Purification of the Thy-1 molecule from rat brain

Barclay, A. Neil; Letarte-Muirhead, M; Williams, Alan F.

doi:10.1042/bj1510699

Cited by 112 publications

(47 citation statements)

References 28 publications

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“…3B). Since Thy-1 is Ͼ10-fold more abundant than PrP (assessed by the degree of purification required from brain; (42)(43)(44)), the lipids associated with 20% of PrP present in the Thy-1 DRMs would be a minor contaminant when compared with those associated with the more abundant Thy-1.…”

Section: Separation Of Prp and Thy-1 On The Membrane And Inmentioning

confidence: 99%

The Membrane Domains Occupied by Glycosylphosphatidylinositol-anchored Prion Protein and Thy-1 Differ in Lipid Composition

Brügger

Graham

Leibrecht

et al. 2004

Journal of Biological Chemistry

151

156

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Glycosylphosphatidylinositol-anchored prion protein and Thy-1, found in adjacent microdomains or "rafts" on the neuronal surface, traffic very differently and show distinctive differences in their resistance to detergent solubilization. Monovalent immunogold labeling showed that the two proteins were largely clustered in separate domains on the neuronal surface: 86% of prion protein was clustered in domains containing no Thy-1, although 40% of Thy-1 had a few molecules of prion protein associated with it. Only 1% of all clusters contained appreciable levels of both proteins (>3 immunogold label for both). In keeping with this distribution, immunoaffinity isolation of detergent-resistant membranes (DRMs) using the non-ionic detergent Brij 96 yielded prion protein DRMs with little Thy-1, whereas Thy-1 DRMs contained ϳ20% of prion protein. The lipid content of prion protein and Thy-1 DRMs was measured by quantitative nano-electrospray ionization tandem mass spectrometry. In four independent preparations, the lipid content was highly reproducible, with Thy-1 and prion protein DRMs differing markedly from each other and from the total DRM pool from which they were immunoprecipitated. Prion protein DRMs contained significantly more unsaturated, longer chain lipids than Thy-1 DRMs and had 5-fold higher levels of hexosylceramide. The different lipid compositions are in keeping with the different trafficking dynamics and solubility of the two proteins and show that, under the conditions used, DRMs can isolate individual membrane microenvironments. These results further identify unsaturation and glycosylation of lipids as major sources of diversity of raft structure.The separation of membrane lipids into different phases creates diverse microenvironments within a biological membrane (1, 2). In particular, cholesterol is believed to condense with saturated phosphatidylcholine (PC) 1 and sphingomyelin (SM) to form minute patches (40 -100 nm wide) of lipids in a liquid-ordered phase (3-7), creating specialized lipid microenvironments called "rafts" within the disordered fluid phase formed by unsaturated lipids (8). These ordered microdomains control the access and egress of subsets of membrane proteins, regulating signaling systems at the cell surface (9). liquidordered domains resist solubilization in non-ionic detergents (6, 7, 10 -13), enabling them to be isolated as detergent-resistant membranes (DRMs) that float at low density upon gradient centrifugation (14). Lipid-anchored proteins partition into both leaflets of these domains, the glycosylphosphatidylinositol (GPI)-anchored proteins into the outer (surface) layer and the diacylated cytoplasmic proteins into the inner layer (9, 15, 16). The membrane environment of GPI-anchored prion protein (PrP) is of particular interest since it is a candidate for chaperoning the conversion of PrP to the altered pathogenic conformation associated with prion disease (17-19). Immunolabeling shows PrP to be present on the neuronal surface in different, albeit often closely adjacent, d...

