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Structure and interactions of subunits of cytoplasmic dynein /Nyarko, Afua A. January 2005 (has links)
Thesis (Ph.D.)--Ohio University, August, 2005. / Includes bibliographical references (leaves 130-156)
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Identification and characterization of novel protein-protein interactions with the basal transcription factor, TATA-binding proteinPrigge, Justin Robert. January 2006 (has links) (PDF)
Thesis (Ph. D.)--Montana State University--Bozeman, 2006. / Typescript. Chairperson, Graduate Committee: Ed Schmidt. Includes bibliographical references (leaves 88-107).
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Quantitative aspects of affinity adsorptionMayes, Andrew Geoffrey January 1992 (has links)
No description available.
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Protein binding studies by diafiltrationPalmer, Cecily M. January 1972 (has links)
A diafiltration technique was used to study drug-protein interactions. Fraction V human serum albumin and plasma and two drugs (phenylbutazone and bishydroxycoumarin) with a high affinity for these substances were used in this investigation. Preliminary experiments were carried out to check for release of foreign substances and for binding of drug to the Amicon diafiltration apparatus. A binding experiment, in the absence of drug, revealed release of a protein-like, ultraviolet absorbing substance from Fraction V human serum albumin. The most suitable method of purification
for albumin was by diafiltration with Tris buffer.
Binding curves for bishydroxycoumarin - human serum albumin, phenylbutazone
- human serum albumin, and bishydroxycoumarin - plasma interactions were obtained. The r and r/Df [subscript omitted] values were calculated and binding parameters estimated by both graphical extrapolation and by a computer non-linear least squares fit analysis. Binding curves were not independent of human serum albumin concentration, but the cause of this effect was not fully resolved.
Results showed the diafiltration technique can yield precise data, can be used over a wide macromolecule concentration range and produces a binding
curve, from one experiment, over a wide range of molar binding ratios.
Use of the Amicon diafiltration apparatus in desorption (washout) experiments and equilibrium or direct experiments was also investigated.
Attempts were made to obtain binding data by centrifugation (ultrafiltration)
and by a gel filtration technique (Sephadex G-25 batch method). These methods yielded unsatisfactory results which could not be compared with those obtained by diafiltration.
This abstract represents the true contents of the thesis submitted. / Pharmaceutical Sciences, Faculty of / Graduate
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The in vitro characterization of the drug-protein binding of racemic propafenone, and its active metabolite 5-hydroxypropafenone in human serum, and in solutions of isolated human serum proteinsTonn, George Roger January 1990 (has links)
An accurate plasma concentration-response relationship for propafenone (PF), a potent class 1 antiarrhythmic agent, has not yet been defined. A general pharmacological premise suggests that only the free drug is available to contribute to the observed pharmacological response. It has previously been shown that PF is highly bound to α-l-acid glycoprotein (AAG) which results in a low free PF concentration. The correlation of free PF concentration and response failed to adequately describe the dose response relationship. It has subsequently been shown that upon chronic dosing, two active metabolites, namely 5-hydroxypropafenone (5-OH-PF), and n-depropylpropafenone (n-depropyl-PF) accumulate in humans treated with PF. It is highly likely that the free concentration of PF, in addition to those of 5-OH-PF and n-depropyl-PF, contributes to the observed pharmacological effect following administration of PF at steady-state. To date, no accurate estimation of 5-OH-PF binding in serum has been established. This thesis examines the binding characteristics of PF and 5-OH-PF and their interaction in human serum, and in solutions of AAG, human serum albumin (HSA), high density lipoproteins (HDL), low density lipoproteins (LDL), and very low density lipoproteins (VLDL) using equilibrium dialysis.
The binding of PF (2.0 μg/mL) and 5-OH-PF (0.5 μg/mL) was examined in serum when both drug and metabolite were present. The free fraction (FF) of PF and 5-OH-PF in serum was 0.063 ± 0.004 and 0.232 ± 0.020, respectively. Both PF and 5-0H-PF were found to bind to a high affinity, low capacity binding site on AAG, in addition PF showed a
second low affinity, high capacity binding site. PF displayed a 10 fold greater affinity for the high affinity binding site on AAG when compared to 5-OH-PF. Both PF and 5-OH-PF showed only one low affinity, high capacity site on HSA of similar affinity. The interaction of PF and 5-OH-PF with HDL, LDL, and VLDL appeared to be due to solubilization, rather than a "true" drug-protein binding interaction, since it correlated well with the concentration of cholesterol within the lipoprotein complex (PF, r²=0.85; 5-OH-PF, r²=0.96). However, PF appeared to show saturable binding to the HDL complex. The uptake of PF and 5-OH-PF was greatest in LDL followed by HDL, and finally VLDL. In serum PF displayed both a high affinity, low capacity, and a low affinity, high capacity binding sites, although a similar observation was expected for 5-OH-PF, only one binding site could be experimentally identified.
The uptake of 5-OH-PF by red blood cells (RBC) appeared to be approximately 5 fold greater than that of PF (i.e. The ratio of PF and 5-OH-PF concentration in the red blood cell/plasma was 0.7 ± 0.1 and 3.2 ± 0.5, respectively).
