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).

scholarly journals Purification and subunit structure of argininosuccinate lyase from Chlamydomonas reinhardi

1977 ◽  
Vol 167 (1) ◽  
pp. 71-75 ◽  
Author(s):  
R F Matagne ◽  
J P Schlösser
Keyword(s):  
Enzyme Activity ◽  
Crude Extract ◽  
Enzyme Preparation ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Activity Analysis ◽  
Disc Electrophoresis ◽  
Subunit Structure ◽  
Argininosuccinate Lyase

Argininosuccinate lyase (EC 4.3.2.1) was purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The final enzyme preparation was purified 46-fold compared with the crude extract. Electrophoresis of this preparation revealed three bands, the major one having the enzyme activity. Analysis of the enzyme by gel filtration and by disc electrophoresis (in two different concentrations of acrylamide) gave mol.wts. of 200000 (+/- 15000) and 190000 (+/- 20000) respectively. Treatment with sodium dodecyl sulphate and mercaptoethanol dissociated the enzyme into subunits of mol.wt. 39000 (+/-2000). The results are indicative of the multimeric structure of the enzyme, which is composed of five (perhaps four or six) identical subunits.

scholarly journals Effects of pronase and neuraminidase treatment on a myelin-associated glycoprotein in developing brain

1976 ◽  
Vol 156 (1) ◽  
pp. 143-150 ◽  
Author(s):  
R H Quarles
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Weight Class ◽  
Developmental Difference ◽  
Adult Rats ◽  
Neuraminidase Treatment

Rats (14 days old) were injected with [14c]fucose and young adult rats with [3H]fucose in order to label the myelin-associated glycoproteins. As previously reported, the major [14C]fucose-labelled glycoprotein in the immature myelin had a higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide gels that the [3H]fucose-labelled glycoprotein in mature myelin. This predominant doubly labelled glycoprotein component was partially purified by preparative gel electrophoresis and converted to glycopeptides by extensive Pronase digestion. Gel filtration on Sephadex G-50 separated the glycopeptides into several clases, which were designted A,B, C AND D, from high to low molecular weight. The 14C-labelled glycopeptides from immature myeline were enriched in the highest-molecular-weight class A relative to the 3H-labelled glycopeptides from mature myelin. Neuraminidase treatment of the glycoprotein before Pronase digestion greatly decreased the proportion of glycopeptides fractionating in the higher-molecular-weight classes and largely eliminated the developmental differences that were apparent by gel filtration. However, neuraminidase treatment did not decrease the magnitude of the developmental difference revealed by electrophoresing the intact glycoprotein on sodium dodecyl sulphate gels, although it did decrease the apparent molecular weight of the glycoprotein from both the 15-day-old and adult rats by an amount comparable in magnitude to that developmental difference. The results from gel filtration of glycopeptides indicate that there is a higher content of large molecular weight, sialic acid-rich oligosaccharide units in the glycoprotein of immature myelin. However, the higher apparent molecular weight for the glycoprotein from 15-day-old rats on sodium dodcyl sulphate gels is not due primarily to its higher sialic acid content.

scholarly journals Inhibition of the thrombin–fibrinogen reaction by a macromolecular factor from rat liver

1972 ◽  
Vol 129 (1) ◽  
pp. 83-89 ◽  
Author(s):  
Ragnar Flengsrud ◽  
Bjarne Østerud ◽  
Hans Prydz
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Competitive Inhibition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Homogenate ◽  
Kinetic Studies ◽  
Disc Electrophoresis ◽  
Nitrobenzoic Acid

1. The supernatant obtained by centrifugation of a rat liver homogenate at 100000g for 1h contained a heat-labile macromolecular inhibitor of the thrombin–fibrinogen reaction. 2. The inhibitor was purified to electrophoretic homogeneity by repeated preparative polyacrylamide disc electrophoresis. Inhibition was observed with purified inhibitor equivalent to about 1μg of protein/ml. 3. The inhibitor had a pI of 3.50–3.75, a molecular weight (from sodium dodecyl sulphate–polyacrylamide-gel electrophoresis) of 72000±3000 and was inactivated by p-hydroxymercuribenzoate or 5,5′-dithiobis-(2-nitrobenzoic acid). 4. Kinetic studies revealed a non-competitive inhibition, with the inhibitor probably acting on the thrombin–fibrinogen complex.

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.

scholarly journals Purification and structural characterization of a cartilage matrix protein

1981 ◽  
Vol 197 (2) ◽  
pp. 367-375 ◽  
Author(s):  
M Paulsson ◽  
D Heinegård
Keyword(s):  
Molecular Weight ◽  
Matrix Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cartilage Matrix ◽  
Sedimentation Equilibrium ◽  
Intact Protein ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Cscl Density Gradient

The cartilage matrix protein is a major non-collagenous protein in bovine cartilage. It was purified from a 5 M-guanidinium chloride extract of bovine tracheal cartilage by sequential CsCl-density-gradient centrifugation, gel chromatography in guanidinium chloride and differential precipitation. The molecular weight of the intact protein is 148 000, determined by sedimentation-equilibrium centrifugation. It was dissociated to three subunits of molecular weight 52 000 by reduction of disulphide bonds. The cartilage matrix protein was insoluble in low-salt solutions and behaved abnormally on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The content of cysteine was high, whereas the contents of aromatic amino acids were low. The carbohydrate content was 3.9% (w/w). Glycopeptides obtained after papain digestion were heterogenous on gel chromatography. Asparagine/aspartic acid was enriched in the purified glycopeptides, indicating the presence of N-glycosidic linkages to protein.

