The Isolation of a Cell Membrane Fraction From Rat Liver

Bovine liver plasma membranes [Rosen, Ehrich, Junger, Bubenzer & Kuhn (1979) Biochim. Biophys. Acta 587, 593-6051 show similar insulin-binding characteristics, as evaluated by Scatchard analysis, to those of membrane systems from other species. However, the dissociation rate of bound insulin cannot be accelerated by the addition of insulin, in contrast with membranes isolated from rat liver. The dissociation rate is strongly dependent on the pH. Although dependent on temperature, the total capacity of binding sites is minimally changed, but the number of high-affinity sites is increased 2-3-fold, by lowering the incubation temperature. These data might be interpreted by assuming a single population of receptors whose distribution between different affinity states depends on temperature. In competition studies, most of the modified insulins examined show a close correlation between binding, determined i'n plasma membranes from bovine liver, and biological activity, measured in adipocytes. The hypothesis that a positive charge on the Al residue may be favourable for binding is supported by experiments with an isosteric pair of insulins modified at this residue ([carbamoylGlyAl]-and [amidino-GlyAllinsulin) and with modified insulins carrying one or more positive charges on the Al residue ([Arg-GlyAl1-, [Arg-Arg-GlyAl1-, [Arg-ArgArg-GlyAl1-and [Lys-Arg-GlyAl]insulin). The latter insulin derivatives show a higher binding activity for plasma membranes from bovine, porcine and rat liver than expected from their biological activities in adipocytes.

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“…Plasma membranes from rat liver were prepared as described by Neville (1960) and those from porcine liver as described by Bubenzer et al (1978).…”

Section: Preparationmentioning

confidence: 99%

Binding of insulin to bovine liver plasma membrane. Use of insulin analogues modified at the A1 residue

Rösen

Simón

Reinauer

et al. 1980

Biochemical Journal

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“…Plasma membranes from bovine thyroid and guinea pig liver and muscle were prepared by sucrose density gradient centrifugation according to the method of Neville (15). Plasma membranes of Harderian gland of guinea pigs were similarly prepared.…”

Section: Methodsmentioning

confidence: 99%

The Binding of [ ³ H]Thyrotropin and an ³ H-Labeled Exophthalmogenic Factor by Plasma Membranes of Retro-orbital Tissue

Winand

Kohn

1972

Proc. Natl. Acad. Sci. U.S.A.

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Plasma membranes of cells from retroorbital tissue have been prepared from the Harderian glands of guinea pigs and have been characterized as being reasonably free of other subcellular structures by electron microscopy and by enzyme-marker analyses. Both bovine thyrotropin and a proteolytic derivative of bovine thyrotropin with exophthalmogenic activity but without thyroid-stimulating activity specifically bind to these membranes. Gammaglobulin from the sera of patients with malignant exophthalmos increases the binding of both pituitary factors, whereas binding is not similarly increased by gammaglobulin from the sera of individuals who are not exophthalmic. The increased binding caused by the gammaglobulin from exophthalmic patients is the same whether the sera are positive or negative for the longacting thyroid stimulator. Present binding experiments do not indicate a direct interaction between the gammaglobulin and the plasma membranes of the cells from Harderian glands. A mechanism for the pathogenesis of human exophthalmos is proposed on the basis of these data.

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“…Contiguous face It has long been recognized on the basis of tnorphological criteria that the sheets of membranes interconnected by desmosomes and gap junctions present in plasma membranes prepared from 'nuclear' pellets by Neville (1960Neville ( , 1968, and by variants of this method, originated from the contiguous face of the hepatocyte. Sheets of membranes and intercellular junctions characterized the three heavy subfractions, and the higher buoyant density of these subfractions probably results from the presence of protein-rich junctional areas.…”

Section: Bile-canicularfacementioning

confidence: 99%

Functional polarity of the rat hepatocyte surface membrane. Isolation and characterization of plasma-membrane subfractions from the blood-sinusoidal, bile-Canalicular and contiguous surfaces of the hepatocyte

Wisher¹,

Evans²

1975

Biochemical Journal

305

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1. Six rat liver plasma-membrane subfractions of different density and morphological, enzymric and chemical properties were prepared from homogenates by a combination of differential, rate-zonal and density-gradient centrifugation. They consisted of three vesicular 'light' subfractions of density 1.12-1.13 and three 'heavy' subfractions of density 1.16-1.18 containing membrane strips and intercellular junctions. 2. All six subfractions contained a basal adenylate cyclase activity. One of the 'light' subfractions that showed the highest glucagon-stimulated adenylate cyclase activity was identified as deriving from the blood-sinusoidal face of the hepatocyte. This subfraction, unlike the others, was contaminated by Golgi components, as indicated by its morphological properties and the presence of galactosyl-and sialyl-transferase activities. 3. All the six subfractions showed high activities of the following plasma-membrane marker enzymes: 5'-nucleotidase, alkaline phosphodiesterase (nucleotide pyrophosphatase), alkaline phosphatase, leucine naphthylamidase and Mg2+-activated adenosine triphosphatase. A 'light' subfraction that showed the highest specific activities of all the above marker enzymes, but lacked a glucagon-stimulated adenylate cyclase activity, was identified as deriving from the bilecanalicular face of the hepatocyte. 4. The 'heavy' subfractions, which showed generally the lowest activities of the above plasma-membrane enzyme markers, and were characterized by the presence of desmosomes and gap junctions, were taken to originate from the contiguous faces of the hepatocyte. 5. The protein composition of the six subfractions was generally similar, as shown by polyacrylamide-gel electrophoresis. Differences in the amounts of various protein and glycoprotein bands among the subfractions correlated with their morphology, enzymic composition and sialic acid content. 6. Hormonal and histochemical evidence supporting the identification of a bile-canalicular subfraction, a blood-sinusoidal subfraction and contiguous-face subfractions is discussed.

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The Isolation of a Cell Membrane Fraction From Rat Liver

Cited by 488 publications

References 10 publications

Binding of insulin to bovine liver plasma membrane. Use of insulin analogues modified at the A1 residue

Binding of insulin to bovine liver plasma membrane. Use of insulin analogues modified at the A1 residue

The Binding of [ ³ H]Thyrotropin and an ³ H-Labeled Exophthalmogenic Factor by Plasma Membranes of Retro-orbital Tissue

Functional polarity of the rat hepatocyte surface membrane. Isolation and characterization of plasma-membrane subfractions from the blood-sinusoidal, bile-Canalicular and contiguous surfaces of the hepatocyte

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The Isolation of a Cell Membrane Fraction From Rat Liver

Cited by 488 publications

References 10 publications

Binding of insulin to bovine liver plasma membrane. Use of insulin analogues modified at the A1 residue

Binding of insulin to bovine liver plasma membrane. Use of insulin analogues modified at the A1 residue

The Binding of [ 3 H]Thyrotropin and an 3 H-Labeled Exophthalmogenic Factor by Plasma Membranes of Retro-orbital Tissue

Functional polarity of the rat hepatocyte surface membrane. Isolation and characterization of plasma-membrane subfractions from the blood-sinusoidal, bile-Canalicular and contiguous surfaces of the hepatocyte

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The Binding of [ ³ H]Thyrotropin and an ³ H-Labeled Exophthalmogenic Factor by Plasma Membranes of Retro-orbital Tissue