The synthesis and initial characterization of an immobilized purinergic receptor (P2Y1) liquid chromatography stationary phase for online screening

Moaddel, Ruin; Calleri, Enrica; Massolini, Gabriella; Frazier, Chester R.; Wainer, Irving W.

doi:10.1016/j.ab.2007.02.014

Cited by 44 publications

(34 citation statements)

References 13 publications

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“…Instead, purinergic signaling was elicited by increasing the concentration of ATP in the bathing solution to a final concentration of 0.5 or 1 mM. Notably, these concentrations are well above the reported affinity of the purinoceptors (1-20 M) (27). Likewise, they are higher than the up to 80 M ATP observed in the bulk extracellular space of human polymorphonuclear leukocytes, Jurkat T cells (10), and stretch-activated or contracting skeletal muscles (22,23).…”

Section: Discussionmentioning

confidence: 99%

Effect of purinergic receptor activation on Na⁺-K⁺ pump activity, excitability, and function in depolarized skeletal muscle

Broch‐Lips

Pedersen

Nielsen

2010

American Journal of Physiology-Cell Physiology

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Section: Discussionmentioning

confidence: 99%

Effect of purinergic receptor activation on Na⁺-K⁺ pump activity, excitability, and function in depolarized skeletal muscle

Broch‐Lips

Pedersen

Nielsen

2010

American Journal of Physiology-Cell Physiology

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“…This is emphasized by the development of a CMAC column containing the P2Y 1 receptor, a GPCR, which was produced using cellular membrane fragments obtained from the 1321N1 astrocytoma cell line. 32 In this case, the detergent was n-octyl β-Dglucopyranoside and it was not necessary to add lipids to the solubilization/immobilization buffer. This may be due to the cell type, the membrane localization of the P2Y 1 receptor, or the fact that n-octyl β-D-glucopyranoside has a different detergent extractability profile than cholate and CHAPS.…”

Section: Discussionmentioning

confidence: 99%

Initial Synthesis and Characterization of an α7 Nicotinic Receptor Cellular Membrane Affinity Chromatography Column: Effect of Receptor Subtype and Cell Type

et al. 2007

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In this study, cellular membrane fragments from SH-EP1-pCEP4-hα7 and α7 HEK-293 cell lines were used to synthesize cellular membrane affinity chromatography (CMAC) columns containing functional α7 nicotinic acetylcholine receptors, CMAC(α7 nAChR) columns. The synthesis of stable columns required the addition of cholesterol to the 2% cholate solubilization/immobilization (s/i) buffer and to the mobile phase. In addition, when membranes from the SH-EP1 cell line were used, L-α-phosphatidylserine and L-α-phosphatidylethanolamine also had to be added to the s/i buffer. A CMAC(α4β2 nAChR) column was prepared using membrane fragments from a SH-EP1-pCEP4-hα4β2 cell line, and this process required the addition of L-α-phosphatidylserine and L-α-phosphatidylethanolamine to the s/i buffer, but not cholesterol. The s/i buffers from the three columns were compared with the s/i buffer utilized in the preparation of a CMAC(α4β2 nAChR) column prepared using an α4β2 HEK-293 cell line, which required no additions to the 2% cholate s/i buffer. The data demonstrate that both cell type and receptor type affect the protocol required to produce a stable CMAC column and that, at the current time, the development of an optimum immobilization protocol is an empirical process. The results are also consistent with the observation that the α7 nAChR is localized in lipid rafts in both of these cell lines and that the cholate detergent removed cholesterol from these microdomains.Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand gated ion channels that are composed of five transmembrane subunits oriented around a central pore. 1,2 To date, 12 different neuronal subunits have been identified, 9 α subunits (α2-α10) and 3 β subunits (β2-β4). These subunits combine to form a wide variety of homomeric α x subtypes where x = 7, 9, 10 and heteromeric α x β y subtypes where x = 2, 3, 4, 5, 6, and y = 2, 3, 4.The homomeric α7 nAChR is found in the mammalian brain, 3 and these receptors appear to be involved in learning and memory 4 and to play a role in Alzheimer's disease. 5,6 The receptor is also involved in schizophrenia through the mediation of the release of GABA by

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“…Thus, the IAM stationary phase has been used to create a series of CMAC columns from a variety of cell lines. The resulting columns have been used to study G-protein coupled receptors (GPCRs), including κ and μ opioid 11 , β 2 -adrenergic 12 and P2Y 1 purinergic 13 receptor, ligand-gated ion channels (LGICs), such as nAChRs 8,14 , and transmembrane drug transporters, such as P-glycoprotein (Pgp) 15 , human organic cation transporter-1 (hOCT1) 16 and human organic anion transporter 1 and 2 (OAT1, OAT2) 17 . A multiple LGIC-CMAC column containing coimmobilized nAChRs, γ-amino-butyric acid (GABA A ) and N -methyl- d -aspartate (NMDA) receptors have also been created through the immobilization of solublized brain tissue 18 .…”

Section: Introductionmentioning

confidence: 99%

“…In addition, this approach can be used to investigate and describe the interactions of small molecules and proteins with the target membrane protein 13–20 . Other methods that can be used to study membrane protein–ligand interactions include filtration assays, binding assays and functional studies.…”

Section: Introductionmentioning

confidence: 99%

The preparation and development of cellular membrane affinity chromatography columns

Moaddel

Wainer

2009

Nat Protoc

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Cellular membrane affinity chromatography is a technique that is based on the immobilization of a target trans-membrane protein onto a stationary phase. The target protein is isolated by homogenization and solubilization of a source (e.g., cell line) followed by immobilization on either the immobilized artificial membrane-phosphatidyl choline (IAM-PC) stationary phase or the surface of an open tubular capillary during a dialysis step. The procedure typically takes 3–4 d for the IAM-PC stationary phase, whereas the open-tubular method takes an extra week for the preparation of the capillary. The resulting columns can then be used to characterize binding sites on the target protein through frontal chromatographic and/or nonlinear chromatographic studies using a wide variety of ligands including small molecules and polypeptides. The columns have been used in drug discovery as well as in the screening of tobacco smoke condensates.

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The synthesis and initial characterization of an immobilized purinergic receptor (P2Y1) liquid chromatography stationary phase for online screening

Cited by 44 publications

References 13 publications

Effect of purinergic receptor activation on Na⁺-K⁺ pump activity, excitability, and function in depolarized skeletal muscle

Effect of purinergic receptor activation on Na⁺-K⁺ pump activity, excitability, and function in depolarized skeletal muscle

Initial Synthesis and Characterization of an α7 Nicotinic Receptor Cellular Membrane Affinity Chromatography Column: Effect of Receptor Subtype and Cell Type

The preparation and development of cellular membrane affinity chromatography columns

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The synthesis and initial characterization of an immobilized purinergic receptor (P2Y1) liquid chromatography stationary phase for online screening

Cited by 44 publications

References 13 publications

Effect of purinergic receptor activation on Na+-K+ pump activity, excitability, and function in depolarized skeletal muscle

Effect of purinergic receptor activation on Na+-K+ pump activity, excitability, and function in depolarized skeletal muscle

Initial Synthesis and Characterization of an α7 Nicotinic Receptor Cellular Membrane Affinity Chromatography Column: Effect of Receptor Subtype and Cell Type

The preparation and development of cellular membrane affinity chromatography columns

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Product

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Effect of purinergic receptor activation on Na⁺-K⁺ pump activity, excitability, and function in depolarized skeletal muscle

Effect of purinergic receptor activation on Na⁺-K⁺ pump activity, excitability, and function in depolarized skeletal muscle