scholarly journals Thiol reduction of human α2-macroglobulin. The subunit structure

1972 ◽  
Vol 127 (1) ◽  
pp. 187-197 ◽  
Author(s):  
J. M. Jones ◽  
J. M. Creeth ◽  
R. A. Kekwick
Keyword(s):  
Molecular Weight ◽  
Large Scale ◽  
Sedimentation Equilibrium ◽  
Subunit Structure ◽  
Experimental Conditions ◽  
A Value ◽  
Α2 Macroglobulin ◽  
A Subunit ◽  
Normal Human ◽  
Ph Range

1. Human α2-macroglobulin was prepared from a fraction obtained during the large-scale separation of normal human plasma proteins for clinical use. 2. Sedimentation-equilibrium measurements indicated a molecular weight of 725000. A value of 18.1S was obtained for s020,w. 3. The dissociation that occurs in the pH range 4.5–2.5 and in the region of neutrality in urea-containing solutions is consistent with a dimeric structure of the molecule. 4. The effects of the thiol reagents mercaptoethanol, mercaptoethylamine and N-acetylcysteine were investigated over a range of experimental conditions. Distinct components having sedimentation coefficients of 15, 12 and 8.5S were identified. 5. Conditions were found under which limited reduction with thiol liberated a subunit with a molecular weight approximately one-quarter of that of the intact molecule. This subunit retains the serological specificity of the whole molecule.

scholarly journals The subunit structure of horse spleen apoferritin: the molecular weight of the oligomer and its stability to dissociation by dilution (Short Communication)

1973 ◽  
Vol 131 (4) ◽  
pp. 855-857 ◽  
Author(s):  
Robert R. Crichton ◽  
Robert Eason ◽  
Allan Barclay ◽  
Charles F. A. Bryce
Keyword(s):  
Molecular Weight ◽  
Short Communication ◽  
Sedimentation Equilibrium ◽  
Subunit Structure ◽  
Subunit Dissociation ◽  
A Value

The oligomer molecular weight of horse spleen apoferritin was determined by sedimentation-equilibrium techniques and a value of 443000 found. It is concluded that the apoferritin molecule consists of 24 subunits. At concentrations as low as 0.01μm there is no evidence of subunit dissociation.

Human Serum Pseudocholinesterases: Molecular Weight Estimation of a Subunit Structure

1971 ◽  
Vol 49 (8) ◽  
pp. 777-779 ◽  
Author(s):  
Denis Boutin ◽  
Jules Brodeur
Keyword(s):  
Molecular Weight ◽  
Human Serum ◽  
Cholinesterase Activity ◽  
Gel Filtration ◽  
Semipermeable Membrane ◽  
Subunit Structure ◽  
Weight Estimation ◽  
Experimental Conditions ◽  
A Subunit

Molecular weight determinations by gel filtration on Sephadex G-200 were performed on fractions of human serum cholinesterases as obtained after ultrafiltration through a semipermeable membrane. The molecular weight of the fraction filtering through a membrane with molecular weight exclusion limits of 100 000 was estimated to be 86 000, whereas that of the fraction retained by the filter was higher than 300 000, approximating the value of 348 000 previously reported in the literature. Both fractions were shown to be interconvertible under the experimental conditions used. These results provide further evidence in favor of the existence of an enzymatically active subunit structure of cholinesterases and suggest that subunits combine into tetramers to form the major component of the cholinesterase activity in human serum.

scholarly journals Physical properties and subunit structure of l-asparaginase isolated from Erwinia carotovora

1972 ◽  
Vol 126 (2) ◽  
pp. 361-379 ◽  
Author(s):  
K. A. Cammack ◽  
D. I. Marlborough ◽  
D. S. Miller
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Molecular Conformation ◽  
Sodium Dodecyl ◽  
Erwinia Carotovora ◽  
Optical Rotatory Dispersion ◽  
Spherical Particles ◽  
Sedimentation Equilibrium ◽  
Subunit Structure ◽  
Equilibrium Ultracentrifugation

