The potential of Ultrogel®, an agarose‐polyacrylamide copolymer, as a matrix for affinity chromatography

Doley, S.G.; Harvey, Michael J.; Dean, P.D.G.

doi:10.1016/0014-5793(76)80627-8

Cited by 21 publications

(4 citation statements)

References 13 publications

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“…Some disadvantages also exist, such as the broader specificity of the triazine dyes, although this can be narrowed by using various spacer molecules, as shown here with poly(ethyleneimine), or by changing the support from agarose to a pellicular support, such as Matrex 201 R, or agarose-polyacrylamide copolymers (Doley et al, 1976). These effects, especially those associated with the introduction of a polyethylene spacer molecule, may be related to the observations of Wilson (1976) on the interaction of nucleotidebinding enzymes with Cibacron Blue F3G-A and the conjugate 'Blue Dextran'.…”

Section: Discussionmentioning

confidence: 99%

The selective retardation of NADP+-dependent dehydrogenases by immobilized procion red HE-3B

Watson¹,

Harvey²,

Dean³

1978

Biochemical Journal

106

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The capacities of Procion Red HE-3B and Cibacron Blue F3G-A immobilized to Sepharose CL-4B and Matrex 201R for NAD+-, NADP+- and NAD(P)+-dependent dehydrogenases were measured. Procion Red HE-3B columns retarded NADP+-dependent dehydrogenases more effectively than NAD+-dependent dehydrogenases, whilst immobilized Cibacron Blue F3G-A retarded NAD+-dependent dehydrogenases more effectively than NADP+-dependent dehydrogenases. The capacity of procion Red HE-3B-Sepharose CL-4B for five dehydrogenases was highest in the region of 70nmol of immobilized ligand/ml of settled gel. The effects of using poly(ethyleneimine) as a spacer for both porous and pellicular supports were also examined. Four NADP+-dependent dehydrogenases were purified from yeast extract by using Procion Red HE-3B-Sepharose CL-4B. Two NAD+-dependent dehydrogenases were purified from the same source using Cibacron Blue F3G-A-Sepharose CL-4B. These results are discussed in relation to the use of immobilized Procion Red HE-3B to purify dehydrogenases. This immobilized dye's chromatograhic behaviour is compared with that of immobilized nucleotides. The most important feature of immobilized tirazine dyes seems to be their high operational capacities when compared with group-specific nucleotide adsorbents.

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Section: Discussionmentioning

confidence: 99%

The selective retardation of NADP+-dependent dehydrogenases by immobilized procion red HE-3B

Watson¹,

Harvey²,

Dean³

1978

Biochemical Journal

106

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“…In these experiments, antigenically active molecules were detected by binding with anti-gpl80 (anti-T-200)--conjugated gel beads. To prepare anti-gpl80conjugated gel beads, rabbit antibody (IgG fraction) raised against gpl80 (34) was added to glutaraldehyde-activated Ultrogel beads (ACA 22; LKB Instruments, Inc.) (46) and incubated at 4"C for 6 h. A small column of these conjugated immunobeads was prepared and samples of NP-40-solubilized, ~zs1labeled PM were passed through. This was followed by extensive washing with 0.…”

Section: Immunobinding Proceduresmentioning

confidence: 99%

A T-lymphoma transmembrane glycoprotein (gp180) is linked to the cytoskeletal protein, fodrin.

