Simultaneous measurement of multiple membrane ATPases in microtiter plates

Sadrzadeh, S.M. Hossein; Vincenzi, Frank F.; Hinds, Thomas R.

doi:10.1016/1056-8719(93)90013-5

Cited by 27 publications

(10 citation statements)

References 26 publications

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“…Membrane ATPases activity were measured simultaneously in multi-well plates (as described by Sadrzadeh et al, 1993). Briefly, membranes were washed with assay buffer following incubation with different agents.…”

Section: Atpase Assaysmentioning

confidence: 99%

Green tea metabolite EGCG protects membranes against oxidative damage in vitro

2004

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Section: Atpase Assaysmentioning

confidence: 99%

Green tea metabolite EGCG protects membranes against oxidative damage in vitro

2004

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“…This indirect measure of activity was performed by measurement of the net amount of inorganic phosphate (P i ) formed (Sadrzadeh et al, 1993). Decreased UV-absorbance (810 nm) of 2.8 h and 3.4 h PMI sample sets compared to 0 h PMI samples denotes decreased Mg 2+ ATPase activity ( Fig.…”

Section: Measurement Of Mg 2+ Atpase Activity In Mouse Synaptosomesmentioning

confidence: 99%

“…Then, 5 µl of 5% SDS and 125 µL of color reagent (0.36 g ascorbic acid mixed with 15 ml of molybdate acid solution) were added to each well. The activity of Mg 2+ ATPase was measured with a UV plate reader as the amount of phosphate produced by the enzyme (Sadrzadeh et al, 1993).…”

Section: Mouse Brain Tissuementioning

confidence: 99%

“…Mg 2+ ATPase activity-This assay was completed according to previous procedures using FVB mice (Castegna et al, 2004;Sadrzadeh et al, 1993). Synaptosome aliquots (7.5 µg) were suspended in ATPase assay buffer (18 mM histidine, 18 mM imidazole, 80 mM NaCl, 15 mM KCl, 3 mM MgCl 2 , 0.1 mM EGTA, pH 7.1) and treated with 0.1 mM of the Na + K + ATPase inhibitor ouabain to a final volume of 100 µl in a microtiter plate.…”

Section: Mouse Brain Tissuementioning

confidence: 99%

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Loss of phospholipid asymmetry and elevated brain apoptotic protein levels in subjects with amnestic mild cognitive impairment and Alzheimer disease

Lange

Cenini

Piroddi

et al. 2008

Neurobiology of Disease

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Oxidative stress, a hallmark of Alzheimer disease (AD), has been shown to induce lipid peroxidation and apoptosis disrupting cellular homeostasis. Normally, the aminophospholipid phosphatidylserine (PtdSer) is asymmetrically distributed on the cytosolic leaflet of the lipid bilayer. Under oxidative stress conditions, asymmetry is altered, characterized by the appearance of PtdSer on the outer leaflet, to initiate the first stages of an apoptotic process. PtdSer asymmetry is actively maintained by the ATP-dependent translocase flippase, whose function is inhibited if covalently bound by lipid peroxidation products, 4-hydroxynonenal (HNE) and acrolein, within the membrane bilayer in which they are produced. Additionally, pro-apoptotic proteins Bax and caspase-3 have been implemented in the oxidative modification of PtdSer resulting in subsequent asymmetric collapse, while antiapoptotic protein Bcl-2 has been found to prevent this process.The current investigation focused on detection of PtdSer on the outer leaflet of the bilayer in synaptosomes from brain of subjects with AD and amnestic mild cognitive impairment (MCI), as well as expression levels of apoptosis-related proteins Bcl-2, Bax, and caspase-3. Fluorescence and Western blot analysis suggest PtdSer exposure on the outer leaflet is significantly increased in brain from subjects with MCI and AD contributing to early apoptotic elevation of pro-and anti-apoptotic proteins and finally neuronal loss. MCI is considered a possible transition point between normal cognitive aging and probable AD. Brain from subjects with MCI is reported to have increased levels of tissue oxidation; therefore, the results of this study could mark the progression of patients with MCI into AD. This study contributes to a model of apoptosis-specific oxidation of phospholipids consistent with the notion that PtdSer exposure is required for apoptotic-cell death.

