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Biomedical subjects

K Gast

Publications and source records attributed to K Gast.

13 recordsLinked to original sources
PubMed1992

Application of dynamic light scattering to studies of protein folding kinetics.

The applicability of dynamic light scattering to studies of the kinetics of unfolding and refolding reactions of proteins is discussed and demonstrated experimentally. The experimental set-up and the data acquisition and data evaluation schemes that have been optimized for kinetic experiments are described. The relationship of the signal-to-noise ratio to the minimum data acquisition time that is needed to obtain results of sufficiently high precision is discussed. It turns out that the attainable time resolution is of the order of a few seconds for proteins with molar masses of about 50,000 g.mol(-1) and concentrations of 1 g.1(-1). Thus, DLS is too slow to follow conformational changes in the subsecond region, but it is useful for studies of unfolding-refolding reactions of proteins that proceed with time constants in the range of seconds or minutes. This is demonstrated by investigations of the kinetics of the cold denaturation of 3-phosphoglycerate kinase from yeast.

Kinetics↗
PubMed1992

Streptokinase is a flexible multi-domain protein.

The structure of streptokinase in solution has been studied by dynamic light scattering, small-angle X-ray scattering and circular dichroism spectroscopy. The Stokes' radius and radius of gyration of the protein monomer are 3.58 nm and 4.03 nm, respectively. The maximum intraparticle distance of the molecule is 14 nm. More than half of the amino acids of the molecule are organized in regular secondary structures. The X-ray scattering curve, the results from dynamic light scattering, and the finding that at least 50% of the amino acid residues are organized in regularly folded secondary structures are consistent with the following structural model. Streptokinase consists of four compact, separately folded, domains linked by mobile segments of the protein chain. The molecule exhibits the conformation of a flexible string-of-beads in solution.

Animals↗
PubMed1991

Acid denatured apo-cytochrome c is a random coil: evidence from small-angle X-ray scattering and dynamic light scattering.

The conformation of a denatured protein has been investigated, since the experimental data on the structure of denatured proteins have been incomplete until now. The Stokes' radius Rs and the radius of gyration Rg of apo-cytochrome c at pH 2.3 have been determined by dynamic light scattering and small-angle X-ray scattering, respectively. The values of these structure parameters, extrapolated to zero protein concentration, are Rs = 3.0 nm and Rg = 4.6 nm. The ratio Rg.Rs-1 is a sensitive indicator of the molecular conformation. The ratio of 1.55 obtained by us is typical for a random-coil polymer. The persistence length--the characteristic of the molecular flexibility--was determined to be a = 1.81 nm. From this results the root-mean-square average end-to-end distance of the molecules [h2] 1/2 = 11.2 nm and the characteristic ratio [h2]/npl2p = 8.43, where np = 104 is the number of amino acid residues and lp the distance between C alpha-atoms. We obtained a second virial coefficient A2 = 8.2.10(-3) mol cm3 g-2. The experimentally determined structure parameters are in approximate agreement with those predicted by Flory and others for an unperturbed, randomly coiled polypeptide. The expansion factor lies between 1.1 and 1.2. In conclusion, we have shown that apo-cytochrome c at pH 2.3 and at low concentrations has the conformation of a perturbed random coil with repulsive potentials between the chain segments.

Animals↗
PubMed1991

Solvent dependence of dimensions of unfolded protein chains.

The radii of gyration of unfolded apo-cytochrome C at pH 2.3 have been determined in three conditions: (i) 20 mM sodium phosphate buffer; (ii) 0.25 M NaCl; and (iii) 6.65 M GuHCl by small-angle X-ray scattering, and (iii) from translational diffusion coefficients measured by dynamic light scattering. The radius of gyration of the unfolded protein chain depends remarkably on the quality of the solvent, decreasing in the order 20 mM sodium phosphate greater than 6.65 M GuHCl greater than 0.25 M NaCl. The value of the radius of gyration in 0.25 M NaCl and also the value estimated for infinite ionic strength are close to the value predicted theoretically for the theta-point. This means that water in the absence of electrostatic interactions is a poor solvent for an unfolded protein while 6.65 M GuHCl is a better solvent.

Animals↗
PubMed1991

Measuring of the in-vitro assembly of microtubules by dynamic light scattering.

The kinetics of in-vitro assembly of microtubule protein (MTP) to microtubules (MTs) was followed under various conditions (temperature, GTP, ultrasonic treatment) by dynamic light scattering (DLS) and turbidimetric measurements. The results of both methods roughly coincide, but DLS additionally allows to differentiate between MTs and aggregates and to follow their growth. The complexity of these investigations, however, causes serious restrictions in the interpretation of the DLS data.

Animals↗
PubMed1990

Changes of the oligomeric structure of legumin from pea (Pisum sativum L.) after succinylation.

The influence of various levels of succinylation on the structure of the legumin from pea seed has been studied by the techniques of sedimentation velocity, viscometry, fluorescence and circular dichroism spectroscopy, as well as dynamic light scattering. The protein dissociates gradually into the 3S subunit forming a 7S intermediate. At a level of 75-80% succinylation, sudden unfolding of the protein occurs characterized by drastic changes in viscometric and spectroscopic properties. The fluorescence spectra point to the formation of a novel organized structure at a moderate degree of modification before the molecular unfolding takes place. The succinylated subunit was shown to have a sedimentation coefficient of 3.2S, a diffusion coefficient of 5.03 x 10(-7) cm2 . s-1 a Stokes' radius of 4.24 nm, a partial specific volume of 0.703 ml/g, an intrinsic viscosity of 0.13 dl/g, a molar mass of 52.2 kDa and a frictional ratio of 1.74.

Circular Dichroism↗
PubMed1990

Insulin aggregation in solution.

