Purpose: It has been hypothesized that short-range, protein-protein interactions of crystallin are necessary for the maintenance of lens transparency. Because of their probable weak nature, it has been difficult to both detect and quantitate the nature of these interactions. To determine if interactions exist between alpha-crystallin and gamma-crystallin under true equilibrium conditions, we have used microequilibrium dialysis.
Methods: Total alpha-crystallin and gamma-crystallin were prepared from soluble proteins of fetal bovine lenses by HPLC and gel filtration chromatography. The proteins were added to one side of a microequilibrium dialysis cell, comprised of two chambers separated by a membrane with 100 kDa molecular weight cut-off. After reaching equilibrium, the amount of free gamma-crystallin and the amount of gamma-crystallin bound to alpha-crystallin was determined by HPLC and reverse phase analysis of both chambers. Selected gamma-crystallin that bound to alpha-crystallin was further purified by ion exchange chromatography, and then incubated with alpha-crystallin, to verify the specificity of their binding.
Results: Analysis of both microequilibrium dialysis chambers incubated at different times at 37 degrees C indicated that equilibrium was reached at 4 days. When total alpha-crystallin and gamma-crystallin were incubated for this time period, significant binding was observed between alpha-crystallin and the IIIA, II, and IVA species of gamma-crystallin. These interactions were confirmed by microequilibrium dialysis determinations containing alpha-crystallin and purified gamma-crystallin species.
Conclusions: These results show that microequilibrium dialysis can be used to demonstrate significant noncovalent interactions of alpha-crystallin and gamma-crystallin under true equilibrium conditions.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1361693 | PMC |
Publication Analysis
Top Keywords
Similar Publications
Screening and Identification of DNA Aptamers to Tyramine Using in Vitro Selection and High-Throughput Sequencing.
ACS Comb Sci
June 2016
Institute of Sciences of Food Production, National Research Council of Italy , via G. Amendola 122/O, 70126 Bari, Italy.
Aptamers are synthetic single-stranded DNA or RNA sequences that can fold into tertiary structures allowing them to interact with and bind to targets with high affinity and specificity. This paper describes the first selection and identification of DNA aptamers able to recognize the biogenic amine tyramine. To successfully isolate aptamers to this challenging small molecule target, the SELEX methodology was adapted by combining a systematic strategy to increase the selection stringency and monitor enrichment success.
View Article and Find Full Text PDFMeasuring unbound versus total vancomycin concentrations in serum and plasma: methodological issues and relevance.
Ther Drug Monit
April 2015
*Department of Laboratory Medicine, Ghent University Hospital; †Department of Clinical Chemistry, Microbiology and Immunology, Ghent University; and ‡Department of Critical Care Medicine, Ghent University Hospital, Belgium.
Background: Studies on the unbound fraction (fu) of vancomycin report highly variable results. Great controversy also exists about the correlation between unbound and total vancomycin concentrations. As differences in (pre-)analytic techniques may explain these findings, we investigated the impact of the procedure used to isolate unbound vancomycin in serum/plasma on fu and the correlation between total and unbound concentrations.
View Article and Find Full Text PDFAge-dependent association of gamma-crystallins with aged alpha-crystallins from old bovine lens.
Mol Vis
May 2008
Division of Biology, Kansas State University, Manhattan, KS, USA.
Purpose: Previous theoretical and experimental studies have predicted that the loss of weak protein interactions between alpha- and gamma-crystallins could result in a decrease in the transparent properties of the aging lens.
Methods: alpha-Crystallins were prepared from the nucleus of old bovine lens, and gamma-crystallins were prepared from whole fetal bovine lens or from the nucleus of old bovine lens. The possible interactions of old alpha-crystallins with either old gamma-crystallins or fetal gamma-crystallins were quantitated at equilibrium using microequilibrium dialysis.
Dexamethasone as a probe for CYP3A4 metabolism: evidence of gender effect.
Cancer Chemother Pharmacol
July 2007
EA3035, Institut Claudius-Regaud, 31000 Toulouse, France.
Background: A study was conducted to evaluate prospectively the correlation between docetaxel clearance and pharmacokinetics of dexamethasone previously obtained in 21 patients.
Patients And Methods: Dexamethasone pharmacokinetics were performed in 17 patients 24 h before docetaxel treatment as monochemotherapy. Dexamethasone and docetaxel plasma concentrations were determined by HPLC methods.
Decreased association of aged alpha crystallins with gamma crystallins.
Exp Eye Res
October 2006
Department of Biology, Ackert Hall, Kansas State University, Manhattan, 66506, USA.
Previous studies have demonstrated non-covalent interactions of alpha crystallins with gamma crystallins, under true equilibrium conditions. These interactions could affect short-range interactions of lens crystallins that are necessary for the transparent properties of the lens. Since the transparent properties of the lens decrease during aging, it is possible that there are corresponding changes in the ability of aged alpha crystallins to interact with gamma crystallins.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!