Sedimentation equilibrium analysis of glycopolymers

2007 ◽  
pp. 54-65 ◽  
Author(s):  
S. E. Harding
Keyword(s):  
Equilibrium Analysis ◽  
Sedimentation Equilibrium

scholarly journals The structure of L-lactate oxidase from Mycobacterium smegmatis

1977 ◽  
Vol 165 (2) ◽  
pp. 375-383 ◽  
Author(s):  
P A Sullivan ◽  
C Y Soon ◽  
W J Schreurs ◽  
J F Cutfield ◽  
M G Shepherd
Keyword(s):  
Electron Microscopy ◽  
Mycobacterium Smegmatis ◽  
Sedimentation Velocity ◽  
Equilibrium Analysis ◽  
Sedimentation Equilibrium ◽  
Cross Linking ◽  
Velocity Analysis ◽  
Approach To Equilibrium ◽  
Lactate Oxidase ◽  
Dimethyl Suberimidate

1. An improved purification was developed for L-lactate oxidase from Mycobacterium smegmatis. 2. The mol.wt. of the native enzyme by a sedimentation-equilibrium analysis was 345 000, and other ultracentrifuge methods gave values in the range 345 000-350 000. 3. An amino acid analysis, determinations of protein and flavin, a sedimentation-velocity analysis and an approach to equilibrium analysis gave values for the subunit mol.wt. in the range 43 500-47 000. 4. It was concluded that L-lactate oxidase contains eight subunits of mol.wt. 43 500. 5. Cross-linking of the subunits with dimethyl suberimidate and electron-microscopy studies were consistent with an octameric structure.

scholarly journals Purification and characterization of goblet-cell mucin of high Mr from the small intestine of sheep

1985 ◽  
Vol 229 (2) ◽  
pp. 419-428 ◽  
Author(s):  
T K S Mukkur ◽  
D L Watson ◽  
K S Saini ◽  
A K Lascelles
Keyword(s):  
Small Intestine ◽  
Analytical Ultracentrifugation ◽  
Gradient Centrifugation ◽  
Goblet Cells ◽  
Immunofluorescence Assay ◽  
Equilibrium Analysis ◽  
Sedimentation Equilibrium ◽  
Equilibrium Density ◽  
Total Carbohydrate ◽  
Protamine Sulphate

Crude soluble mucus from sheep small intestine was freed of nearly all the nucleic acid contaminants by precipitation with protamine sulphate and treatment with nucleases. After removal of non-covalently bound proteins by equilibrium density-gradient centrifugation in CsCl, a high-Mr glycoprotein was isolated by repeated h.p.l.c. from the partially purified mucin. The high degree of purity of the high-Mr mucin was borne out by (a) the observation of a single boundary on analytical ultracentrifugation in the presence of 5M-guanidinium chloride and (b) the observation of apparent monodispersity on sedimentation-equilibrium analysis. The Mr of the highly purified mucin, determined by sedimentation equilibrium, was 5.0 (+/- 0.1) X 10(6) and was concentration-independent. Finally, only goblet cells and the mucus blanket lining the intestinal epithelial cells were immunofluorescent when guinea-pig anti-(highly purified mucin) serum was used in an indirect immunofluorescence assay. The above antiserum reacted with apparently equal strength with goblet cells and with free mucin in abomasum, caecum and colon. The chemical composition of the glycoprotein was 66% carbohydrate and 34% protein, 45% of the latter being composed of valine and threonine. The glycoprotein migrated anodally on immunoelectrophoresis and contained 7.1% (w/w) sulphate. Neutral hexoses accounted for nearly half of the total carbohydrate content, followed by galactosamine and glucosamine. Whereas fucose and sialic acid were present in only small amounts, uronic acid was not detectable in the highly purified mucus glycoprotein.

scholarly journals Molecular Weight Distribution Studies on Heparin

1977 ◽  
Author(s):  
Grant H. Barlow
Keyword(s):  
Molecular Weight ◽  
Molecular Weight Distribution ◽  
Weight Distribution ◽  
Gel Filtration ◽  
Equilibrium Analysis ◽  
Resolving Power ◽  
Sedimentation Equilibrium ◽  
Distribution Data ◽  
Molecular Weights ◽  
Distribution Studies

The determination of molecular weight distribution using the sedimentation equilibrium analysis developed for polymers by T. Scholte (J. Polymer Sei, 6, 111, 1968) has been adapted for heparin analysis. Pork mucosal heparin separated into molecular weight subfractions by gel filtration on Ultrogel AcA44 (L.K.B.) was used to test the validity and resolving power of the method. Results indicate that the method is able to differentiate molecular weight distribution satisfactorily. Comparisons have been made of molecular weight distribution of samples from different species, organs and manufacturers. Average molecular weights for most samples center around 15,000 Dal tons, but samples show considerable variation in their distribution data. Results suggest that variations between manufacturers is more pronounced than the specie and organ difference indicating the importance of the purification procedure.

