PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “ULTRACENTRIFUGATION”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,225 records · Page 68Linked to original sources
PubMed1994

Effects of molecular crowding on protein self-association: a potential source of error in sedimentation coefficients obtained by zonal ultracentrifugation in a sucrose gradient.

Theoretical and experimental studies have illustrated a potential source of error in sedimentation coefficients obtained by sucrose density gradient centrifugation of proteins undergoing reversible self-association. The error stems from the excluded volume (molecular crowding) effect of the sucrose on the activity coefficients of monomeric and polymeric states. The consequent displacement of the equilibrium position in favor of polymeric state(s) is a function of sucrose concentration, and can therefore result in failure to detect the equilibrium coexistence of monomer if 5% sucrose suffices to displace the equilibrium completely toward dimer. In less extreme situations, it may result in the evaluation of an average sedimentation coefficient whose magnitude is a function of sucrose concentration and hence of the distance migrated into the sucrose gradient. These features are illustrated by the results of computer-simulated sedimentation of reversibly dimerizing systems in a sucrose gradient, and by conventional sedimentation velocity experiments on yeast enolase.

Centrifugation, Zonal↗
PubMed1982

Physicochemical studies of pregnancy-specific beta 1-glycoprotein: unusual ultracentrifugal and circular dichroic properties.

The molecular properties of native and modified pregnancy-specific beta 1-glycoprotein (SP1) from human placenta were evaluated by sedimentation equilibrium, gel electrophoresis, and circular dichroic measurements. Native SP1 contained 6.2% N-acetylneuraminic acid (NANA), 5.8% galactose (Gal), 13% N-acetylglucosamine, 6.5% mannose, and 1.1% fucose but no detectable N-acetylgalactosamine. Treatment with mixed exoglycosidases and alpha-mannosidase removed 79% of the carbohydrate including all of the NANA and Gal. The intensity of the circular dichroic spectrum of SP1 in the far ultraviolet was quite low with a positive maximum at 235 nm and a negative maximum at 215 nm. The 235-nm band was lost upon treatment with reducing agents or with guanidinium chloride (GdmCl), but not by treatment with neuraminidase. Treatment of SP1 with neuraminidase, or with mixed exoglycosidases and alpha-mannosidase, resulted in decreases of the apparent molecular weight obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Neither exposure of SP1 to GdmCl nor its reduction and alkylation resulted in the appearance of subunits on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The partial specific volume of SP1 determined experimentally by comparing sedimentation equilibrium profiles in H2O and D2O was 0.695 +/- 0.007 mL/g. The molecular weight of SP1 in 6 M GdmCl (in the presence or absence of reducing agents) by equilibrium sedimentation was 42 300 +/- 400. In the absence of denaturing agents, SP1 existed in the form of aggregates (at least as high as trimeric SP1) that dissociated only slowly upon dilution. The presence of these aggregates may contribute to the reported molecular heterogeneity of SP1.

Alkylation↗
PubMed1984

Separation of active tubulin and microtubule-associated proteins by ultracentrifugation and isolation of a component causing the formation of microtubule bundles.

A new method for separating microtubule-associated proteins (MAPs) and tubulin, appropriate for relatively large-scale preparations, was developed. Most of the active tubulin was separated from the MAPs by centrifugation after selective polymerization of the tubulin was induced with 1.6 M 2-(N-morpholino)ethanesulfonate (Mes) and GTP. The MAPs-enriched supernatant was concentrated and subsequently clarified by prolonged centrifugation. The supernatant (total soluble MAPs) contained almost no tubulin, most of the nucleosidediphosphate kinase activity of the microtubule protein, good activity in promoting microtubule assembly in 0.1 M Mes, and proteins with the electrophoretic mobility of MAP-1, MAP-2, and tau factor. The pellet, inactive in supporting microtubule assembly, contained denatured tubulin, most of the ATPase activity of the microtubule protein, and significant amounts of protein with the electrophoretic mobility of MAP-2. Insoluble material at this and all previous stages, including the preparation of the microtubule protein, could be heat extracted to yield soluble protein active in promoting microtubule assembly and containing MAP-2 as a major constituent. The total soluble MAPs were further purified by DEAE-cellulose chromatography into bound and unbound components, both of which induced microtubule assembly. The bound component (DEAE-MAPs) contained proteins with the electrophoretic mobility of MAP-1, MAP-2, and tau factor. The polymerization reaction induced by the unbound component (flow-through MAPs) produced very high turbidity readings. This was caused by the formation of bundles of microtubules. Although the flow-through MAPs contained significantly more ATPase, tubulin-independent GTPase, and, especially, nucleosidediphosphate kinase activity than the DEAE-MAPs, preparation of a MAPs fraction without these enzymes required heat treatment.

