Search results for "Biotin"
showing 10 items of 127 documents
Controlling quaternary structure assembly: subunit interface engineering and crystal structure of dual chain avidin.
2006
Dual chain avidin (dcAvd) is an engineered avidin form, in which two circularly permuted chicken avidin monomers are fused into one polypeptide chain. DcAvd can theoretically form two different pseudotetrameric quaternary assemblies because of symmetry at the monomer-monomer interfaces. Here, our aim was to control the assembly of the quaternary structure of dcAvd. We introduced the mutation I117C into one of the circularly permuted domains of dcAvd and scanned residues along the 1-3 subunit interface of the other domain. Interestingly, V115H resulted in a single, disulfide locked quaternary assembly of dcAvd, whereas I117H could not guide the oligomerisation process even though it stabilis…
Chicken Avidin-related Protein 4/5 Shows Superior Thermal Stability when Compared with Avidin while Retaining High Affinity to Biotin
2003
The protein chicken avidin is a commonly used tool in various applications. The avidin gene belongs to a gene family that also includes seven other members known as the avidin-related genes (AVR). We report here on the extremely high thermal stability and functional characteristics of avidin-related protein AVR4/5, a member of the avidin protein family. The thermal stability characteristics of AVR4/5 were examined using a differential scanning calorimeter, microparticle analysis, and a microplate assay. Its biotin-binding properties were studied using an isothermal calorimeter and IAsys optical biosensor. According to these analyses, in the absence of biotin AVR4/5 is clearly more stable (T…
8-N(3)-3'-biotinyl-ATP, a novel monofunctional reagent: differences in the F(1)- and V(1)-ATPases by means of the ATP analogue.
2001
A novel photoaffinity label, 8-N(3)-3'-biotinyl-ATP, has been synthesized. The introduction of an additional biotin residue is advantageous for easy detection of labeled proteins. This could be first tested by reaction with the F(1)-ATPase from the thermophilic bacterium PS3 (TF(1)). UV irradiation of TF(1) in the presence of 8-N(3)-3'-biotinyl-ATP results in a nucleotide-dependent binding of the analogue in the noncatalytic alpha and the catalytic beta subunits of TF(1), demonstrating the suitability of this analogue as a potential photoaffinity label. Reaction with the V(1)-ATPase, however, led to labeling of subunit E, which has been suggested as a structural and functional homologue of …
Staphylococcus aureus alpha-toxin. Production of functionally intact, site-specifically modifiable protein by introduction of cysteine at positions 6…
1993
Staphylococcal alpha-toxin, the prototype of an oligomerizing, pore-forming cytotoxin, is sensitive to biochemical modifications and cannot be labeled with biotin or fluorescein under preservation of its biological activity. In this study, we have used site-directed mutagenesis to introduce cysteine residues at positions 69, 130, and 186. Each mutant was fully and rapidly reactive with several sulfhydryl-specific reagents, indicating superficial location. Coupling of SH-groups with fluorescein-maleimide or biotin-maleimide was tolerated without loss of hemolytic activity at position 130, and the formed hexamers were visible on target cells by fluorescence microscopy and could be detected on…
Sequence features and evolutionary mechanisms in the chicken avidin gene family
2001
The chicken avidin gene family comprises the avidin gene (avd) and several homologous avidin-related genes (avrs). The sequences of the avr genes are nearly identical to each other but exhibit nonrandomly distributed, frequently nonsynonymous nucleotide substitutions compared to avd. In this study, we determined the genetic distances and the phylogeny of the avd and avr genes and found differences between different exons and introns. Our results suggest the involvement of biased gene conversion in the evolution of the genes. Furthermore, one of the genes was identified as a putative fusion gene. The occurrence of both gene conversion and recombination supports the models suggesting a common…
Uptake of an Endocytic Marker by Rice Cells: Variations Related to Osmotic and Saline Stress
2003
Saline and osmotic stress are the main abiotic factors limiting the productivity of rice and other crop plants. Although both coincide in generating water deficit and affect many aspects of plant growth and development similarly, some effects of salinity are distinctively related to the ionic component of salt stress. At the cellular level, dessication tolerance is largely dependent on the cell's ability for osmotic adjustment. Here, we have studied the effects of saline and osmotic stress on endocytosis by rice cells, to investigate the common and distinctive effects of saline-generated stress and osmotically generated stress, and the possible involvement of endocytosis in tolerance mechan…
MCC1019, a selective inhibitor of the Polo-box domain of Polo-like kinase 1 as novel, potent anticancer candidate
2019
Polo-like kinase (PLK1) has been identified as a potential target for cancer treatment. Although a number of small molecules have been investigated as PLK1 inhibitors, many of which showed limited selectivity. PLK1 harbors a regulatory domain, the Polo box domain (PBD), which has a key regulatory function for kinase activity and substrate recognition. We report on 3-bromomethyl-benzofuran-2-carboxylic acid ethyl ester (designated: MCC1019) as selective PLK1 inhibitor targeting PLK1 PBD. Cytotoxicity and fluorescence polarization-based screening were applied to a library of 1162 drug-like compounds to identify potential inhibitors of PLK1 PBD. The activity of compound MC1019 against the PLK1…
Biotin-Labelled and Photoactivatable Aldosterone and Progesterone Derivatives as Ligands for Affinity Chromatography, Fluorescence Immunoassays and P…
1996
New derivatives of progesterone and aldosterone were synthesized and functionally tested with commercially available antibodies. The covalent labelling of antibodies specific for aldosterone and progesterone was detected by SDS/PAGE analysis and subsequent autoradiography after using 3-(O-carboxymethyl)-oximino-(3-[125I]iodo-4-azidosalicylamidobu tylamine) derivatives of aldosterone and progesterone, respectively, as photoactivatable radioligands. Labelling was not observed in the presence of an excess of the unlabelled steroid. Aldosterone was labelled with biotin and used as a tracer in a time-resolved fluorescence immunoassay. The nonradioactive tracer is highly selective for its antibod…
Biotin-Containing Reduced Graphene Oxide-Based Nanosystem as a Multieffect Anticancer Agent: Combining Hyperthermia with Targeted Chemotherapy
2015
Among the relevant properties of graphene derivatives, their ability of acting as an energy-converting device so as to produce heat (i.e., thermoablation and hyperthermia) was more recently taken into account for the treatment of solid tumors. In this pioneering study, for the first time, the in vitro RGO-induced hyperthermia was assessed and combined with the stimuli-sensitive anticancer effect of a biotinylated inulin-doxorubicin conjugate (CJ-PEGBT), hence, getting to a nanosystem endowed with synergic anticancer effects and high specificity. CJ-PEGBT was synthesized by linking pentynoic acid and citraconic acid to inulin. The citraconylamide pendants, used as pH reversible spacer, were …
Two amino acid residues determine the low substrate affinity of human cationic amino acid transporter-2A.
2003
Mammalian cationic amino acid transporters (CAT) differ in their substrate affinity and sensitivity to trans-stimulation. The apparent Km values for cationic amino acids and the sensitivity to trans-stimulation of CAT-1, -2B, and -3 are characteristic of system y+. In contrast, CAT-2A exhibits a 10-fold lower substrate affinity and is largely independent of substrate at the trans-side of the membrane. CAT-2A and -2B demonstrate such divergent transport properties, even though their amino acid sequences differ only in a stretch of 42 amino acids. Here, we identify two amino acid residues within this 42-amino acid domain of the human CAT-2A protein that are responsible for the apparent low af…