Change In Sedimentation Coefficient Research Articles

Studies on Rat Liver Phenylalanyl Transfer Ribonucleic Acid Synthetase: I. PURIFICATION, STABILIZATION, AND COMPLEX FORMATION

Abstract The phenylalanyl-tRNA synthetase (l-phenylalanine: tRNA ligase [AMP] EC 6.1.1.6) from rat liver was purified 500-fold by phosphocellulose and Sephadex G-100 chromatography. The behavior of this enzyme during purification suggests that intracellularly it exists as a complex with its specific transfer ribonucleic acid (tRNAphe). The sedimentation coefficient in sucrose gradients of the purified enzyme is 7.7 S while that of the complex with yeast tRNAphe is 11.6 S. This change in sedimentation coefficient is most compatible with the binding of 4 tRNAphe molecules to a synthetase of about 180,000 molecular weight although alternative explanations cannot be fully discounted. Similar changes in sedimentation coefficient were observed on binding rat liver tRNAphe and yeast tRNAphe from which the Y base had been removed. Heat inactivation of the synthetase, as measured by its decrease in ability to aminoacylate yeast tRNAphe, proceeds with apparent first order kinetics between pH 6 and pH 8. At pH 6.3 the rate constant for inactivation, ki, increases markedly with increasing ionic strength. However, at pH 7.5, ki decreases somewhat with increasing ionic strength. An inflection point in the plot of ki in 0.2 m KCl versus pH occurs at pH 6.4 and corresponds to the known pK of histidyl residues. Glycerol substantially protects against heat inactivation under all conditions of ionic strength and pH examined.

Conformational changes in proteins as measured by difference sedimentation studies. I. A technique for measuring small changes in sedimentation coefficient.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTConformational changes in proteins as measured by difference sedimentation studies. I. Technique for measuring small changes in sedimentation coefficientMarc W. Kirschner and H. K. SchachmanCite this: Biochemistry 1971, 10, 10, 1900–1919Publication Date (Print):May 11, 1971Publication History Published online1 May 2002Published inissue 11 May 1971https://doi.org/10.1021/bi00786a027RIGHTS & PERMISSIONSArticle Views141Altmetric-Citations66LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (5 MB) Get e-Alerts Get e-Alerts

The detection of a conformational change in the antibody molecule upon interaction with hapten

The technique of differential sedimentation is used to detect any change in sedimentation coefficient and in frictional coefficient of the γ-G immunoglobulin molecule upon interaction with hapten. Interaction of anti-lactoside antibody with the univalent haptens, lactose and p-(p-dimethylaminobenzeneazo)-phenyl β-lactoside, results in a slight increase in sedimentation coefficient of the antibody molecule, which corresponds to a decrease in frictional coefficient. The interpretation is that the antibody molecule assumes a more compact configuration upon interaction with univalent hapten.

Ultracentrifuge tudies of histone fractions from calf thymus deoxyribonucleoprotein.

Molecular weights and sedimentation coefficients of four major fractions of calf thymus histones were measured. The minimum molecular weights were determined in concentrated solutions of guanidine hydrochloride. The results indicate that, with the possible exception of fraction F3, the fractions are heterogeneous. Comparisons in 0.1m-sodium chloride suggest that fraction F1 does not aggregate and show that fractions F2(a) and F3 aggregate to form larger complexes than does fraction F2(b). The degree of aggregation of each fraction is independent of pH in the range pH1-7. Detailed studies with fraction F2(b) have confirmed that the change in sedimentation coefficient observed as the sodium chloride concentration of the solution is increased results from increases in the apparent molecular weight of the sedimenting units. It has been found that the molecules of fraction F2(b) are present as single molecules only in sodium chloride solutions of 33mm or less. At these low concentrations the effects of charge greatly increase the concentration dependence of the sedimentation rate; the results can, however, be interpreted by using the theory developed by Alexandrowicz & Daniel (1963) and Daniel & Alexandrowicz (1963).

Thermodynamic properties of the three conformational transitions of alanine specific transfer RNA from yeast

The melting behavior of alanine specific tRNA was studied by a modified differential absorption technique. It was found that the melting proceeds in three resolvable steps, 1, 2, 3. Steps 1 and 3 were interpreted as single processes, step 2 as two superimposed transitions. The corresponding reaction enthalpies were determined to be ΔH (1) = 40 ± 10, ΔH (2) = 2 · (65 ± 10) and ΔH (3) = 70 ± 10 kcal/mole, and are discussed on the basis of the “clover-leaf” model. Only the ionic strength dependence of steps 2 and 3 agrees with other double-stranded nucleic acids. Step 1 produces a smaller change in sedimentation coefficient than step 2.

Oxidation of pyrimidine nucleosides and nucleotides by osmium tetroxide.

1. Pyrimidine nucleosides such as thymidine, uridine or cytidine are oxidized readily at 0 degrees by osmium tetroxide in ammonium chloride buffer. There is virtually no oxidation in bicarbonate buffer of similar pH. Oxidation of 1-methyluracil yields 5,6-dihydro-4,5,6-trihydroxy-1-methyl-2-pyrimidone. 2. Osmium tetroxide and ammonia react reversibly in aqueous solution to form a yellow 1:1 complex, probably OsO(3)NH. A second molecule of ammonia must be involved in the oxidation of UMP since the rate of this reaction is approximately proportional to the square of the concentration of unprotonated ammonia. 3. 4-Thiouridine reacts with osmium tetroxide much more rapidly than does uridine. The changes of absorption spectra are different in sodium bicarbonate buffer and in ammonium chloride buffer. They occur faster in the latter buffer and, under suitable conditions, cytidine is a major product. 4. Polyuridylic acid is oxidized readily by ammoniacal osmium tetroxide, but its oxidation is inhibited by polyadenylic acid. Pyrimidines of yeast amino acid-transfer RNA are oxidized more slowly than the corresponding mononucleosides, especially the thymine residues. Appreciable oxidation can occur without change of sedimentation coefficient.

Sedimentation studies of the size and shape of DNA

Analytical zone sedimentation (Vinograd, Bruner, Kent & Weigle, 1963) is shown to be a sensitive and reliable method for detecting differences in conformation and for determining the molecular weight of homogeneous phage DNA's. Changes in sedimentation coefficient as a function of pH and ionic strength reveal transitions between native and denatured DNA and between different forms of denatured DNA. In alkaline solution, denatured DNA appears to exist in a random coil conformation, whereas at neutral pH it is contracted by intrastrand base-base interactions to a degree determined by the ionic strength. The denatured forms appear to be the single strands of the native DNA double helix. Calibrations of sedimentation coefficient against molecular weight are derived for single -stranded DNA in neutral and alkaline molar sodium chloride and for native DNA in molar sodium chloride. Degradation of homogeneous DNA is sensitively detected, and endonuclease I of Escherichia coli is shown to make double-strand breaks in native DNA, as distinct from pancreatic DNase which makes single-strand breaks.

Molecular association behavior of apomyoglobin I from the seal ( Phoca vitulina)

In the present paper the ultracentrifugal, electrophoretic, and optical rotation behavior of apomyoglobin as a function of pH and ionic strength is reported. It has been observed that polymerization occurs between pH 3 and 5.5, with maximum association between pH 4 and 5. At pH 8, the isoelectric point of apomyoglobin I, no change in sedimentation coefficient with different ionic strengths was observed. In free electrophoresis at pH 5 and 0.1 ionic strength, only a single rather symmetric peak was seen. The rotatory dispersion constant decreases with a change of pH from 5 to 3, thus indicating an unfolding of the molecules at pH 3. The nature of the polymerization reaction is discussed.