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 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 A novel low-molecular weight chondroitin sulphate proteoglycan isolated from cartilage

1981 ◽  
Vol 197 (2) ◽  
pp. 355-366 ◽  
Author(s):  
D Heinegård ◽  
M Paulsson ◽  
S Inerot ◽  
C Carlström
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Chondroitin Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Partial Specific Volume ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Cscl Density Gradient

Proteoglycans were isolated from cartilage by extraction with 4M-guanidinium chloride followed by direct centrifugation in 4M-guanidinium chloride/CsCl at a low starting density, 1.34 g/ml. N-Ethylmaleimide was included in the extraction solvent as a precaution against contamination of proteoglycans with unrelated proteins mediated by disulphide exchange. A novel, discrete, low-buoyant-density proteoglycan (1.40-1.35 g/ml) was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Its proteoglycan nature was revealed by the shift in the molecular size observed on gel electrophoresis after treatment with chondroitinase ABC. The core protein was monodisperse. The proteoglycan was further purified by gel chromatography with and without addition of hyaluronate. The proteoglycan constitutes less than 2% (by weight) of the total extracted proteoglycans and is not capable of interacting with hyaluronate. The same proteoglycan was purified in larger quantities by sequential associative and dissociative CsCl-density-gradient centrifugation, zonal rate sedimentation in a sucrose gradient and gel chromatography on Sepharose CL-4B. The pure proteoglycan had a molecular weight of 76 300 determined by sedimentation-equilibrium centrifugation and an apparent partial specific volume of 0.59 ml/g. It contained about 25% protein (of dry weight) and had remarkably high contents of leucine and cysteine as compared with other proteoglycans. The proteoglycan contained two to three large chondroitin sulphate chains and some oligosaccharides.

scholarly journals Detection in milk of a 60,000 Mr mouse and a 68,000 Mr human phosphorylated glycoprotein by histochemical analysis on polyacrylamide gels.

1983 ◽  
Vol 31 (6) ◽  
pp. 709-716 ◽  
Author(s):  
M R Green
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Periodic Acid ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Blue Staining ◽  
Polyacrylamide Gels ◽  
Periodic Acid Schiff ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

Proteins in colostrum and skimmed milk from humans and mice were separated by electrophoresis on polyacrylamide gels and stained with Coomassie blue (CB), Ethyl-Stains-all (ESA), and periodic acid-Schiff (PAS) to investigate changes that may occur in milks throughout lactation. In mouse colostrum but not in mature mouse milk, a PAS-positive protein of apparent molecular weight of 60,000 stained prominently blue with ESA. A protein in human milk with a molecular weight of 68,000 stained similarly but was present throughout lactation. The intensity of blue staining of these minor proteins in milk approached that obtained with casein phosphoproteins. The metachromatic dye ESA stains phosphoproteins and sialic acid-rich glycoproteins blue to blue-green. Removal of phosphorus from the former and sialic acid from the latter results in those proteins staining red with ESA. The intensity of blue staining of the 60,000 and 68,000 Mr proteins was diminished but not lost following treatment with phosphatase. It was eliminated following neuraminidase digestion of the mouse protein and mild acid hydrolysis of the human protein. Coomassie blue staining of the proteins was not affected by these procedures. Following electrophoresis of milk and milk fractions in a non-sodium dodecyl sulfate-containing system, the proteins were identified by their characteristic staining properties with ESA and isolated.

scholarly journals Purification and subunit structure of legumin of Vicia faba L. (broad bean)

1974 ◽  
Vol 141 (2) ◽  
pp. 413-418 ◽  
Author(s):  
David J. Wright ◽  
Donald Boulter
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Broad Bean ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Subunit Structure ◽  
Isoelectric Precipitation ◽  
Molecular Weights

Zonal isoelectric precipitation was shown to be an effective method for the preparation of legumin which was homogeneous as judged by ultracentrifugation and polyacrylamide-gel electrophoresis. The subunit structure of legumin was investigated by preparative sodium dodecyl sulphate–polyacrylamide-gel electrophoresis and ion-exchange chromatography in urea. Five distinct subunits, of which two were acidic (α) and had a molecular weight of 37000, and three were basic (β) with molecular weights of 20100, 20900 and 23800, were identified. The α and β subunits were present in equimolar amounts in the legumin molecule and, in view of this and molecular-weight considerations, an α6β6 subunit model was proposed for legumin.

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