Isolation and Characterization of Plasma Membranes from Human Blood Platelets

Barber, A.J.; Jamieson, G.A.

doi:10.1016/s0021-9258(18)62618-3

Cited by 411 publications

(30 citation statements)

References 53 publications

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“…Preliminary studies using the method of Barber and Jamieson [12] yielded similar results. However, we have found sonication to result in less cytosotic contamination as determined by measurements of lactate dehydrogenase than the glycerol lysis method (data not shown).…”

Section: Discussionsupporting

confidence: 52%

“…The availability of a purified plasma membrane preparation for the nicotinic eholinergie [10] receptor system has facilitated biochemical and mechanistic studies of that receptor. Human platelets provide a ready source of a 2adrenergic receptors [8.9,11] and many methods for purifying plasma membranes from human platelets have been reported [11][12][13]. One study of Ctz-agonist binding to a partially purified plasma membrane preparation has been published [11].…”

Section: Introductionmentioning

confidence: 99%

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Large-scale purification of α2-adrenergic receptor-enriched membranes from human platelets. Persistent association of guanine nucleotides with nonpurified membranes

Neubig

Szamraj

1986

Biochimica et Biophysica Acta (BBA) - Biomembranes

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A simple large-scale purification of alpha 2-adrenergic receptor-enriched membranes from human platelets is described. Binding of the antagonist [3H]yohimbine is enriched 3-5-fold compared to a crude membrane fraction. Binding of low concentrations of the partial agonist 3-H-rho-aminoclonidine is increased 15-20-fold due to a higher binding affinity for the purified membranes. A soluble inhibitor of 3H-rho-aminoclonidine binding to purified membranes is found even in thrice-washed crude platelet membranes. The guanine nucleotides GDP and GTP are found to account for this inhibitory activity. Forskolin-stimulated adenylate cyclase activity is also enriched in the purified membrane fraction. Adenylate cyclase activity is inhibited by alpha 2-agonist to a comparable extent in all membrane fractions. This membrane preparation should prove useful in studies of alpha 2-adrenergic receptor mechanisms.

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Section: Discussionsupporting

confidence: 52%

Section: Introductionmentioning

confidence: 99%

Large-scale purification of α2-adrenergic receptor-enriched membranes from human platelets. Persistent association of guanine nucleotides with nonpurified membranes

Neubig

Szamraj

1986

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…Platelets were purified from platelet concentrates, and the plasma membranes isolated by the glycerol lysis technique, as described by Barber & Jamieson (1970). Yields were 1.4 + 0.2 mg of plasma-membrane proteins per blood unit and the phosphodiesterase activity was 0.245 mol of p-nitrophenol/h per mg of protein.…”

Section: Isolation Of Gpiib and Gpiiiamentioning

confidence: 99%

“…Human platelets from outdated platelet concentrates (72 h after blood collection) were subjected to hypoosmotic lysis after intracellular loading with glycerol (Barber & Jamieson, 1970). The lysate was loaded on top of a discontinuous 30-50% (w/v) sucrose gradient, and centrifuged at 85000 g (ray.…”

Section: Platelet Subceliular Fractionationmentioning

confidence: 99%

New isolation procedure and further biochemical characterization of glycoproteins IIb and IIIa from human platelet plasma membrane

Eirin¹,

Calvete²,

González-Rodrı́guez³

1986

Biochemical Journal

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We describe a new procedure for isolation of glycoproteins IIb (GPIIb) and IIIa (GPIIIa) from human platelet plasma membrane with high yields (2.7 mg of GPIIb and 3.3 mg of GPIIIa per 100 mg of starting platelet membrane proteins), equivalent to a recovery of 35% and 55% respectively of the total GPIIb and GPIIIa of the membrane. The procedure involves Triton X-100 differential extraction of platelet membranes, SDS solubilization of the 4%-Triton X-100 supernatant, zonal centrifugation in a sucrose density gradient, and preparative high-performance size-exclusion chromatography. The weight percentage of sugar is 15.7% for GPIIb and 12.5% for GPIIIa. Neuraminic acid is present in both glycoproteins, representing 30% and 15% respectively of the total sugar weight of GPIIb and GPIIIa. Mannose, galactose and glucosamine account for 45%, 13% and 28% respectively of the sugars of GPIIIa, whereas galactosamine was not detected. Mannose, galactose, glucosamine and galactosamine represent 17%, 21%, 24% and 10% respectively of the sugar content of GPIIb. The molar percentages of half-cystine and methionine are 4-fold and 2-fold higher respectively in GPIIIa than in GPIIb. From the amino acid and sugar compositions we confirmed the acidic nature of both glycoproteins. The Mr values obtained, 136,500 for GPIIb and 91,500 for GPIIIa, are in very good agreement with those obtained by physical methods. The apparent lack of free thiol groups in both glycoproteins indicates that the tertiary structure of GPIIIa is maintained by 21 intrachain disulphide bonds, and that there are eight intrachain and interchain disulphide groups in GPIIb.

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“…Plasma-membrane and endoplasmic-reticulum fractions were recovered in good yield and high purity. Barber & Jamieson (1970) devised a method for homogenizing platelets that involved the uptake of glycerol by these cells from a 4.2M solution followed by transfer of the glycerol-loaded cells to a relatively hypo-osmotic solution of 0.25 M-sucrose: the osmotic gradient generated across the plasma membrane caused the platelets to swell and burst. The present paper is concerned with the large-scale application of this method to Lettr6e cells and the subsequent fractionation of the released membranes in a zonal rotor.…”

mentioning

confidence: 99%

Tissue-culture Cell fractionation. Zonal centrifugation of Lettrée cells homogenized by glycerol-induced lysis: subfractionation of membranes in sucrose gradients

Graham¹,

Coffey²

1979

Biochemical Journal

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1. Lettrée cells were grown intraperitoneally in MF-1 mice. 2. Cells that were loaded with glycerol were swollen in 0.1 M-sucrose and disrupted by Dounce homogenization. 3. Early-passage Lettrée cells were more easily disrupted than late-passage cells by this method, and the former produced larger fragments of plasma membrane. 4. The membranes were fractionated initially in sucrose gradients (on the basis of sedimentation rate) in a BXIV zonal rotor. 5. Fractions from this gradient were further resolved in isopycnic sucrose gradients. 6. Plasma-membrane and endoplasmic-reticulum fractions were recovered in good yield and high purity.

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Isolation and Characterization of Plasma Membranes from Human Blood Platelets

Cited by 411 publications

References 53 publications

Large-scale purification of α2-adrenergic receptor-enriched membranes from human platelets. Persistent association of guanine nucleotides with nonpurified membranes

Large-scale purification of α2-adrenergic receptor-enriched membranes from human platelets. Persistent association of guanine nucleotides with nonpurified membranes

New isolation procedure and further biochemical characterization of glycoproteins IIb and IIIa from human platelet plasma membrane

Tissue-culture Cell fractionation. Zonal centrifugation of Lettrée cells homogenized by glycerol-induced lysis: subfractionation of membranes in sucrose gradients

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