Bourguignon¹,

Suchard²,

Nagpal³

et al. 1985

The Journal of Cell Biology

116

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A major mouse T-lymphoma surface glycoprotein (gp180) has been identified by labeling cells with 1251 and [3H]glucosamine. After ligand-induced receptor patching and/or capping, the amount of gp180 in the membrane-associated cytoskeleton fraction increases in direct proportion to the percentage of patched/capped cells. There is a parallel increase in the amount of fodrin in the membrane-associated cytoskeleton fraction. Evidence is presented that gp180 is the same as or very similar to the T-lymphocyte-specific glycoprotein T-200. An immunobinding assay of Nonidet P-40-solubilized plasma membrane selectively co-isolates gp180 and fodrin. After induction of receptor rearrangement, double-label immunofluorescence reveals that fodrin accumulated directly beneath gp180 patches and caps. Membrane extraction with Triton X-114 followed by sucrose gradient centrifugation permits isolation of a gp180-fodrin complex with a 1:1 molar ratio and sedimentation coefficient(s) of approximately 20. This complex remains stable during isoelectric focusing and exhibits a pl in the range of 5.2-5.7. On the basis of our results we conclude that gp180, an intergral membrane glycoprotein, and fodrin, a component of the membrane-associated cytoskeleton, are closely associated into a complex. Furthermore, we contend that, through fodrin's association with actin, this complex is of functional significance in ligand-induced patching and capping of gp180. We also propose that, through lateral interactions in the plane of the membrane, the gp180-fodrin complex might be responsible for linking other surface receptors to the intracellular microfilament network during lymphocyte patching and capping.The involvement of contractile microfilaments in the redistribution of cell surface receptors during patching and capping was first described by Taylor et al. in 1971 (1). Since then, numerous reports have confirmed this observation for a variety of different surface receptors of mouse and human lymphocytes (2, 3). Double-label immunofluorescence microscopy reveals that cytoplasmic actin and myosin accumulate directly beneath patches and caps; that is, beneath aggregated surface receptors (4-7). More conclusively, isolation of the plasma membrane (PM) t and associated cytoskeletal elements with non-ionic detergents demonstrates that, during capping, the surface receptors form a specific association with the actin-containing cytoskeleton (8, 9). However, the nature of the linkage between membrane surface receptors and the Abbreviations used in this paper." IEF, isoelectric focusing; NP-40, Nonidet P-40; PBES, phosphate-buffered Earle's balanced salt solution; PM, plasma membrane. cytoskeleton is not understood.Recently, analogs to proteins of the erythrocyte membranecytoskeleton complex (such as spectrin, ankyrin, and glycophorin) have been identified in nonerythroid cells (10)(11)(12)(13)(14). Two distinct, spectrin-like proteins have been identified: fodrin (15-18) isolated from brain ; and TW 260/240 (16,17) isolated from epithelial cells...

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“…(14) at 4°C for 6 h to prepare myosin-conjugated gel beads. Antisera against human uterus smooth muscle myosin (IgG fraction) was then passed through a small column of myosin-conjugated Ultrogel, and the eluent not binding to the myosin-column was used as the antimyosin-free or preabsorbed reagent in control experiments related to the immunoprecipitation studies.…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation of myosin light chain during capping of mouse T-lymphoma cells.

Bourguignon¹,

Nagpal²,

Hsing³

1981

The Journal of Cell Biology

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Colchicine induces the clustering of at least three different T-lymphoma surface antigens (T200, Thy-1, and gp 69/71) into a cap structure in the absence of any external ligand . In addition, colchicine induces the intracellular accumulation of actin and myosin directly beneath the surface cap structure . We have discovered that myosin molecules (both heavy and light chains) are closely associated with the plasma membrane of T-lymphoma cells. Most importantly, we have found that the 20,000-dalton light chain of lymphocyte myosin is both phosphorylated and preferentially accumulated in the plasma membrane of colchicine-induced capped cells. It is proposed that myosin light chain is directly involved in the activation of membrane-associated actomyosin required for the collection of surface proteins into a cap structure (analogous to muscle cell sliding filament contraction).Redistribution of lymphocyte surface receptors can be induced either with or without the addition of external ligands (15,18,23,29,30,37,41,(46)(47)(48) . During the process of receptor movement, surface components first form small clusters (patches), which then aggregate at one pole of the cell into socalled caps .In the case of ligand-dependent capping, the ligand and its receptors appear to be clustered by a cross-linking event at the cell surface that is highly dependent on the valency of the ligand (37). For example, divalent antibodies against surface immunoglobulin (Ig) are required for the induction of Ig capping; monovalent anti-Ig is unable to cause any redistribution of the Ig receptor (30) . In addition, capping induced by the lectin, concanavalin A (Con A), requires the tetrameric form of Con A (at the proper concentration) since the dimer form (e .g ., succinyl Con A) is not active in cap formation (15,18) . Information concerning ligand-independent capping is very limited . Recently, it has been found that hypertonic media, mitogens, trypsin, and cholinergic drugs are each able to induce surface receptors to form cap structures in the absence of any externally added ligand (29,(46)(47)(48). The molecular mechanisms underlying both ligand-dependent and ligand-independent capping are not yet understood .Previously, we have used a double immunofluorescence labeling technique to simultaneously determine the distribution of certain surface receptors and intracellular actin and myosin in human skin fibroblasts grown in monolayers (9) or in murine lymphoid cells grown in suspension cultures (7,10,11) . The THE JOURNAL of CELL BIOLOGY -VOLUME 91 DECEMBER 1981 889-894

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The potential of Ultrogel®, an agarose‐polyacrylamide copolymer, as a matrix for affinity chromatography

Cited by 21 publications

References 13 publications

The selective retardation of NADP+-dependent dehydrogenases by immobilized procion red HE-3B

The selective retardation of NADP+-dependent dehydrogenases by immobilized procion red HE-3B

A T-lymphoma transmembrane glycoprotein (gp180) is linked to the cytoskeletal protein, fodrin.

Phosphorylation of myosin light chain during capping of mouse T-lymphoma cells.

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