Sedimentation Equilibrium Analysis of Recombinant Human Tropoelastin Isoform

2000 ◽  
Vol 28 (5) ◽  
pp. A470-A470
Author(s):  
P. Toonkool ◽  
M. B. Morris ◽  
A. S. Weiss
Keyword(s):  
Equilibrium Analysis ◽  
Sedimentation Equilibrium

scholarly journals Oligomeric Structure of the Human Immunodeficiency Virus Type 1 Envelope Protein on the Virion Surface

2002 ◽  
Vol 76 (15) ◽  
pp. 7863-7867 ◽  
Author(s):  
Rob J. Center ◽  
Richard D. Leapman ◽  
Jacob Lebowitz ◽  
Larry O. Arthur ◽  
Patricia L. Earl ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Envelope Protein ◽  
Gel Filtration ◽  
Equilibrium Analysis ◽  
Sedimentation Equilibrium ◽  
Oligomeric Structure ◽  
Immunodeficiency Virus

ABSTRACT The envelope protein (Env) of human immunodeficiency virus type 1 forms homo-oligomers in the endoplasmic reticulum. The oligomeric structure of Env is maintained after cleavage in a Golgi compartment and transport to the surfaces of infected cells, where incorporation into budding virions takes place. Here, we use biophysical techniques to assess the oligomeric valency of virion-associated Env prior to fusion activation. Virion-associated Env oligomers were stabilized by chemical cross-linking prior to detergent extraction and were purified by immunoaffinity chromatography. Gel filtration revealed a single predominant oligomeric species, and sedimentation equilibrium analysis-derived mass values indicated a trimeric structure. Determination of the masses of individual Env molecules by scanning transmission electron microscopy demonstrated that virion-associated Env was trimeric, and a triangular morphology was observed in 20 to 30% of the molecules. These results, which firmly establish the oligomeric structure of human immunodeficiency virus virion-associated Env, parallel those of our previous analysis of the simian immunodeficiency virus Env.

scholarly journals Evidence for the formation of a heterotrimeric complex of leukaemia inhibitory factor with its receptor subunits in solution

1997 ◽  
Vol 325 (3) ◽  
pp. 693-700 ◽  
Author(s):  
Jian-Guo ZHANG ◽  
Catherine M. OWCZAREK ◽  
Larry D. WARD ◽  
Geoffrey J. HOWLETT ◽  
Louis J. FABRI ◽  
...  
Keyword(s):  
Interleukin 6 ◽  
Equilibrium Analysis ◽  
Inhibitory Factor ◽  
Sedimentation Equilibrium ◽  
Leukaemia Inhibitory Factor ◽  
Mouse Serum ◽  
Stable Complex ◽  
Resonance Spectroscopy ◽  
High Affinity ◽  
Α Chain

Leukaemia inhibitory factor (LIF) is a polyfunctional cytokine that is known to require at least two distinct receptor components (LIF receptor α-chain and gp130) in order to form a high-affinity, functional, receptor complex. Human LIF binds with unusually high affinity to a naturally occurring mouse soluble LIF receptor α-chain, and this property was used to purify a stable complex of human LIF and mouse LIF receptor α-chain from pregnant-mouse serum. Recombinant soluble human gp130 was expressed, with a FLAG® epitope (DYKDDDDK) at the N-terminus, in the methylotropic yeast Pichia pastoris and purified using affinity chromatography. The formation of a trimeric complex in solution was established by native gel electrophoresis, gel-filtration chromatography, sedimentation equilibrium analysis, surface plasmon resonance spectroscopy and chemical cross-linking. The stoichiometry of this solution complex was 1:1:1, in contrast with that of the complex of interleukin-6, the interleukin-6-specific low-affinity receptor subunit and gp130, which is 2:2:2.

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