scholarly journals The isolation and partial characterization of the major glycoprotein (LGP-I) from the articular lubricating fraction from bovine synovial fluid

1977 ◽  
Vol 161 (3) ◽  
pp. 473-485 ◽  
Author(s):  
D A Swann ◽  
S Sotman ◽  
M Dixon ◽  
C Brooks
Keyword(s):  
Amino Acids ◽  
Synovial Fluid ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Galactose Oxidase ◽  
Sedimentation Equilibrium ◽  
Molar Ratio ◽  
Side Chains ◽  
Total Carbohydrate ◽  
Intact Molecule

The articular lubricating fraction from bovine synovial fluid was prepared by repeated fractionation in three consecutive CsCl density gradients to remove completely traces of hyaluronic acid. The major glycoprotein consituent (LGP-I) was then isolated by repeated gel-permeation chromatography. The yield of the LGP-I component was about 20 mg/litre of synovial fluid. Sedimentation-equilibrium measurements showed that this glycoprotein was homogeneous and the mol.wt. was calculated to be 227500. Amino acids represented 43% (w/w) and carbohydrate constituents 44% (w/w) of the molecule. Threonine, glutamic acid, proline and lysine (224, 127, 242 and 128 residues/1000 residues respectively) were the major amino acids. Galactosamine, galactose and N-acetylneuraminic acid (202, 162 and 114 residues/molecule of LGP-I component respectively) accounted for 98% of the total carbohydrate residues present. Small amounts of mannose and glucosamine (1 and 9mol respectively/mol of LGP-I component) were also present. After treatment of LGP-I component with alkali and NaB3H4 radioactivity was incorporated into alpha-aminobutyric acid and alanine in a molar ratio of 4:1, and radioactive galactosaminitol was isolated by ion-exchange chromatography from a cleaved oligosaccharide fraction. These data demonstrate the presence of threonine and serine -O-GalNAc linkages, but only 25% of the theoretical likages involving threonine were cleaved by a beta-elimination reaction. Digestion of LGP-I component with Pronase followed by chromatography on DEAE-cellulose yielded glycopeptide fractions with a similar amino acid and carbohydrate composition to the intact molecule. Treatment of desialylated and intact LGP-I component with galactose oxidase followed by reduction with NaB3H4 revealed the presence of 52mol of terminal galactose in the intact molecule and 153mol of galactose/mol of LGP-I component after treatment with neuraminidase. The data indicate the LGP-I component is composed of a single polypeptide chain containg more than 150 oligaosaccharide side chains composed of O-GaINAc-Gal distributed over the length of the peptide chain and that terminal sialic acid residues are linked to galactose in two-thirds of these side chains.

scholarly journals Physical, immunochemical, and functional properties of Acanthamoeba profilin.

1984 ◽  
Vol 98 (1) ◽  
pp. 214-221 ◽  
Author(s):  
P C Tseng ◽  
M S Runge ◽  
J A Cooper ◽  
R C Williams ◽  
T D Pollard
Keyword(s):  
Fluorescent Antibody ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Sedimentation Equilibrium ◽  
Binding Assays ◽  
Cytoplasmic Matrix ◽  
Unbound Fraction ◽  
Antibody Staining ◽  
High Concentrations ◽  
Equilibrium Ultracentrifugation

Acanthamoebe profilin has a native molecular weight of 11,700 as measured by sedimentation equilibrium ultracentrifugation and an extinction coefficient at 280 nm of 1.4 X 10(4) M-1cm-1. Rabbit antibodies against Acanthamoeba profilin react only with the 11,700 Mr polypeptide among all other ameba polypeptides separated by electrophoresis. These antibodies react with a 11,700 Mr polypeptide in Physarum but not with any proteins of Dictyostelium or Naeglaria. Antibody-binding assays indicate that approximately 2% of the ameba protein is profilin and that the concentration of profilin is approximately 100 mumol/liter cells. During ion exchange chromatography of soluble extracts of Acanthamoeba on DEAE-cellulose, the immunoreactive profilin splits into two fractions: an unbound fraction previously identified by Reichstein and Korn (1979, J. Biol. Chem., 254:6174-6179) and a tightly bound fraction. Purified profilin from the two fractions is identical by all criteria tested. The tightly bound fraction is likely to be attached indirectly to the DEAE, perhaps by association with actin. By fluorescent antibody staining, profilin is distributed uniformly throughout the cytoplasmic matrix of Acanthamoeba. In 50 mM KCl, high concentrations of Acanthamoeba profilin inhibit the elongation rate of muscle actin filaments measured directly by electron microscopy, but the effect is minimal in KCl with 2 MgCl2. By using the fluorescence change of pyrene-labeled Acanthamoeba actin to assay for polymerization, we confirmed our earlier observation (Tseng, P. C.-H., and T. D. Pollard, 1982, J. Cell Biol. 94:213-218) that Acanthamoeba profilin inhibits nucleation much more strongly than elongation under physiological conditions.

