Purification of bovine striatal dopamine D-2 receptor by affinity chromatography.

Ramwani, Jai; Mishra, Ram K.

doi:10.1016/s0021-9258(19)84466-6

Cited by 49 publications

(3 citation statements)

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“…Membrane Antigen Preparation. Membrane antigens were prepared as described by Ramwani and Mishra (29). In brief, trophozoites were harvested by centrifugation, washed three times with 150 mM NaCl, and finally resuspended in 100 mM sodium phosphate, 1 mM EDTA, and 5 mM iodoacetamide, pH 7.2.…”

Section: Methodsmentioning

confidence: 99%

“…Four female BALB/c mice (Forschungsinstitut für Versuchstierzucht, Himberg, Austria) were immunized intraperitoneally starting at the age of 8 wk. For the initial dose, 50-75 g membrane preparation (29) from E. histolytica strain SFL-3 in 150 l of 0.9% (wt/vol) NaCl was mixed with 150 l of complete Freund's adjuvant. On days 27 and 35, animals received the same amount of membrane preparation in incomplete Freund's adjuvant.…”

Section: Methodsmentioning

confidence: 99%

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Protection against Invasive Amebiasis by a Single Monoclonal Antibody Directed against a Lipophosphoglycan Antigen Localized on the Surface of Entamoeba histolytica

Marinets

Zhang

Guillén

et al. 1997

The Journal of Experimental Medicine

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A panel of monoclonal antibodies was raised from mice immunized with a membrane preparation from Entamoeba histolytica, the pathogenic species causing invasive amebiasis in humans. Antibody EH5 gave a polydisperse band in immunoblots from membrane preparations from different E. histolytica strains, and a much weaker signal from two strains of the nonpathogenic species Entamoeba dispar. Although the exact chemical structure of the EH5 antigen is not yet known, the ability of the antigen to be metabolically radiolabeled with [32P]phosphate or [3H]glucose, its sensitivity to digestion by mild acid and phosphatidylinositol-specific phospholipase C, and its specific extraction from E. histolytica trophozoites by a method used to prepare lipophosphoglycans from Leishmania showed that it could be classified as an amebal lipophosphoglycan. Confocal immunofluorescence and immunogold labeling of trophozoites localized the antigen on the outer face of the plasma membrane and on the inner face of internal vesicle membranes. Antibody EH5 strongly agglutinated amebas in a similar way to concanavalin A (Con A), and Con A bound to immunoaffinity-purified EH5 antigen. Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A. The protective ability of antibody EH5 was tested in a passive immunization experiment in a severe combined immunodeficient (SCID) mouse model. Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess. Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Protection against Invasive Amebiasis by a Single Monoclonal Antibody Directed against a Lipophosphoglycan Antigen Localized on the Surface of Entamoeba histolytica

Marinets

Zhang

Guillén

et al. 1997

The Journal of Experimental Medicine

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“…Dopaminergic neurotransmissions in the central nervous system have been intensively studied and linked to several neurological disorders including Parkinsonism, schizophrenia, hyperprolactemia, and hypertension. The D-2 receptor has been extensively characterized at the molecular level and purified to homogeneity (Gorissen & Laduron, 1979;Hall et al, 1983; Kilpatrick & Caron, 1983; Lew & Goldstein, 1984; Ramwani & Mishra, 1986). Although D-l receptors have also been the subject of intensive investigation, very little is known about the molecular or physiological properties of this receptor.…”

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confidence: 99%

Solubilization and reconstitution of the D-1 dopamine receptor: potentiation of the agonist high-affinity state of the receptor

Sidhu¹

1988

Biochemistry

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The D-1 dopamine receptor was extracted from rat striatal membranes with sodium cholate and NaCl in the presence of a specific agonist and phospholipids. The soluble receptor then was reconstituted into phospholipid vesicles by further addition of phospholipids prior to detergent removal. Of the total membrane receptors, up to 48% were extracted and 36% were reconstituted into phospholipid vesicles. Yields were greatly reduced if the agonist was omitted or replaced with an antagonist. The solubilized and reconstituted D-1 receptors retained the pharmacological properties of the membrane-bound receptors, including the ability to discriminate between active and inactive enantiomers of specific agonists and antagonists. In this regard, the affinity of the reconstituted receptors for the D-1 specific antagonist 125I SCH 23982 was similar to that of the membrane-bound receptors with a Kd of 1.5 nM. Both the soluble and reconstituted forms of the D-1 receptor exhibited two affinity states for the D-1 specific agonist SK&F R-38393. In contrast to the low proportion of the receptors that had a high affinity for the agonists in striatal membranes (less than 6%), there was a dramatic increase following solubilization (22%) and reconstitution (40%). Similar results were obtained by using dopamine; the proportion of high-affinity sites increased from 4% (membrane-bound) to 48% (reconstituted) of the total receptor population. These high-affinity sites were coupled to G proteins, as guanyl nucleotides completely abolished them. Addition of guanyl nucleotides prior to solubilization or to reconstitution, however, had no effect on the subsequent yield of the reconstituted receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

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Characterization of rat NCA/CD9 cell surface antigen and its expression by normal and malignant neural cells

et al. 1996

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Purification of bovine striatal dopamine D-2 receptor by affinity chromatography.

Cited by 49 publications

References 20 publications

Protection against Invasive Amebiasis by a Single Monoclonal Antibody Directed against a Lipophosphoglycan Antigen Localized on the Surface of Entamoeba histolytica

Protection against Invasive Amebiasis by a Single Monoclonal Antibody Directed against a Lipophosphoglycan Antigen Localized on the Surface of Entamoeba histolytica

Solubilization and reconstitution of the D-1 dopamine receptor: potentiation of the agonist high-affinity state of the receptor

Characterization of rat NCA/CD9 cell surface antigen and its expression by normal and malignant neural cells

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