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“…Radioactivity in the column flow-through, reflecting [ 3 H]argininosuccinate, was added to In-Flow BD liquid scintillant (IN/US Systems Inc.) and quantified by liquid scintillation spectrometry. Additionally, purified AS activity was assessed by measuring the formation of inorganic phosphate by the method of Fiske and Subbarow (36), as previously adapted to the 96-well microtiter plate format (37). Incubation mixtures were identical to above, except they were scaled to 100 l total volume, the ATP concentration was increased to 300 M, and the ATP regenerating system was omitted (i.e.…”

Section: Preparation Of Smooth Muscle Cell Cytosol and Membranementioning

confidence: 99%

Argininosuccinate Synthetase Overexpression in Vascular Smooth Muscle Cells Potentiates Immunostimulant-induced NO Production

Xie¹,

Gross²

1997

Journal of Biological Chemistry

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Immunostimulants trigger vascular smooth muscle cells (VSMC) to express both the inducible isoform of NO synthase (iNOS) and argininosuccinate synthetase (AS).With constitutively expressed argininosuccinate lyase (AL), AS confers cells with an Arg/Cit cycle that can sustain NO production via continuous regeneration of the NOS substrate, L-arginine (Arg), from the NOS coproduct, L-citrulline (Cit). To assess whether NO synthesis can be rate-limited by Arg recycling, we tested whether AS-overexpressing cells have an enhanced capacity for immununostimulant-induced NO synthesis. Rat VSMC were stably transfected with human AS cDNA in a eukaryotic cell expression vector, driven by a strong viral promoter. AS activity in transfected VSMC exceeded that induced in untransfected cells treated for 24 h with a combination of bacterial lipopolysaccharide and interferon-␥ (LPS/IFN). AS activity was predominantly associated with membranes but was also found in cytosol. Recombinant AS was purified from cytosol and possessed a specific activity exceeding that reported for native AS. Western blotting verified the basal expression of AS antigen in membranes from untreated AStransfected VSMC and from untransfected VSMC after 24 h exposure to LPS/IFN. Epifluorescence histochemistry revealed a punctate distribution of AS antigen in transfected cells, consistent with a predominant membrane localization. Remarkably, on a per cell basis, LPS/ IFN-induced NO production was 3-4-fold greater in AStransfected cells than untransfected VSMC. In untransfected VSMC, maximal NO production during 48 h required millimolar Arg; notably, Cit was needed at Ϸ3-fold higher concentrations than Arg for a comparable NO synthesis rate. In contrast, AS-transfected VSMC utilized Arg and Cit equi-effectively and at much lower concentrations; 100 M of either precursor supported a maximal rate of NO synthesis for 48 h. The enhanced ability of AS-transfected cells to produce NO, compared with untransfected cells, could not be ascribed to differences in iNOS protein content or LPS/IFN potency for immunoactivation. We conclude that transfection with AS provides a continuous flux of Arg which drives NO synthesis in immunoactivated VSMC. Arg regeneration by AS is rate-limiting to NO synthesis and apparently provides iNOS with a preferred cellular source of Arg. In accord with the reported "channeling" of substrates by urea cycle enzymes, we hypothesize that the Arg/Cit cycle sequesters a discrete pool of recyclable substrate that sustains high-output NO synthesis.

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Simultaneous measurement of multiple membrane ATPases in microtiter plates

Cited by 27 publications

References 26 publications

Green tea metabolite EGCG protects membranes against oxidative damage in vitro

Green tea metabolite EGCG protects membranes against oxidative damage in vitro

Loss of phospholipid asymmetry and elevated brain apoptotic protein levels in subjects with amnestic mild cognitive impairment and Alzheimer disease

Argininosuccinate Synthetase Overexpression in Vascular Smooth Muscle Cells Potentiates Immunostimulant-induced NO Production

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