A simple and rapid method for the preparation of plasma membranes

Metabolism of natriuretic peptides is regulated by two degradative pathways: uptake by the clearance receptor (natriuretic peptide receptor C -NPR-C) and hydrolysis by neutral endopeptidase (NEP). Affinity studies favour a dominant role of NPR-C in hormone degradation in several species but do not account for the efficacy of NEP inhibitors in vivo, nor the uniquely prolonged half life (t ½ ) of human brain natriuretic peptide (hBNP). Postulating that (1) delayed metabolism of hBNP reflects resistance to NEP and (2) interactions between NPR-C and NEP increase enzyme activity, we have used purified ovine and human NEP, plus ovine lung plasma membranes to study the relative importance of receptor and enzyme pathways. We have also related the findings to hormone metabolism in vivo. Binding affinities of atrial natriuretic peptide (ANP), hBNP and ovine BNP (oBNP) to oNPR-C were similar (K d =8-16 pM). In contrast, unlike ANP and oBNP, hBNP was not significantly degraded by purified oNEP or plasma membranes. Despite similar (and high) affinity of oNPR-C for oBNP and hBNP, the t ½ of hBNP (12·7 min) was more than fourfold that of oBNP (2·6 min). Although we found no evidence for receptorenzyme interaction, our results show that the delayed metabolism of hBNP reflects resistance to NEP. These findings have important implications for future treatment strategies in human disease.

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“…Ovine lung membranes were prepared according to the method of Maeda (Maeda et al 1983). In brief, tissue was homogenised in two volumes (wt/vol.)…”

Section: Preparation Of Ovine Lung Plasma Membranesmentioning

confidence: 99%

Delayed metabolism of human brain natriuretic peptide reflects resistance to neutral endopeptidase

Smith¹,

Espiner²,

Yandle³

et al. 2000

Journal of Endocrinology

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“…Plasma membrane of ascitic fibrosarcoma cells were isolated over a 41% sucrose solution according to Maeda et al [16]. Liver plasma membranes from the 37% to 41% sucrose interface and brain plasma membranes from the 0.8 to 1.2 M sucrose interface and fibrosarcoma plasma membranes as white interfacial band were collected and washed with 0.01 M phosphate-buffered saline (PBS), pH 7.2 and stored at -7O'C.…”

Section: Febslettersmentioning

confidence: 99%

Evidence for the existence of hyaluronectin‐binding proteins in the plasma membranes

Deb

Datta

1993

FEBS Letters

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This report documents for the tirst time the existence of spechic binding proteins for hyaluronic acid binding protein (hyaluronectin) in the plasma membranes of normal and transformed cells. Firstly, we showed the specific binding of hyaluronic acid binding protein to the cell surface of normal rat heart fibroblasts (NRHF) by saturation and competition methods using '*'I-labeled hyaluronic acid binding protein and calculated the binding dissociation constant (0.43 x lo-" M). In order to identify hyahtronectin-binding protein on the cell surface, plasma membranes isolated from rat brain, liver and fibrosarcoma were separated by SDS-PAGE and transferred to nitrocellulose paper by electroblotting. Incubation of the transferred membrane proteins with '251-lab&d hyaluroneotin in the presence of non-ionic as well as ionic detergents revealed two prominent bands of approximate molecular mass of 37 kDa and 40 kDa in brain, liver and fibrosarcoma. The specificity of the binding ['251Jhyaluronectin to 37-kDa and 40-kDa membrane proteins was further conIirmed, as the intensity of the bands was reduced in the presence of a 20-fold excess of unlabeled hyaluronectin. We discuss our observations on hyalmonectin-binding membrane proteins in the context of hyalmonectin-mediated cellular functions.

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“…The homogenate was centrifuged at 800 x g for 10 min and the pellet discarded. Crude plasma membranes were isolated from the 800 x g supernatant, which was layered over 1.2 M sucrose and centrifuged at 95000 x g for 60 min (Maeda et al, 1983). Thc interfacial layer containing plasma membranes was collected, washed and suspended in 0.25 M sucrose and 2 m M dithiothreitol, pH 7.4.…”

Section: Subcellular Fractionation Of Livermentioning

confidence: 99%

Oleate stimulation of diacylglycerol formation from phosphatidylcholine through effects on phospholipase D and phosphatidate phosphohydrolase

Siddiqui

Exton

1992

European Journal of Biochemistry

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Hydrolysis of exogenous phosphatidylcholine (PtdCho) to 1,2-diacyIglycerol by rat liver plasma membranes was stimulated by oleate concentrations as low as 0.1 mM. In the presence of 75 mM ethanol, the falty acid also enhanced phosphatidylethanol (PtdEtOH) formation from PtdCho. These effects were also observed with linoleate and arachidonate, but not with saturated fatty acids or detergents, and were minimal in microsomes or mitochondria. Kelease of [3H]choline from exogenous Pt~l[~H]Cho was stimulated by oleate, whereas ph~sphoryl[~H]choline formation was inhibited. Oleate and other unsaturated, but not saturated, fatty acids also stimulated the conversion of exogenous ['4C]phosphatidic acid to ['4C]diacylglycerol. These data are consistent with stimulatory effects of these fatty acids on both phospholipase D and phosphatidate phosphohydrolase in liver plasma membranes. The stimulatory effect of guanosine 5'-0-[3-thio]triphosphate) (20 pM) on PtdEtOH and diacylglycerol formation from PtdCho was enhanced by low concentrations of oleate. Phospholipase A2 also stimulated PtdEtOH and diacylglycerol formation from exogenous PtdCho. It is proposed that unsaturated fatty acids may play a physiological role in the regulation of diacylglycerol production through activation of phospholipasc D and phosphatidate phosphohydrolase.

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A simple and rapid method for the preparation of plasma membranes

Cited by 183 publications

References 10 publications

Delayed metabolism of human brain natriuretic peptide reflects resistance to neutral endopeptidase

Delayed metabolism of human brain natriuretic peptide reflects resistance to neutral endopeptidase

Evidence for the existence of hyaluronectin‐binding proteins in the plasma membranes

Oleate stimulation of diacylglycerol formation from phosphatidylcholine through effects on phospholipase D and phosphatidate phosphohydrolase

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