Polymerization of proteins with glutaraldehyde. Soluble molecular-weight markers

The lectin of the Indian bean or lablab (Dolichos lablab L.) was purified by affinity chromatography on two types of affinity carriers: O-a-D-mannopyranosyl-Separon and Separon-bound ovomucoid. The lectin is homogeneous in the ultracentrifuge: s2O, w= 6.14S, Mr= 110000; the molecule appears to comprise two pairs of two types of subunits (Mr 16000 and 40000), and contains 2% neutral sugar and 0.2 Mn and 0.5 Zn atom respectively. The lectin agglutinates human erythrocytes non-specifically with regard to ABO grouping at a limit concentration of 8,ug/ml, and this activity is inhibited most effectively by N-acetyl-D-glucosamine, methyl a-D-mannopyranoside and ovomucoid, but not by free D-mannose.The presence of a haemagglutinin in seeds of the Indian bean or lablab (Dolichos lablab L.) has been known for a long time (Tobis'ka, 1964). Extracts of D. lablab seeds were found also to be mitogenic (Aurich et al., 1971). To our knowledge, however, little attention, in comparison with other lectins, has been paid to the isolation and characterization ofthis lectin. Rao et al. (1976) used for its purification conventional methods of protein chemistry and described some of its properties.Though the erythroagglutinating activity of the D. lablab lectin was inhibited most effectively by methyl a-D-mannoside, in common with many other lectins specific for D-glucose and D-mannose [e.g. concanavalin A, Pisum sativum (pea) and Lens esculenta (lentil) lectins], further inhibition studies showed that the sugar specificity of the D. lablab lectin is quite different. Therefore it seemed worthwhile to seek a suitable affinity carrier for isolation of the lectin and to study its properties in more detail. Materials and methodsThe seeds of D. lablab L. were collected at Marmaris in the southern part of Turkey.Separon H-1000 was a product of Laboratorni Abbreviations used: SDS, sodium dodecyl sulphate; t.l.g.c., thin-layer gel chromatography. * This is paper 56 of the series 'Studies on lectins'. § To whom correspondence and requests for reprints should be sent. pristroje n.p., Praha, Czechoslovakia; it was glycosylated with free D-mannose or N-acetyl-D-glucosamine in dioxan with dry HCI as catalyst as described by Filka et al. (1978). Ovomucoid isolated from egg white (Lineweaver & Murray, 1947) or concanavalin A were bound to periodate-oxidized D-glucopyranosyl-Separon H-1000 containing 12.4% D-glucose (Filka et al., 1979). The watersoluble O-glycosyl-polyacrylamide copolymers used for affinity electrophoresis were prepared as described by Horejsi et al. (1978).

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“…( (Steward et al, 1965) or acidic (Reisfeld et al, 1962) (Payne, 1973). Reduction of the presumed -S-Sbond was performed with 2-mercaptoethanol.…”

Section: Methodsmentioning

confidence: 99%

Isolation and properties of a lectin from the seeds of the Indian bean or lablab (Dolichos lablab L.)

Güran¹,

Tichá²,

Filka³

et al. 1983

Biochemical Journal

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“…'The stability of the HF conjugate may be explained by the extensive cross-linkage introduced by glutaraldehyde, as a result of stable Michael-type additions formed between amino groups and a, ,ß-unsaturated aldehydes (23,31).…”

Section: Hf Conjugatesmentioning

confidence: 99%

Histamine receptors of the microvascular endothelium revealed in situ with a histamine-ferritin conjugate: characteristic high-affinity binding sites in venules.