1. l-Asparaginases from Erwinia carotovora and Escherichia coli (EC2 enzyme) are both capable of inhibiting and eliminating certain types of tumour cells. The Er. carotovora enzyme is a more basic protein, however, and in contrast with the EC2 enzyme it contains neither tryptophan nor cystine, and disulphide bonds are therefore absent. The molecule is very stable in solution from pH3.0 to about pH12.0, and is somewhat more stable at alkaline pH than is the Esch. coli enzyme. Calculations based on a s020,w 7.43S and a sedimentation-equilibrium molecular weight of 135000±10000 give a frictional ratio (f/f0) of 1.08. The molecular conformation is therefore very compact in solution, and the electron microscope shows the negatively stained molecules as almost spherical particles with a diameter of 7.2±0.7nm. 2. Sedimentation-velocity and equilibrium ultracentrifugation, in 5–8m solutions of urea and guanidinium chloride, and also electrophoresis in sodium dodecyl sulphate–polyacrylamide gel, reveal a dissociation of the native protein molecule into four subunits of similar molecular weight in the range 32500–38000. The enzymically inactive subunits can be physically reassembled into an active tetramer when urea is removed by dialysis. Although the subunit structures of the Er. carotovora enzyme and the Esch. coli enzyme molecules are similar, the secondary bonding forces holding the subunits together in the tetramer are somewhat stronger in the Er. carotovora enzyme. 3. The optical-rotatory-dispersion (o.r.d.) parameters that characterize the Cotton effects arising from ordered structure in the molecule are [m′]233=−3522±74° and [m′]200=9096±1700°. These show very marked changes as the secondary structure is disrupted and the molecule dissociates into subunits. A correlation pathway was traced on the basis of o.r.d. parameters and enzyme activity as the polypeptide chains were denatured and renatured (and reconstituted) into active molecules after the dilution of solutions in urea. Subunits resulting from treatment with sodium dodecyl sulphate do not show the typically disordered o.r.d. profile, but nevertheless they are inactive.

scholarly journals The composition and proposed subunit structure of egg-white β-ovomucin. The isolation of an unreduced soluble ovomucin

1975 ◽  
Vol 147 (1) ◽  
pp. 55-62 ◽  
Author(s):  
D S Robinson ◽  
J B Monsey
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Sialic Acid ◽  
Sodium Dodecyl ◽  
Egg White ◽  
Disc Electrophoresis ◽  
Subunit Structure ◽  
Sedimentation Analysis ◽  
Guanidinium Chloride ◽  
A Subunit

1. New preparations of reduced carboxymethylated β-ovomucin (S-carboxymethyl-β-ovomucin) were homogeneous by sedimentation analysis, analytical sedimentation to equilibrium in CsCl gradients, and disc electrophoresis in sodium dodecyl sulphate. 2. Degradation of S-carboxymethyl-β-ovomucin with either CNBr or trypsin indicated the presence of a subunit (approx. mol. wt. 112300). 3. Electron microscopy showed that S-carboxymethyl-β-ovomucin consisted of chains of globular units (approx. mol. wt. 103 000). IN 6M-guanidinium chloride S-carboxymethyl-β-ovomucin existed mainly as an aggregate (mol. wt. 720 000). 4. S-Carboxymethyl-β-ovomucin contained ester sulphate (4.24%, W/W) and carbohydrate (60%, W/W), which consisted of large amounts of galactose (22%, W/W), galactosamine (8.9%, W/W) and sialic acid (10.6%, W/W). 5. An unreduced soluble fibrous component (component SGH) extracted from crude ovomucin precipitate with 5M-guanidinium chloride contained β-ovomucin (approx. 70%, W/W). By using the Scheraga-Mandelkern equation the molecular weight of component SGH was calculated to be 11.5 times 10(6).

Affinity Chromatography of Human Factor VIII Using Human and Rabbit Antibodies to Factor VIII

1979 ◽  
Vol 42 (04) ◽  
pp. 1306-1315 ◽  
Author(s):  
Janet L Lane ◽  
H Ekert ◽  
A Vafiadis
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Affinity Chromatography ◽  
Protein Concentration ◽  
Gel Filtration ◽  
Subunit Structure ◽  
Molecular Weights ◽  
Human Factor Viii ◽  
Sds Page ◽  
Normal Human

