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 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.

The Different Forms of Antithrombin III in Serum

1977 ◽  
Vol 38 (02) ◽  
pp. 0494-0503 ◽  
Author(s):  
D. S Pepper ◽  
D Banhegyi ◽  
J. D Cash
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Human Serum ◽  
Degradation Product ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Free Form ◽  
Polymeric Form ◽  
At Iii ◽  
Human Thrombin

SummaryAntithrombin III (AT III) complexes were isolated from human serum by affinity chromatography and gel filtration. In the first step of the preparation, using heparin-agarose chromatography, we observed that the complexed form of AT III bound less strongly to the gel than the free form and that about half of the AT III was free. With further purification a 2.5 × 105 molecular weight complex was isolated. Using 125I labelled human thrombin, this complex was radioactive indicating the presence of thrombin. Only in a synthetic thrombin-AT III system was a 9 × 104 molecular weight complex detected, but not in serum. These facts suggest that in serum AT III complexes may exist in a polymeric form. Also, an AT III antigen derived from the original AT III molecule, but not complexed, was isolated which may be a degradation product.Abbreviations used: AT-III, antithrombin III. Hepes, N-2-Hydroxyethylpiperazine-N-2-Ethanesulphonic acid.

Purification and studies of some physicochemical properties of glutamine synthetase of Neurospora crassa

1978 ◽  
Vol 56 (10) ◽  
pp. 927-933 ◽  
Author(s):  
W. S. Lin ◽  
M. Kapoor
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Glutamine Synthetase ◽  
Neurospora Crassa ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Affinity Column ◽  
Subunit Structure ◽  
Α Helix

Glutamine synthetase (EC 6.3.1.2) of Neurospora crassa was purified to near homogeneity by chromatography on a glutamate–Sepharose affinity column. Its properties, including molecular weight, subunit structure, amino acid composition, and approximate α-helix content, have been examined. In the native state, this enzyme has been demonstrated by gel filtration to be an octamer of molecular weight 360 000 and as having a sedimentation coefficient of 13.2 S by sedimentation velocity measurements. Circular dichroism spectra in the far ultraviolet range suggest an approximate α-helix content of 23–24%. The subunit generated by treatment with urea was found to be 45 000 daltons by gel filtration methods and a molecular weight of 46 000 was calculated for the monomer obtained by sodium dodecyl sulphate (SDS) treatment and electrophoresis in SDS-polyacrylamide gels. Interprotomeric cross-linking experiments, using diimidoesters, suggest the presence of two noncovalently linked tetramers comprising the native octameric structure. Amino acid analyses revealed the presence of six tryptophans, four half cystines, and nine methionine residues per monomer of 45 000 daltons.

Cytoplasmic Carboxylesterases of Human and Domestic Animal Liver: Aggregation, Dissociation and Molecular Weight Estimation

1972 ◽  
Vol 50 (1) ◽  
pp. 9-15 ◽  
Author(s):  
D. J. Ecobichon
Keyword(s):  
Molecular Weight ◽  
Human Liver ◽  
Gel Filtration ◽  
Domestic Animal ◽  
Starch Gel Electrophoresis ◽  
Weight Estimation ◽  
Animal Liver ◽  
Starch Gel ◽  
Species Specific ◽  
Two Peaks

The cytoplasmic carboxylesterases of bovine, ovine, equine and human liver were fractionated by starch gel electrophoresis and by gel filtration on Sephadex. While species-specific, heterogeneous bands were observed in starch gel, the esterases of the bovine, ovine and equine liver were eluted from Sephadex G-100 as single peaks of activity, each with a characteristic elution volume. Gel filtration of human liver extracts yielded two peaks of activity, one containing electrophoretically slow esterases, the other electrophoretically fast esterases. Extracted equine and human hepatic carboxylesterases aggregated readily on storage or concentration, forming larger units which could be dissociated by a combination of acidic pH and high salt concentration. Molecular weight estimates of the hepatic esterases by gel filtration on Sephadex G-100 and G-200 yielded values of 65 000 for ovine, 55 000 for bovine, 96 000 and 70 000 for equine variants and 180 000 and 65 000 for human variants. The observations suggested that the cytoplasmic enzymes in relatively crude hepatic extracts had a lower molecular weight than those in concentrated or partially purified preparations which formed stable dimers or trimers.

scholarly journals Rabbit Tamm–Horsfall urinary glycoprotein. Chemical composition and subunit structure

1971 ◽  
Vol 122 (5) ◽  
pp. 623-631 ◽  
Author(s):  
Anne M. S. Marr ◽  
A. Neuberger ◽  
Wendy A. Ratcliffe
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Chemical Composition ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Subunit Structure ◽  
Terminal Amino

1. Tamm–Horsfall glycoprotein from rabbit urine has been isolated and characterized. The homogeneity of the preparation has been established by a variety of procedures including disc gel electrophoresis and ultracentrifugation in aqueous solution, sodium dodecyl sulphate and formic acid. 2. The chemical composition has been determined and a carbohydrate content of approx. 31% was obtained. The relative contents of the amino acids were shown to be very similar to those in human Tamm–Horsfall glycoprotein. A trace of lipid was also detected. 3. Leucine was identified as the only N-terminal amino acid. 4. The subunit structure was investigated in the presence of sodium dodecyl sulphate by gel filtration and disc gel electrophoresis. These studies indicated that the subunit possessed a molecular weight of approx. 84000±6000. A similar value was obtained after reduction and S-alkylation of the glycoprotein indicating that the disulphide bonds were all intrachain. 5. A minimum value for the chemical molecular weight of 85000±6000 was obtained from the number of N-terminal amino acids released by cyanogen bromide cleavage of the glycoprotein. 6. The immunological properties of the glycoprotein were studied. Cross reactivity was demonstrated between human Tamm–Horsfall glycoprotein and a guinea-pig anti-rabbit Tamm–Horsfall antiserum.

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.

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