Heltianu¹,

Simionescu²,

Simiónescu³

1982

The Journal of Cell Biology

135

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Histamine covalently bound to glutaraldehyde-activated ferritin was prepared as either monomers or as small aggregates of -0 .05 to 0.15 p,m Diam, suitable for electron microscopic detection of histamine cellular binding sites. The histamine-ferritin conjugates (MF) maintain the histamine capability to induce the opening of endothelial junctions in venules. To investigate the distribution of histamine receptors in the vascular endothelium, monomers or aggregates of MF were perfused in situ (mice), and various vascular beds, particularly that of the diaphragm, were fixed and processed for electron microscopy . The conjugate was preferentially bound on restricted areas of luminal endothelial cell plasmalemma especially in regions rich in filaments, and near the junctions between endothelial cells. The density of histamine binding sites was characteristically high in venules; it occurred to a much lesser extent in arterioles, veins, and muscular arteries whereas capillaries and aorta showed the lowest values . A similar distribution was obtained after perfusion of H, or H 2 receptor agonists coupled to ferritin (2-pyridylethylamine-ferritin [PF], or 4-methylhistamine-ferritin [MF], respectively) . The binding specificity was assessed through control experiments with either native or activated ferritin or by competition with histamine. The findings suggest that histamine receptors are largely represented in the cell membrane of the vascular endothelium, particularly in venules. Experiments using specific H, and H2 receptor agonists (PF and MF) and antagonists (mepyramine and cimetidine) indicate that the venular endothelium contains mainly H 2 receptors.

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“…wt. estimation were lysozyme, chymotrypsinogen, bovine serum albumin, phosphorylase a, /3-galactosidase and polymers of lysozyme and bovine serum albumin prepared as described by Payne (1973).…”

Section: Methodsmentioning

confidence: 99%

Lactoperoxidase-catalysed iodination of surface proteins on human melanoma cells

Roberts¹

1978

Br J Cancer

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Summary.-The cell-surface proteins of 6 different melanoma cell cultures have been labelled with 125I using lactoperoxidase-catalysed iodination. Fractionation of the proteins was achieved using 5-22.5% polacrylamide-gradient gel electrophoresis in the presence of sodium dodecyl sulphate (SDS) and the proteins were detected by autoradiography. Up to 24 labelled proteins were detected in the individual cell cultures, but the proteins labelled differed considerably in the 6 cultures examined. A possible reason for this, involving variation in the glycosylation of cell-surface glycoproteins is discussed. Cells of the same melanoma line had similar cell-surface proteins at different passage levels, but changes in the labelled proteins occurred when the culture conditions were altered. The cell-surface proteins of high molecular weight were cleaved by trypsin, but most of the low mol. -wt. proteins were resistant to trypsin. The "large external transformation sensitive" (LETS) protein detected as a major protein on fibroblasts in culture was not a dominant protein on the melanoma cells. It was detected on only 4/6 cell cultures. Possible relationships of the cell-surface proteins described in this study to morphology, immunological properties and proteolytic activity of human melanoma cells are discussed.CELLS interact with each other and with their environment by way of their surfaces, and surface structures are thought to be involved in a variety of phenomena such as tumour metastases, escape from immunological attack, growth regulation, and differentiation (Nicolson et al., 1975). An understanding of these phenomena would be facilitated by detailed information on cell surfaces. In an approach to this problem, we have studied the cell-surface proteins of human melanoma cells by a radioiodination technique, followed by separation of the labelled proteins by polyacrylamide-gel electrophoresis and detection of the proteins by autoradiography. MATERIALS AND METHODSCells. Melanoma and fibroblast cell cultures were developed from operative biopsies and cultured as described by Whitehead (1976). The medium used was McCov's 5A (GibcoBiocult Ltd., Paisley) containing 150/0 foetal calf serum (FCS), 2 mm glutamine, 2 ,ug/ml insulin, 15 mm HEPES, 10% MEM vitamins, 50 ,tg/ml penicillin, 50 ,ug/ml streptomycin and 20 mm NaHCO3. In some experiments the FCS was replaced by 500 human serum.Two of the melanoma cell cultures (HTCs 163 and 447) were derived from nodular primary tumours and 4 (HTCs 312, 364, 367 and 436) from secondary lymphnode deposits. All the cell cultures were from Caucasians, 2 of whom were males (HTCs 163 and 436). Melanoma cell lines HTC 163 and 312 have been described by Whitehead (1976

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Polymerization of proteins with glutaraldehyde. Soluble molecular-weight markers

Cited by 168 publications

References 25 publications

Isolation and properties of a lectin from the seeds of the Indian bean or lablab (Dolichos lablab L.)

Isolation and properties of a lectin from the seeds of the Indian bean or lablab (Dolichos lablab L.)

Histamine receptors of the microvascular endothelium revealed in situ with a histamine-ferritin conjugate: characteristic high-affinity binding sites in venules.

Lactoperoxidase-catalysed iodination of surface proteins on human melanoma cells

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