Animals↗
PubMed1981

An ultracentrifuge study of C-phycocyanin aggregation.

The molecular weight of C-phycocyanin has been determined as a function of protein concentration in a sodium acetate buffer solution of pH 4.8 at 21.2 degrees C by the Yphantis method. The higher aggregate, the dodecamer (19 S), is first removed by dissociation simply by dialysis against pH 3.9 acetate buffer solution. The molecular weight data are best interpreted by the simultaneous presence of monomers, trimers, and hexamers. Assuming a monomer K13 in equilibrium trimer K36 in equilibrium hexamer equilibrium system, the equilibrium constants have been calculated, and the values are K13 = 1.4 x 10(12) (L/mol)2 and K36 = 6.1 x 10(5) L/mol. The corresponding free energies for each step have been calculated to be -16.3 kcal/mol (delta G degrees 13) and -7.76 kcal/mol (delta G degrees 36). With these values of K13 and K36, the weight-average molecular weight as a function of concentration is calculated. Satisfactory agreement is obtained between the calculated curve and the experimental data. Sedimentation velocity studies performed with a band-forming centerpiece, on the same protein solutions used for the sedimentation equilibrium studies, confirm the presence of three species: monomers, trimers, and hexamers.

Acetates↗
PubMed1977

Ultracentrifugal studies of human luteinizing hormone and its subunits: dependence on protein concentration and ionic strength.

Purified human pituitary luteinizing hormone (hLH) and its alpha and beta subunits have been examined by physical methods. Freshly prepared hLH showed three closely adjacent bands on electrophoresis in polyacrylamide gels under alkaline conditions, but on standing even in the freeze-dried condition additional bands appeared. alpha and beta subunits gave bands which were quite different from the main hLH bands but comparable with the additional bands, as well as traces of hLH bands. hLH was investigated at three ionic strengths (0.1, 0.2, 0.5) at pH 5.9 +/- 0.1. Sedimentation velocity experiments demonstrated a complex system of association-dissociation which was further investigated by sedimentation equilibrium. Association occurred at the higher protein concentrations at each ionic strength, but to a significantly higher level at I = 0.5. Only at I = 0.1, pH 5.8, was there a clear indication over a range of protein concentration of the occurrence of a species of molecular weight 32 000 +/- 2000, in fair agreement only with the sum of the molecular weights of the alpha and beta subunits. At higher ionic strengths, there were indications of dissociation at low protein concentration (less than 0.10 g/100 mL) as well as association at higher values (greater than 0.20 g/100 mL). In view of the occurrence of molecular weights less than 28 000 at I = 0.2, HLH was treated in terms of a monomer of molecular weight 14 000, and some evidence was obtained for tetramer formation (4M = M4). At higher ionic strength this model does not apply and it is thought that indefinite association may be occurring to some extent also. The alpha subunit gave indications of association from its sedimentation coefficient vs. concentration plot, and sedimentation equilibrium (at pH 5.9 I = 0.1) demonstrated molecular weights increasing with increasing concentration. Evidence for tetramer formation also was obtained. The beta subunit, in spite of an apparently simple sedimentation coefficient-concentration curve, showed molecular weights varying from well below 14 000 to beyond 20 000. There is evidence to suggest that the isolated alpha and beta subunits, even on standing as dry solid, are not stable but give rise to lower molecular weight products. Aged freeze-dried hLH did not show such impurity.

Humans↗
PubMed1996

Thermodynamic analysis of the effects of small inert cosolutes in the ultracentrifugation of noninteracting proteins.

Considerations of the effect of a small cosolute on the sedimentation equilibrium distribution for a noninteracting protein have led to the development of a simple procedure for evaluating both the molecular weight of the protein and the second virial coefficient describing the excluded volume interaction between protein and cosolute. Its application is illustrated by analysis of sedimentation equilibrium distributions for bovine thyroglobulin and horse liver alcohol dehydrogenase in the presence of a range of sucrose concentrations, and also of those for aldolase in the presence of urea to obtain the subunit molecular weight of this tetrameric enzyme. The effects of sucrose concentration on the sedimentation coefficients of thyroglobulin, catalase, and horse liver alcohol dehydrogenase are also examined to demonstrate that the second virial coefficients for protein-cosolute excluded volume interaction may be determined, albeit with less precision, from the cosolute concentration required to render the sedimentation coefficient zero by virtue of its effect on the buoyancy term. These findings serve to reinforce the fact that the effects of small cosolutes usually ascribed to changes in "protein solvation" are envisaged more realistically in terms of excluded volume.

Alcohol Dehydrogenase↗