A rapid method for the isolation of rat liver plasma membranes using an aqueous two-phase polymer system

Lesko, Lawrence J.; Donlon, Mildred A.; Marinetti, G.V.; Hare, J.D.

doi:10.1016/0005-2736(73)90264-2

Cited by 170 publications

(43 citation statements)

References 9 publications

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“…Membranes were isolated using a two-phase polymer system, as previously described [39]. Using this method, about 33-40 mg membrane protein/rat liver can be isolated.…”

Section: Liver Plasma Membrane Preparationmentioning

confidence: 99%

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Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

et al. 2005

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Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteinsA model system for selective solubilization and fast separation of proteins from the rat liver membrane fraction and purified rat liver plasma membranes for their further proteomic analysis is presented. For selective solubilization, high-pH solutions and a concentrated urea solution, combined with different detergents, are used. After extraction, proteins are separated by anion-exchange chromatography or a combination of anion-and cation-exchange chromatography with convective interaction monolithic supports. This separation method enables fast and effective prefractionation of membrane proteins based on their hydrophobicity and charge prior to one-dimensional (1-D) and 2-D electrophoresis and mass spectrometry. By use of this sample preparation method, the less-abundant proteins can be detected and identified.

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“…Membranes were isolated using a two-phase polymer system, as previously described [39]. Using this method, about 33-40 mg membrane protein/rat liver can be isolated.…”

Section: Liver Plasma Membrane Preparationmentioning

confidence: 99%

“…Further experiments were performed using plasma membranes purified with a two-phase polymer system [39]. Using this method, about 100-120 mg protein in highly purified plasma membranes, out of three rat livers (50-60 g wet weight) could be isolated.…”

Section: Fractionation Of Purified Liver Plasma Membranesmentioning

confidence: 99%

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

et al. 2005

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“…I M Tris-HCl buffer (PH 7) and the conce~tration determined as in 1151. The purity of the plasma membrane preparation was examined by the enzyme markers: 5'-monon~cleotidase, Mg2+-stimulated ATPase and (Na" -t Kf)-activated ATPase by the method in [13] and the Pi liberated was assayed as in [16]. Glucose 6-phosphatase was assayed as in 1171 …”

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

In vitro studies on some parameters of the binding of the rat hemopexin—heme complex with the hepatic membrane reeptor

Tran-Quang¹,

Bernard²,

Higa³

et al. 1983

FEBS Letters

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“…(Na' ,K')ATPase was assayed by measuring the liberation of Pi from ATP (3 mM) in the presence of 5 mM Mg2+, 150 mM Na' ,15 mM K' ,50 mM Tris (pH 7.5) and in the presence and absence of ouabain (3 mM). 5'-Nucleotidase and glucose-6-phosphatase were measured by following the liberation of Pi from AMP (5 mM) according to Lesko et al [27] and from glucose 6-phosphate (100 mM), respectively. Pi was measured by the method of Baginski et al [28].…”

Section: Calculationsmentioning

confidence: 99%

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na⁺ transport mediated by (Na⁺, K⁺)ATPase or Na⁺/H⁺ exchanger of rat liver plasma membrane vesicles

Jakubowski¹,

Jakob²

1990

European Journal of Biochemistry

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Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/ H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase‐mediated transport was determined from initial velocities of vanadate‐sensitive uptake of 22Na+, the Na+‐dependence of Na+/H+ exchange from initial velocities of amiloride‐sensitive uptake. By studying vanadate‐sensitive Na+ transport, high‐affinity binding sites for ATP with an apparent Km(ATP) of 15 ± 1 μM were observed at low concentrations of Na+ (1 mM) and K+ (1 mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 ± 25 μM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate‐sensitive transport by 3.8‐fold and 1.9‐fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 ± 7 μM and 76 ± 10 μM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6 – 1.7‐fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride‐sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6‐fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13‐fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride‐sensitive Na+ transport. The results show that hormone receptor‐mediated effects on (Na+,K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.

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A rapid method for the isolation of rat liver plasma membranes using an aqueous two-phase polymer system

Cited by 170 publications

References 9 publications

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

In vitro studies on some parameters of the binding of the rat hemopexin—heme complex with the hepatic membrane reeptor

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na⁺ transport mediated by (Na⁺, K⁺)ATPase or Na⁺/H⁺ exchanger of rat liver plasma membrane vesicles

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A rapid method for the isolation of rat liver plasma membranes using an aqueous two-phase polymer system

Cited by 170 publications

References 9 publications

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

In vitro studies on some parameters of the binding of the rat hemopexin—heme complex with the hepatic membrane reeptor

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles

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Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na⁺ transport mediated by (Na⁺, K⁺)ATPase or Na⁺/H⁺ exchanger of rat liver plasma membrane vesicles