The process of insulin aggregation in neutral solutions was studied by dynamic light scattering. Solutions of different concentrations were subjected to thermal and mechanical stress (37 degrees, rotation) for a period of 4 weeks. The starting solutions contained exclusively one particle distribution of insulin in the association equilibrium with hexamers as the largest structures. After a lag period of about 8 days the solutions showed continuously increasing scattering intensities but did not evolve perceptible turbidity within the experimental period. A more rapid increase in scattering intensity was observed in diluted than in concentrated solutions. The analysis of scattering data unexpectedly revealed that insulin species did not grow continuously. After the lag period one additional relatively restricted size distribution with particles of a mean radius of about 100 nm was found, the amount of which increased continuously with time. The occurrence of these particles seems to be related to adsorption phenomena of insulin to the solid interface. We assume the 100 nm-class of aggregates to be a transient state in the physical destabilization process of insulin solutions.

Humans↗
PubMed1990

Combination oral contraceptives and cancer risk.

Substantial evidence exists to suggest that the use of oral contraceptives alters the risk for some types of cancer. Use of oral contraceptives for one year or more will reduce the risk of endometrial cancer and epithelial ovarian cancer by 50%, with the protective effect lasting for at least 10 years. The risk for developing cervical cancer in women who have used oral contraceptives appears to be slightly increased, although two independent studies actually found a protective effect associated with oral contraceptive use. The protective effect was probably related to the increased screening frequency found in oral contraceptive users and not related to a biologically protective effect. Therefore, women should be encouraged to undergo regular Pap tests. Data regarding breast cancer, in general, show no increased risk associated with oral contraceptive use. The latency associated with the development of breast cancer does not allow a definitive conclusion, and further study will be required. Oral contraceptives appear to increase the risk for developing benign hepatocellular adenoma, but not hepatocellular carcinoma.

Breast Neoplasms↗
PubMed1988

Preparation and properties of large octylglucoside dialysis/adsorption liposomes.

The suitability and capacity of the polystyrene resin Wofatit EP 60 for the adsorption of octylglucoside, Triton X-100, Cholate, and CHAPS was studied. Optimal detergent/bead ratios and the maximum capacity of Wofatit EP 60 for the four detergents were determined as prerequisites for optimal application of the beads in liposome preparations. It is shown that large unilamellar liposomes can be prepared easily, quickly and cost-effectively using a combined dialysis/adsorption method with octylglucoside as detergent and Wofatit EP 60 and adsorbing polystyrene beads. Structure, composition, size, homogeneity, lamellarity, stability, internal volume, and residual octylglucoside concentration were studied by gel chromatography, radioactive assay, dynamic light scattering and electron microscopy. Vesicle size and homogeneity depend on lipid concentration, lipid composition, cholesterol content, and the rate of octylglucoside removal, but not on the detergent/lipid ratio. The reliability of the method and the properties of the vesicles are compared with those of other methods and researchers.

Adsorption↗
PubMed1986

Shape and quaternary structure of alpha-globulin from sesame (Sesamum indicum L.) seed as revealed by small angle x-ray scattering and quasi-elastic light scattering.

The alpha-globulin from sesame seed has a molar mass of 2.7 X 10(5) g mol-1, determined by x-ray scattering, and (2.8 +/- 0.3) 10(5) g mol-1, determined by quasi-elastic light scattering. The radius of gyration RG amounts to (4.1 +/- 0.1) nm and (3.9 +/- 0.2) nm as determined by Guinier approximation and from the distribution function D(x), respectively. The molecule has a Stokes radius Rs of (5.4 +/- 0.15) nm and a maximum dimension L of (11 less than L less than 15) nm. The translational diffusion coefficient D0(20),w and the ratio of fractional coefficients f/fmin amount to (3.95 +/- 0.12) X 10(-7) cm2 s-1 and 1.25, respectively. The quaternary structure of the protein molecule is approximated by a model consisting of six spherical subunits situated at the vertices of an octahedron having the symmetry 32.

Alpha-Globulins↗
PubMed1986

Comparison of the structure of ribosomal 5S RNA from E. coli and from rat liver using X-ray scattering and dynamic light scattering.

The structure of eukaryotic ribosomal 5S RNA from rat liver and of prokaryotic 5S RNA from E. coli (A-conformer) have been investigated by scattering methods. For both molecules, a molar mass of 44,500 +/- 4,000 was determined from small angle X-ray scattering as well as from dynamic light scattering. The shape parameters of the two rRNAs, volume Vc, surface Oc, radius of gyration Rs, maximum dimension of the molecule L, thickness D, and cross section radius of gyration Rsq, agree within the experimental error limits. The mean values are Vc = 57 +/- 3 nm3, Oc = 165 +/- 10 nm2, Rs = 3.37 +/- 0.05 nm, L = 10.8 +/- 0.7 nm, D = 1.57 +/- 0.07 nm, Rsq = 0.92 +/- 0.01 nm. Identical structures for the E. coli 5S rRNA and the rat liver 5S rRNA at a resolution of 1 nm can be deduced from this agreement and from the comparison of experimental X-ray scattering curves and of experimental electron distance distribution functions. The flat shape model derived for prokaryotic and eukaryotic 5S rRNA shows a compact region and two protruding arms. Double helical stems are eleven-fold helices with a mean base pair distance of 0.28 nm. Combining the shape information obtained from X-ray scattering with the information about the frictional behaviour of the molecules, deduced from the diffusion coefficients D020, w = (5.9 +/- 0.2) X 10(-7) cm2 s-1 and (6.2 +/- 0.2) X 10(-7) cm2 s-1 for rat liver 5S rRNA and E. coli 5S rRNA, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