When the binding of PF and 5-OH-PF was considered separately, the binding profiles were similar, that is, both drugs showed high affinity binding to AAG, and low affinity binding and/or non-specific binding to other serum proteins such as HSA, HDL, LDL, and VLDL. However, when both drug and metabolite were present, the binding of 5-OH-PF to AAG was found to be reduced. This is thought to occur as a result of the displacement of 5-OH-PF by PF from AAG. Thus, the binding of 5-OH-PF
was noted to be more dependent on HSA, and lipoproteins when compared to PF. On the other hand, the binding of PF (2.0 μg/mL), even with the addition of 5-OH-PF, was dependent largely on the concentration of AAG. Although the binding of 5-OH-PF was apparently not altered by the addition of PF in serum, a decrease in the binding of 5-OH-PF by the addition of PF was observed. It is hoped that the understanding gained from this thesis will provide information regarding the relative importance of free PF and 5-OH-PF plasma concentration in future pharmacodynamic studies of PF. / Pharmaceutical Sciences, Faculty of / Graduate
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Characterization of Functional Domains of Cul3, an E3 Ubiquitin Ligase, Using Chimeric AnalysisMitchell, Jennifer Anne 03 September 2014 (has links)
Modification of cellular proteins with molecules of ubiquitin is an important process that regulates the activity of cellular proteins. Cullin RING ligases (CRLs) are multi-subunit complexes that act in concert with E2 enzymes to attach molecules of ubiquitin to protein substrates. There are seven CRLs in mammalian cells (Cul1, Cul2, Cul3, Cul4A, Cul4B, Cul5, and Cul7) that are highly homologous in sequence and structure. CRLs possess a highly conserved C- terminal domain that interacts with E2 enzymes, and a more variable N- terminal domain which recruits substrates through distinct substrate adapter molecules. Despite the structural similarity, these CRLs recognize distinct substrates and carry out unique functions in cells.
In order to characterize the functional domains of cullins that are responsible for their unique activity, we generated cullin chimeras for expression and analysis in mammalian cells. These chimeras are Cul3 mutants in which the C- terminal domain or N- terminal domain of Cul3 has been replaced by that of Cul1 or Cul2, respectively. These chimeras were cloned into a mammalian expression vector for the purpose of experimentation in cultured cells.
The chimeric cullin constructs provided a valuable tool for investigating how different functional domains of CRLs contribute to their specific functions in cells. In this study, we first investigated if the chimeras that we engineered were able to interact with their respective substrate adapters. We performed co- immunoprecipitation experiments in which we tested the ability of wild type, chimeric, or mutant cullin proteins to bind to three different substrate adapter proteins. We found that the chimera possessing the C- terminus of Cul1 and the N- terminus of Cul3 retains the ability to interact with the BTB substrate adapters Ctb57 and KLHL3. We also found that the chimera that possesses the C- terminus of Cul3 and the N- terminus of Cul1 was unable to interact with BTB proteins. Lastly, we found that the Cul1 adapter Skp1 was able to bind to Cul1, but did not bind to Cul3 or either chimera. We concluded that the chimera possessing the N- terminus of Cul3 likely retains the functional binding abilities of Cul3 at the N- terminus and would therefore be useful for conducting experiments.
In this study, we also used the cullin chimeras to investigate the binding interactions between E2 enzymes and cullin RING ligases. We performed co- immunoprecipitation assays to examine the interactions between E2 enzymes and wild type, mutant or chimeric cullin proteins. We found that E2 enzyme UbE2E1 selectively binds to Cul3 and not to Cull. Notably, the BTB binding region at the N- terminus of Cul3 is required for binding to UbE2E1. Furthermore, we found that UbE2E1 also binds to Cul3 substrate adapter protein Ctb57. These experiments revealed a novel interaction between and E2 enzyme and the N- terminus of Cul3, as well as with a Cul3 substrate adapter protein. In conclusion, the chimeras generated in this study have provided valuable information regarding what regions of CRLs are important for interactions with other proteins, and will continue to be a useful tool for investigating CRL structure and function.
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A Method for Estimation of Plasma Protein Binding Using Diffusion Ordered NMR Spectroscopy (DOSY)Taylor, Rachel, Wilkinson, D., Swift, Thomas, Afarinkia, Kamyar 30 July 2024 (has links)
Yes / The plasma protein binding (PPB) of a drug plays a key role in both its pharmacokinetic and pharmacodynamic properties. During lead optimisation, medium and high throughput methods for the early determination of PPB can provide important information about potential PKPD profile within a chemotype or between different chemotype series. Diffusion ordered spectroscopy (DOSY) is an NMR spectroscopic technique that measures the diffusion of a molecule via applying varying magnetic field gradients, where the diffusion is primarily affected by its molecular size/weight and solution viscosity. Here, we describe the use of DOSY for a rapid and straight forward method to evaluate the PPB of drug molecules, using their binding to bovine serum albumin (BSA) as a model. / The full-text of this article will be released for public view at the end of the publisher embargo on 11 Jun 2025.
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Collagen binding proteins of intestinal Lactobacillus reuteri characterisation, purification and cloning /Aleljung, Pär. January 1994 (has links)
Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.
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Collagen binding proteins of intestinal Lactobacillus reuteri characterisation, purification and cloning /Aleljung, Pär. January 1994 (has links)
Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.
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The purification and characterization of a specific 3-methylcholanthrene-binding protein (SBP)Arnold, P. S. January 1987 (has links)
No description available.
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