Heterogeneity of dog-liver glutathione S-transferases. Evidence for a unique temperature dependence of the catalytic process

A method has been developed for the simultaneous isolation of basolateral plasma membrane vesicles from surface and crypt cells of rabbit distal colon epithelium by sequential use of differential sedimentation, isopycnic centrifugation and Ficoll 400 barrier centrifugation. The protein yield was high (total 0.81 mg/g mucosa) and surface and crypt cell-derived basolateral membrane fractions have been purified 34- and 9-fold with respect to the homogenate. The pattern of marker enzyme enrichments revealed only minor contamination by subcellular organelles. Latency of ouabain-sensitive (Na+,K+)-ATPase activity prior and after trypsin treatment of membranes indicated a vesicle configuration of sealed right side-out: sealed inside-out: leaky of approximately 2:1:1. The presence of sealed vesicles was also evident from the osmotic sensitivity of the D-[1-14C] mannitol equilibrium space determined with either fraction. Although considerably different in protein profile, surface and crypt basolateral membranes were similar in cholesterol to phospholipid molar ratio and membrane fluidity as determined by steady-state fluorescence polarization. Stopped-flow light scattering experiments revealed a rather low water permeability of the membranes with a permeability coefficient of 6 microns/sec at 35 degrees C, which is one order of magnitude lower than reported for small intestinal plasma membranes. Both membrane fractions have been shown to effectively generate outward uphill potassium ion gradients, a process that is energized by ATP and inhibited by the membrane-permeant cardiac-glycoside digitoxin. These characteristics are consistent with the activity of a (Na+,K+) pump operating in inside-out vesicles.

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“…Protein analysis of membrane fractions was carried out by SDS-polyacrylamide slab gel electrophoresis [21] as described elsewhere [39].…”

Section: Chemical Analysismentioning

confidence: 99%

Rabbit distal colon epithelium: I. Isolation and characterization of basolateral plasma membrane vesicles from surface and crypt cells

Wiener

Turnheim

1989

J. Membrain Biol.

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“…GSTs have been identified in a number of canine tissues, including liver [24] and lens [25]. As in man, the liver has a high concentration of hGST and canine liver cytosolic GSTs have been purified, characterised and found to contain class v, h and y subunits, together with a possible q class subunit [26].…”

Section: Introductionmentioning

confidence: 99%

Glutathione S-transferases as biomarkers of organ damage: applications of rodent and canine GST enzyme immunoassays

Kilty

Doyle

Hassett

et al. 1998

Chemico-Biological Interactions

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The cytosolic glutathione S-transferase (GST) enzymes serve as ideal biomarkers of organ damage as they exhibit many of the required characteristics, i.e. specific localisation, high cytosolic concentration and relatively short half-life. The role of GSTs as early indicators of organ damage is applicable to both human and animal models. Because of the regio-specific localisation of the different isoforms of GST in liver and kidney, simultaneous monitoring of classes of GSTs in biological matrices permits the identification of specific areas of damage within a particular organ. Immunoassays have been developed which quantify canine aGST and rodent vGST (Yb1). The immunoassays are solid phase EIAs, where GST in the sample or standard is captured by a specific anti-GST antibody coated onto the solid phase. After washing, a specific enzyme-labelled IgG conjugate is added which binds to the captured GST. After a further washing step, substrate is added and a colour developed. The absorbance is measured on an ELISA plate reader and is directly proportional to the amount of GST present in the sample. The assays are performed at room temperature and can be completed within 3 h. The immunoassays are specific for each GST and have a range of 0-100 vg/l. A range of assay parameters were investigated to validate the EIAs for GST detection. The assays are sensitive and reproducible. CV for inter-and intra-assay variation were below 9% for Yb1 assay and below 20% for the canine aGST EIA. Recovery of spiked GST over the standard curve range was 102 and 99%, respectively. No prozone effect was observed and samples exhibited linearity of dilution in both assays. Validation has shown that using these enzyme immunoassay, Yb1 and canine aGST can be measured accurately

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Plant Polyphenols as Epigenetic Modulators of Glutathione S-Transferase P1 Activity

Thakur

Gupta

2013

Epigenetics and Cancer

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Heterogeneity of dog-liver glutathione S-transferases. Evidence for a unique temperature dependence of the catalytic process

Cited by 9 publications

References 48 publications

Rabbit distal colon epithelium: I. Isolation and characterization of basolateral plasma membrane vesicles from surface and crypt cells

Rabbit distal colon epithelium: I. Isolation and characterization of basolateral plasma membrane vesicles from surface and crypt cells

Glutathione S-transferases as biomarkers of organ damage: applications of rodent and canine GST enzyme immunoassays

Plant Polyphenols as Epigenetic Modulators of Glutathione S-Transferase P1 Activity

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