| 1. |
- Nahálková, Jarmila, et al.
(författare)
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Affinity analysis of lectin interaction with immobilized C- and O-gylcosides studied by surface plasmon resonance assay
- 2002
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Ingår i: Journal of Biochemical and Biophysical Methods. - 0165-022X .- 1872-857X. ; 52:1, s. 11-18
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Tidskriftsartikel (refereegranskat)abstract
- A biosensor based on the surface plasmon resonance (SPR) principle was used for kinetic analysis of lectin interactions with different immobilized saccharide structures. A novel affinity ligands beta-D-glycopyranosylmethylamines derived from common D-aldohexoses linked to the carboxymethyl dextran layer of the SPR sensor surface served for interactions with a wide range of lectins. The method of preparation and use of the beta-D-mannopyranosyl glycosylated sensor surface was described. The results of affinity analysis of lectin-ligand interactions were evaluated and compared with data obtained from measurements using commercially available p-aminophenyl alpha-D-glycopyranosides. Possible applications and advantages of C- and O-glycosylated SPR biosensors are discussed.
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| 2. |
- Lagerquist Hägglund, Christine, et al.
(författare)
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Centrifugal and chromatographic analyses of tryptophan and tyrosine uptake by red blood cells and GLUT1 proteoliposomes with permeability estimates and observations on dihydrocytochalasin B
- 2003
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Ingår i: Journal of Biochemical and Biophysical Methods. - 0165-022X .- 1872-857X. ; 55:2, s. 127-140
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Tidskriftsartikel (refereegranskat)abstract
- We analyzed transport into liposomes and proteoliposomes, separated the free and internalized radioactively labeled substrates by size-exclusion chromatography (SEC) and observed a net influx owing to nonfacilitated diffusion across the lipid bilayers during the separation. The permeabilities (10(-9) cm/s) of glucose transporter (GLUT1) proteoliposomes were estimated to be 4.6, 1.0, 1.4 and 2.1 for D-glucose, L-glucose, L-Tyr and L-Trp, respectively; 15, 3.3, 5.1 and 2.1 times higher than the corresponding permeabilities of liposomes. These values indicated that GLUT1 did not transport Tyr or Trp, or transported Tyr, and only Tyr, slowly. This interpretation was supported by further analyses. Dihydrocytochalasin B inhibited the transport of Tyr and, partially, Trp into human red blood cells (centrifugal analyses). It did not inhibit Tyr and Trp influx into GLUT1 proteoliposomes, but partitioned strongly into the bilayers and seemed to make them fragile. The GLUT1 inhibitor cytochalasin B and the GLUT1 substrate 2-deoxy-D-glucose did not inhibit Tyr transport into the cells. Upon immobilized biomembrane affinity chromatography, Trp decreased the cytochalasin B retardation by GLUT1 only at levels far above the physiological Trp concentration. Ethanol (commonly added to aqueous solutions for enhancing a compound's solubility) halved the retardation at 4% (v/v) concentration. Drastic modification of the SEC method is required to allow permeability measurements with nonlabeled and highly permeable substrates.
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| 3. |
- Nordstrom, T., et al.
(författare)
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Method for one-step preparation of double-stranded DNA template applicable for use with Pyrosequencing (TM) technology
- 2002
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Ingår i: Journal of Biochemical and Biophysical Methods. - 0165-022X .- 1872-857X. ; 52:2, s. 71-82
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Tidskriftsartikel (refereegranskat)abstract
- A new one-step method for fast and efficient preparation of double-stranded DNA template, suitable for use with Pyrosequencing(TM) technology, has been developed. In the new method, two different types of oligonucleotides were used to prevent reannealing of remaining PCR primers to the template: oligonucleotides complementary to the PCR primers and 3'-end modified oligonucleotides with the same sequence as the PCR primers. Advantages with the new strategy are: (i) faster and simpler template preparation procedure (one-step); (ii) no need for exonuclease I treatment; and (iii) less problem with unspecific priming from loop structures and dimers. By careful oligonucleotide design, and/or by addition of single-stranded DNA-binding protein, problems with unspecific sequence signals due to mispriming can be reduced. The new method was used for analysis of genotype variations within the renin-angiotensin-aldosterone system.
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| 4. |
- Hedin, Eva M. K., et al.