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Section: Separation Of Prp and Thy-1 On The Membrane And Inmentioning

confidence: 99%

The Membrane Domains Occupied by Glycosylphosphatidylinositol-anchored Prion Protein and Thy-1 Differ in Lipid Composition

Brügger

Graham

Leibrecht

et al. 2004

Journal of Biological Chemistry

151

156

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“…Thy-i antigen and construction ofinhibition assays 125I-labelled antibody was incubated with thymocyte Thy-i antigen purified as described by LetarteMuirhead et al (1975) or with brain Thy-i antigen as described by Barclay et al (1975), with the modification that lipid was removed by chloroform/ methanol (2: 1, v/v) extraction and that an affinity column of MRC OX 7 monoclonal antibody was used rather than lectin affinity chromatography (A. F. Williams, unpublished work). The pure Thy-i antigen was precipitated with ethanol to remove deoxycholate and solubilized in 3mM-NaN3.…”

Section: Measurement Of Rate Of Dissociation With Solublementioning

confidence: 99%

The kinetics of antibody binding to membrane antigens in solution and at the cell surface

Mason¹,

Williams²

1980

Biochemical Journal

Self Cite

522

194

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The reaction kinetics of 251I-labelled mouse monoclonal antibodies binding to three cell-surface antigens of rat thymocytes (Thy-1.1, W3/13 and W3/25) were studied. The differences between bivalent and univalent interactions were determined by using antibody in the F(ab')2 or Fab' form and by using antigen in polymeric or monomeric forms. Association rate constants (k+ ), dissociation rate constants (k-) and equilibrium constants were determined. Also, the dissociation kinetics of rabbit antibodies against rat Thy-i antigen were studied. The major findings were as follows. (i) With F(ab')2 antibody there was no simple relationship between antigen density at the cell surface and extent of bivalent binding. Extensive univalent binding was observed unless the antibody had a high k-, for the univalent interaction, in which case all binding was bivalent.(ii) k+1 values were similar for F(ab')2 or Fab' antibody, and for the different antibodies were in the range 0.8 x 105-1.1 x 106M-1s-1. These differences were sufficient to affect the interpretation of serological assays with the different antibodies. (iii) Antibody bound bivalently dissociated much more slowly than that bound univalently.However, the k-, values for the univalently bound antibody were sufficiently low in most cases that the lifetime of the univalent complex was similar to or greater than the time needed for the assay. Thus the results could be interpreted on the basis of irreversible reactions. The overall conclusion from the study is that for an understanding of the binding of antibody to cell-surface antigens the kinetics of the interaction are of major importance and theories based on equilibrium binding are inappropriate.

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“…The antigenic specificity for Thy-1 isolated from both nervous and lymphoid tissue ap pears to reside in a glycoprotein of approxi-mately 25,000 daltons [5,23,24,46]. Although the amino acid composition of Thy-1 from brain and thymus appears to be the same, Williams et al [43] have found substantial dif ferences in their carbohydrate compositions.…”

Section: Introductionmentioning

confidence: 99%

Distribution of Thy-1 Differentiation Alloantigen in the Rat Nervous System and in a Cell Line Derived from a Rat Peripheral Neurinoma

Acton¹,

Pfeiffer²

1978

Dev Neurosci

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The expression of the Thy-1 ''differentiation alloantigen'' was analyzed in rat tissues of two strains. Cerebrum was shown to express an amount of Thy-1 equivalent to the thymus. The cerebellum, brain stem and spinal cord expressed approximately one-half this amount, and the olfactory bulb expressed 10- to 15-fold less. Of the peripheral nervous system tissues examined, only cauda equina expressed substantial amounts of Thy-1, although minor amounts were found on the lumbar roots and trigeminal nerve. Kidney, spleen, liver, salivary gland and testes expressed little or no Thy-1. Several cell lines derived from nervous system tumors were also examined. RN-1, a line established from a rat peripheral neurinoma, expressed Thy-1 at levels comparable to thymus.

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Purification of the Thy-1 molecule from rat brain

Cited by 112 publications

References 28 publications

The Membrane Domains Occupied by Glycosylphosphatidylinositol-anchored Prion Protein and Thy-1 Differ in Lipid Composition

The Membrane Domains Occupied by Glycosylphosphatidylinositol-anchored Prion Protein and Thy-1 Differ in Lipid Composition

The kinetics of antibody binding to membrane antigens in solution and at the cell surface

Distribution of Thy-1 Differentiation Alloantigen in the Rat Nervous System and in a Cell Line Derived from a Rat Peripheral Neurinoma

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