹,

Anzani²,

Lado³

1994

Physiol Plant

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Plasma Membrane Atpase Isolated From Green Bell Pepper Fruit by Two-Phase Partitioning or Sucrose Density Gradient

et al. 1994

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Plasma membrane was isolated from mature green bell pepper fruit by two-phase partitioning or by sucrose density gradient. The yield of plasma membrane was higher from the sucrose density gradient, but the two-phase system was less contaminated by other membranes, particularly those from chloroplasts and mitochondria. In the two-phase partitioned membranes, ATPase activity was stimulated by Triton X-100 by 100% and in sucrose density gradient membranes by 40%. Plasma membranes from two-phase partitioning exhibited simultaneous proton pumping and ATP hydrolysis, while the sucrose density purified membranes did not. Immunoblotting with ATPase antibody showed enrichment of plasma membrane ATPase in both the U3 phase of the two-phase system, and the 34% sucrose fraction of the sucrose gradient. However, the two-phase partitioned membranes were superior to those prepared by sucrose density for investigating functions of the ATPase.

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Enhanced K⁺‐stimulated pyrophosphatase activity in NaCl‐adapted cells of Acer pseudoplatanus

Zingarelli

Anzani

Lado

1994

Physiologia Plantarum

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Cell suspension cultures of Acer pseudoplatanus L. (Bligny cell line) adapted to growth in the presence of NaCl, are a useful tool for investigating mechanisms for cellular salt tolerance. We compared the activities of vanadate‐sensitive (plasma membrane) and nitrate‐sensitive (tonoplast) ATPases, and tonoplast K+‐stimulated PPase in microsomal fractions (8000–108 000 g) from unadapted and NaCl‐adapted (80 mM) cells of A. pseudoplatanus. Since NaCl reduces the growth rate of the culture, the two cell lines were harvested and compared at both the same cellular density and at the same growth phase (middle exponential phase or beginning of the stationary phase). The ATPase activity of the plasma membrane (expressed both on the basis of protoplast number and in relation to protein content) was not affected by the adaptation to salinity. The two enzyme activities of the tonoplast (mainly as expressed on a protein basis) were higher in adapted than in unadapted cells. However, a preferential increase in PPase activity took place, although the pH dependence, ionic requirements, and apparent Km of the PPase activity were the same in the two cell lines. The three enzyme activities showed different sensitivities to detergents such as Triton X‐100, Brij 58 and lysophosphatidylcholine (LPC). The stimulation of K+‐stimulated PPase activity by detergents was higher in adapted than in unadapted cells. This suggests that the salt‐induced enhancement of the PPase activity might partially depend on a modification of the lipid component of the tonoplast.

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Characteristics of vanadate-sensitive ATPase activities migrating to different densities on a sucrose gradient in microsomes from Acer pseudoplatanus cells

Cited by 3 publications

References 23 publications

Enhanced K+-stimulated pyrophosphatase activity in NaCl-adapted cells of Acer pseudoplatanus

Enhanced K+-stimulated pyrophosphatase activity in NaCl-adapted cells of Acer pseudoplatanus

Plasma Membrane Atpase Isolated From Green Bell Pepper Fruit by Two-Phase Partitioning or Sucrose Density Gradient

Enhanced K⁺‐stimulated pyrophosphatase activity in NaCl‐adapted cells of Acer pseudoplatanus

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Characteristics of vanadate-sensitive ATPase activities migrating to different densities on a sucrose gradient in microsomes from Acer pseudoplatanus cells

Cited by 3 publications

References 23 publications

Enhanced K+-stimulated pyrophosphatase activity in NaCl-adapted cells of Acer pseudoplatanus

Enhanced K+-stimulated pyrophosphatase activity in NaCl-adapted cells of Acer pseudoplatanus

Plasma Membrane Atpase Isolated From Green Bell Pepper Fruit by Two-Phase Partitioning or Sucrose Density Gradient

Enhanced K+‐stimulated pyrophosphatase activity in NaCl‐adapted cells of Acer pseudoplatanus

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Enhanced K⁺‐stimulated pyrophosphatase activity in NaCl‐adapted cells of Acer pseudoplatanus