(författare)
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Low microwave-amplitude ESR spectroscopy : Measuring spin-relaxation interactions of moderately immobilized spin labels in proteins
- 2004
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Ingår i: Journal of Biochemical and Biophysical Methods. - : Elsevier BV. - 0165-022X .- 1872-857X. ; 60:2, s. 117-138
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Tidskriftsartikel (refereegranskat)abstract
- Electron spin resonance (ESR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful tool for determining protein structure, dynamics and interactions. We report here a method for determining interactions between spin labels and paramagnetic relaxation agents, which is performed under subsaturating conditions. The low microwave-field amplitude employed (h(1) < 0.36 G) only requires standard, commercially available ESR equipment. The effect of relaxation enhancement on the spin-spin-relaxation time, T-2e, is measured by this method, and compared to classical progressive power saturation performed on a free spin label, (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl)methanethiosulfonate (MTSL), and a spin-labeled protein (Thermomyces lanuginosa lipase, TLL-1252C), employing the water-soluble relaxation agent chromium(III) oxalate (Crox) in concentrations between 0-10 mM. The low-amplitude theory showed excellent agreement with that of classical power saturation in quantifying Crox-induced relaxation enhancement. Low-amplitude measurements were then performed using a standard resonator, with Crox, on 11 spin-labeled TLL mutants displaying rotational correlation times in the motional narrowing regime. All spin-labeled proteins exhibited significant changes in T-2e. We postulate that this novel method is especially suitable for studying moderately immobilized spin labels, such as those positioned at exposed sites in a protein. This method should prove useful for research groups with access to any ESR instrumentation.
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| 5. |
- Masarova, Jana, et al.
(författare)
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Novel peptide surface for reversible immobilization of concanavalin A
- 2004
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Ingår i: Journal of Biochemical and Biophysical Methods. - : Elsevier BV. - 0165-022X .- 1872-857X. ; 60:2, s. 163-170
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Tidskriftsartikel (refereegranskat)abstract
- Concanavalin A (Con A) was spontaneously adsorbed on polymyxin B surface. This peptide-lectin interaction was strong, KD=1.9×10 -10, based predominantly on creation of hydrophobic bonds, and was completely reversible. Concanavalin A on polymyxin B (PmB) retained higher binding capacity for yeast mannan, compared with covalently immobilized lectin. Kinetics of mannan-concanavalin A interaction were significantly different in dependence on type of concanavalin A immobilization. © 2004 Elsevier B.V. All rights reserved.
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| 6. |
- Bradoo, Sapna, et al.
(författare)
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Microwave-assisted rapid characterization of lipase selectivities.
- 2002
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Ingår i: Journal of Biochemical and Biophysical Methods. - 0165-022X. ; 51:2, s. 115-120
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Tidskriftsartikel (refereegranskat)abstract
- A rapid screening procedure for characterization of lipase selectivities using microwaves was developed. The rate of reaction of various commercial lipases (porcine pancreas, Mucor miehei, Candida rugosa, Pseudomonas cepacia) as well as lipases from laboratory isolates-Bacillus stearothermophilus and Burkholderia cepacia RGP-10 for triolein hydrolysis was 7- to 12-fold higher in a microwave oven as compared to that by pH stat. The esterification of sucrose/methanol and ascorbic acid with different fatty acids was also achieved within 30 s in a microwave using porcine pancreas, B. stearothermophilus SB-1 and B. cepacia RGP-10 lipases. The relative rates and selectivity of the lipases both for hydrolytic and synthesis reactions remains unaltered. However, the rate of reaction was dynamically enhanced when exposed to microwaves. Microwave-assisted enzyme catalysis can become an attractive procedure for rapid characterization of large number of enzyme samples and substrates, which otherwise is a cumbersome and time-consuming exercise.
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| 7. |
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| 8. |
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| 9. |
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| 10. |
- Dolby, Viveka, et al.
(författare)
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Effects of pH, salt and time on ligand binding properties of overexpressed melanocortin 4 receptor.
- 2004
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Ingår i: Journal of Biochemical and Biophysical Methods. - : Elsevier BV. - 0165-022X. ; 58:3, s. 195-205
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Tidskriftsartikel (refereegranskat)abstract
- The G-protein coupled melanocortin 4 receptor (MC4r) plays an important role in the energy metabolism. We overexpressed the MC4r in CHO cells and performed characterisation studies on the cell membranes to determine functional stability and ligand binding properties of the receptor. The affinity for the ligands [Nle4, Image-Phe7]-αMSH and MTII was lost below pH 6 but could be restored by returning to physiological pH. Increasing NaCl concentration up to 1 M had little influence on the binding of either ligand. At neutral pH, physiological salt concentration and 4 °C the ligand affinity of the receptor was stable for up to 6 days. These findings will facilitate design of purification methods for the receptor.
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