SummaryFactor VIII, purified by gel filtration on Sepharose 2B, has an 8 band multiple subunit structure, with molecular weights ranging from 30,000 to 230,000, on reduction and SDS-PAGE at a protein concentration of 400 μg/gel. Affinity chromatography of this factor VIII preparation with insolubilized haemophilic antibody to factor VIII showed that 45-81% VIII:C and 0-33% VIILRag were attached to the column. Elution of the column with 0.25 M CaCl2 did not show VIII:C or VIILRag in the eluate. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the dissociated protein, showed that 95 % of the protein bound by haemophilic antibody had a molecular weight similar to the low molecular weight subunits of the reduced factor VIII.In control experiments with normal Human IgG, 3% of VIII:C and 5% of VIILRag were attached to the column. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the protein, showed 2 faint bands with molecular weight consistent with heavy and light chains of IgG.Similar experiments with antibody to factor VIII showed that 67-83% of VIILC and 61-76% of VIII:Rag were attached to the column. Elution of the column with 0.25 M CaCl2 showed 10% of the applied VIII:C, but no VIII:Rag in the eluate. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the dissociated protein, showed an 8 band subunit structure similar to the reduced factor VIII.

Subunit Structure and some Properties of Pyruvate Kinase of Neurospora

1975 ◽  
Vol 53 (2) ◽  
pp. 109-119 ◽  
Author(s):  
M. Kapoor
Keyword(s):  
Molecular Weight ◽  
Pyruvate Kinase ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Sedimentation Equilibrium ◽  
Phosphoryl Group ◽  
Subunit Structure ◽  
Equilibrium Studies ◽  
Variable Extent ◽  
High Concentrations

Pyruvate kinase isolated from Neurospora and purified to homogeneity has been shown to be a tetramer of molecular weight around 242 000 by gel filtration studies and 239 000 daltons by sedimentation equilibrium measurements. The monomer produced by treatment with guanidine hydrochloride is found to be 51 000–52 000 daltons by sedimentation equilibrium studies; a molecular weight of 62 000 was determined for the monomer generated by SDS treatment by electrophoresis in SDS–polyacrylamide gels. The enzyme has an isoelectric point of 6.35–6.41. Substrate saturation kinetics of PEP show a variable extent of cooperativity depending upon the buffer ions employed in the assay. ADP is the most effective phosphoryl group acceptor, GDP and IDP being poor substitutes. A divalent cation, Mg2+, is required for activity. At low concentrations, Ca2+ acts as an activator of pyruvate kinase but it is inhibitory at high concentrations. Fructose 1,6-diphosphate is the most potent allosteric activator, fructose 6-phosphate being next in order of effectiveness. Valine is a powerful inhibitor. Phenylalanine, tyrosine, and tryptophan are without any effect individually, but their simultaneous presence results in a considerable activation. Alanine does not affect this enzyme appreciably.

scholarly journals Erythrocruorin from the water-flea Daphnia magna. Quaternary structure and arrangement of subunits

1982 ◽  
Vol 207 (2) ◽  
pp. 297-303 ◽  
Author(s):  
E Ilan ◽  
E Weisselberg ◽  
E Daniel
Keyword(s):  
Molecular Weight ◽  
Daphnia Magna ◽  
Quaternary Structure ◽  
Slow Component ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Sedimentation Equilibrium ◽  
Subunit Structure ◽  
Single Chain

The subunit structure of erythrocruorin from the cladoceran Daphnia magna was studied. The native protein was found to have a sedimentation coefficient (S2(20), w) of 17.9 +/- 0.2 S and a molecular weight, as determined by sedimentation equilibrium, of 494 000 +/- 33 000. Iron and haem determinations gave 0.312 +/- 0.011% and 3.84 +/- 0.04%, corresponding to minimal molecular weights of 17900 +/- 600 and 16 100 +/- 200 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave one band with mobility corresponding to a molecular weight of 31 000 +/- 1 500. The molecular weight of the polypeptide chain determined by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol is 31 100 +/- 1300. On a molecular-weight basis, Daphnia erythrocruorin is composed of 16 identical polypeptide chains carrying two haem groups each. The native structure is stable between pH5 and 8.5. At alkaline and acidic pH, a gradual decrease in the sedimentation coefficient down to 9.8S occurs. Above pH 10 and below pH4, a slow component with S20, w between 2.7S and 4.0S is observed. The 2.7S, 4.0S and 9.8S species are identified as single-chain subunits, subunit dimers and half-molecules respectively. We propose a model for the molecule composed of 16 2.7S subunits grouped in two layers stacked in an eclipsed orientation, the eight subunits of each layer occupying the vertices of a regular eight-sided polygon. Support for this arrangement is provided from electron microscopy and from analysis of the pH-dissociation pattern.

scholarly journals Physicochemical properties of Planorbis corneus erythrocruorin

