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Biomedical subjects

A Silberstein

Publications and source records attributed to A Silberstein.

10 recordsLinked to original sources
PubMed1999

Membrane destabilization assay based on potassium release from liposomes.

Inorganic ions are highly suitable markers for monitoring release of the inner content of liposomes. In the present study, a potassium (K(+)) selective electrode was used to evaluate the rate of K(+) release from large unilamellar vesicles (LUV). The developed method is highly sensitive, reproducible and inexpensive. Since the K(+) ion is smaller than other markers conventionally used, the method described is more sensitive than one of the standard methods that uses ANTS/DPX. In addition, the method allows us to expand the set of molecules used as inner content markers to a lower size range. The experimental protocol we described contains improvements on the method of Breukink et al. (Biochemistry, 36 (1997) 6968). Our developed method was applied to compare the destabilizing activities of two amphipathic peptides of natural origin (Melittin and HIV env seg I, 827-851) and of two artificial peptides (Hels 7:11 and 9:9) synthesized de novo by Kiyota et al. (Biochemistry, 35 (1996) 13196). The tested peptides released 20% of the liposomal K(+) in 1 min at peptide-to-lipid ratio of a few mmol per mol of total lipids (LUV sized to 0.2 micrometer, molar composition is POPC:POPS:Chol 2:2:1).

Detergents↗
PubMed1998

Conformational analysis of amphotericin B--cholesterol channel complex.

A molecular model of ionic channel formed by flexible molecules of amphotericin B and cholesterol is proposed. Complexes with axial symmetry from 5 to 11 were simulated. In contrast to the model of the channel formed from rigid molecules, flexible molecules form a tightly packed structure consolidated by both dispersive forces and intermolecular hydrogen bonds. Contributions of a lactone ring, polar heads, cholesterol and lipid environments to the global energy of the complex formation are discussed. Among the complexes capable of ionic transport, that of axial symmetry eight is preferable. Two types of complexes, differing by the number of intramolecular hydrogen bonds, are shown to be possible.

Amphotericin B↗
PubMed1985

Why does Coomassie Brilliant Blue R interact differently with different proteins? A partial answer.

Dimethyl sulfoxide was found to be effective for extraction of Coomassie Brilliant Blue R-250 (Coomassie R) from stained proteins on polyacrylamide gel slices. A good correlation was found between the ability of different proteins to bind Coomassie R and their capacity for interaction with Coomassie Brilliant Blue G-250 (Coomassie G) in solution. Scatchard analysis showed that the number of Coomassie R ligands bound to each protein molecule is approximately proportional to the number of positive charges on the protein, about 1.5-3 dye molecules/charge.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

Quantitative determination of Coomassie R bound to proteins in polyacrylamide gel. A facilitated method for multi-spot analysis of electrograms.

Dimethylsulfoxide (DMSO) was found to be an efficient solvent for extraction of Coomassie Blue R 250 (Coomassie R) from stained proteins on polyacrylamide gels. Kinetic measurements show that the extraction of the dye from a 2-D gel reached equilibrium in 48 h. Staining of E. coli ribosomal proteins by Coomassie R dissolved in trichloroacetic acid exhibited two types of dye-protein complexes, the majority of them yield a blue-purple colour, while the rest are stained with a light-blue tone and fluorescent appearance as well. The absorbance spectra of the complexes in the gel matrix differ significantly from each other. However, the DMSO-extracted Coomassie show identical absorbance profiles with lambda max at 602 nm, thus the amount of the bound dye can easily be measured spectrophotometrically.

Electrophoresis, Polyacrylamide Gel↗
PubMed1979

Relief of polarity in E. coli depleted of 30S ribosomal subunits.

Escherichia coli was depleted of ribosomes by a thermal shock at 47 degrees C which quantitatively destroyed the 30S ribosomal subunits. During recovery in minimal medium at 30 degrees C RNA is synthesized while protein synthesis resumes only after about 90 min. It is shown that lac mRNA is synthesized in the complete absence of ribosomal activity and hence RNA synthesis is not coupled to protein synthesis. Lac mRNA from a series of lac nonsense mutants was examined in both heated and untreated cells. It was found that the polar effect of nonsense mutation is relieved in the absence of ribosomes and that this relief is due to the synthesis of larger mRNA molecules. Since Rho remained active in thermally treated cells, premature termination at secondary signals within the lac operon must also depend on the presence of active ribosomes.

Escherichia coli↗
PubMed1979

Synthesis and degradation of lac mRNA in E. coli depleted of 30S ribosomal subunits.

Escherichia coli was depleted of active ribosomes by a thermal shock at 47 degrees C which quantitatively destroyed the 30S ribosomal subunits. During recovery, RNA is synthesized while protein synthesis resumes only after about 90 minutes. It is shown that lac mRNA is synthesized in the complete absence of ribosomal activity and hence RNA synthesis is not coupled to protein synthesis. Transcription time and average transcript length were slightly less than in untreated cells. lac mRNA was degraded much more slowly in bacteria depleted of ribosomes. In E. coli W both functional half life (T 1/2 = 28 min vs. 2.25 in untreated cells) and chemical stability. The analysis of rna and pnp mutants showed that polynucleotide phosphorylase is involved in lac mRNA degradation in heat treated cells but that RNase I is not. The functional T 1/2 was increased in pnp mutants and was 95 min during the recovery period. The rate of chemical decay is so slow that the half-life cannot be accurately determined.

Bacterial Proteins↗
PubMedDate not supplied

A new method for stoichiometric analysis of proteins in complex mixture--reevaluation of the stoichiometry of E. coli ribosomal proteins.

A novel way is presented for determination of the stoichiometry of ribosomal proteins in the ribosome. The 70S E. coli r-proteins, completely separated on a two-dimensional gel system, were used throughout our experiments. The method is based on our previous observation that the amount of Coomassie R bound to a protein molecule is directly proportional to the number of positive charges on that protein. By plotting the amount of bound Coomassie as a function of the number of positive charges of each r-protein, and relating the experimental amount of the dye bound to each r-protein to the value obtained from the linear regression line based on all (a total of some 50 proteins), one can obtain the molar concentration of every protein in the ribosome. A parallel experiment can be carried out, which relates the radioactivity contributed by 3H-labeled amino acid in each r-protein to its amino acid content in that molecule. The two sets of data, which are completely independent of each other, are well correlated. Further verification of the validity of our procedure is provided by the fact that we found the known proportions of four copies of L7/L12 and one copy of S6 per ribosome. The rationale behind the present study was our finding that recalculation of Hardy's data (Hardy, S.J.S. (1975) Mol. Gen. Genet. 140, 253-274), with the accurate molecular weight value of the r-proteins provided by Giri et al. (Adv. Protein Chem. (1984) 36, 1-78), raises some doubt with regard to his experimental results, although we agree with his final conclusion that E. coli ribosome is homogeneous with respect to its proteins.

Bacterial Proteins↗