1975 ◽  
Vol 149 (2) ◽  
pp. 437-445 ◽  
Author(s):  
E J Wood ◽  
L J Mosby
Keyword(s):  
Molecular Weight ◽  
High Speed ◽  
Sedimentation Coefficient ◽  
Sedimentation Equilibrium ◽  
Alkaline Conditions ◽  
Planorbis Corneus ◽  
A Subunit ◽  
Partial Dissociation ◽  
Equilibrium Ultracentrifugation ◽  
Intermediate Value

The erythrocruorin from the snail Planorbis corneus had a sedimentation coefficient, s020,w, of 33.5 ± 0.31S, and a molecular weight of 1.65 × 10(6) ± 0.04 × 10(6) by high-speed sedimentation-equilibrium ultracentrifugation. The amino acid composition and absorption spectrum of the protein are reported. A very low number of half-cystine residues was found, corresponding to 0.4 residue per haem group. The haem content was 2.76 ± 0.22%, corresponding to a protein molecular weight of about 22300. Under both acid and alkaline conditions partial dissociation took place to yield mixtures of products that could not be identified. A subunit corresponding to that containing one haem group was not obtained under any of the dossociating conditions tried. Electron microscopy revealed a ring-shaped molecule about 12.2 ± 0.5 nm in diameter. The native erythrocruoerin bound O2 co-operatively, the intermediate value of h in Hill plots having values between 1.7 and 3.4 depending on the conditions.

Molecular Weight of Legumin From Pisum sativum

1980 ◽  
Vol 7 (3) ◽  
pp. 221 ◽  
Author(s):  
RJ Blagrove ◽  
GG Lilley ◽  
R Davey
Keyword(s):  
Molecular Weight ◽  
Recent Literature ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sedimentation Equilibrium ◽  
Pumpkin Seed ◽  
Subunit Molecular Weight ◽  
A Value ◽  
Oligomeric Protein ◽  
Physicochemical Studies ◽  
Pea Seed

There have been many physicochemical studies of legumin, one of the major storage globulins isolated from pea seed. The more recent literature values for the molecular weight of this protein are in the range 390 000-420 000. These results are not consistent with the subunit molecular weight of legumin determined by dodecyl sulfate-polyacrylamide gel electrophoresis, if a hexameric model is assumed. We have measured the molecular weight of a highly purified sample of Pisum legumin by meniscus depletion sedimentation equilibrium and have found a value of 350 000 � 10 000. Since the oligomeric protein is homogeneous with respect to molecular weight, the heterogeneity reported for the subunit polypeptides, using various conditions of electrophoresis, presumably reflect differences in charge and amino acid composition. The molecular weight of legumin is significantly greater than the value of 325 000 found for cucurbitin, the equivalent crystalline protein isolated from pumpkin seed.

scholarly journals Platelet-derived growth factor. Isolation by a large-scale procedure and analysis of subunit composition

1981 ◽  
Vol 193 (3) ◽  
pp. 907-913 ◽  
Author(s):  
C H Heldin ◽  
B Westermark ◽  
A Wasteson
Keyword(s):  
Molecular Weight ◽  
Growth Factor ◽  
Gel Electrophoresis ◽  
Large Scale ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
High Specific Activity ◽  
Equilibrium Analysis ◽  
Platelet Derived Growth Factor ◽  
Sedimentation Equilibrium

Platelet-derived growth factor was purified from fresh platelets by a large-scale procedure not involving the use of SDS (sodium dodecyl sulphate). The product, 0.5 mg of platelet-derived growth factor, obtained from about 3 × 10(13) platelets migrated as a single component in analytical gel electrophoresis in the presence of SDS and showed no inhomogeneity on sedimentation-equilibrium analysis in the ultracentrifuge. It had a high specific activity, 2 ng of platelet-derived growth factor/ml (70pM) being equivalent to 1% (v/v) human serum in an assay for multiplication-stimulating activity. Amino acid analysis revealed that platelet-derived growth factor contains all the common amino acids, except tryptophan, but no hexosamine. The molecular weight of platelet-derived growth factor, as determined by sedimentation-equilibrium analysis, was about 33 000. A similar value was obtained by gel electrophoresis in SDS under non-reducing conditions. In the presence of reducing agents the factor molecule was converted into two distinct components of lower molecular weight (17 000 and 14 000 respectively), as demonstrated by protein staining. The molecular model implicated by these findings is that platelet-derived growth factor consists of two different polypeptides chains, linked by disulphide bridges.

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