scholarly journals Isolation and properties of hyaluronic acid from bovine heart valves

1969 ◽  
Vol 113 (3) ◽  
pp. 559-563 ◽  
Author(s):  
F. A. Meyer ◽  
B. N. Preston ◽  
D. A. Lowther
Keyword(s):  
Hyaluronic Acid ◽  
Synovial Fluid ◽  
Heart Valves ◽  
Glucuronic Acid ◽  
Molecular Size ◽  
Sedimentation Equilibrium ◽  
Molar Ratio ◽  
Soluble Extract ◽  
Caesium Chloride ◽  
Bovine Heart

1. A soluble extract of bovine heart valves was obtained after the tissue had been pulverized at liquid-nitrogen temperatures in a mill. 2. Hyaluronic acid was isolated from the crude extract by sedimentation equilibrium in a caesium chloride density gradient (Franek & Dunstone, 1966). 3. Analysis of the product indicated that it contained 15% of protein and the molar ratio of glucuronic acid to glucosamine was 1·27. 4. Its physicochemical properties, as determined by lightscattering, viscosity and sedimentation studies, suggested that its molecular size and configuration were similar to those of hyaluronic acid isolated from ox synovial fluid (Preston, Davies & Ogston, 1965).

scholarly journals Studies on the structure and activity of rabbit C1q (a subcomponent of the first component of complement)

1974 ◽  
Vol 143 (2) ◽  
pp. 265-272 ◽  
Author(s):  
Diane M. Lowe ◽  
Kenneth B. M. Reid
Keyword(s):  
Amino Acids ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Haemolytic Activity ◽  
Subunit Structure ◽  
Carboxypeptidase A ◽  
Rapid Loss ◽  
Collagenase Digestion ◽  
Polypeptide Chains ◽  
Structure And Activity

1. The subunit structure of rabbit subcomponent C1q was examined in a previous publication (Reid et al., 1972). The present paper describes some aspects of the structure of the polypeptide chains derived from the molecule. 2. The three polypeptide chains, produced by performic oxidation, of rabbit subcomponent C1q were isolated by ion-exchange chromatography in 8m-urea on DEAE-cellulose. 3. Each chain was found to contain 15–18% glycine and significant amounts of the amino acids hydroxyproline and hydroxylysine. 4. By means of collagenase digestion it was shown that all three chains of rabbit subcomponent C1q contain collagen-like sequences of amino acids which constitute about 40% of each chain. 5. By use of carboxypeptidase A it was established, indirectly, that the collagen-like sequences, in one of the chains, are probably located near, or at, the N-terminal end of the chain. 6. Collagenase digestion and heating at 52°C (but not at 49°C) caused rapid loss of native rabbit subcomponent C1q haemolytic activity.

Veränderungen der Hämolymphe vor der Verpuppung von Cerura vinula L.: Der Gehalt an Eiweiß, Aminosäuren, Ommochrom-Vorstufen und Ommochromen

1966 ◽  
Vol 21 (12) ◽  
pp. 1184-1195 ◽  
Author(s):  
Detlef Bückmann ◽  
Axel Willig ◽  
Bernt Linzen
Keyword(s):  
Amino Acids ◽  
Ion Exchange ◽  
Free Amino ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Total Carbohydrate ◽  
Protein Nitrogen ◽  
Metabolic Alterations ◽  
Total Free Amino Acids ◽  
Free Aminoacids

Ommochrome synthesis at the beginning of lepidopteran metamorphosis is correlated to morpholytic and morphogenetic activity. In order to examine the metabolic alterations involved, data on respiration, haemolymph volume and on concentrations of total carbohydrate, soluble protein and non-protein nitrogen have been collected. Free aminoacids were separated by ion exchange chromatography and the levels of tryptophan, 3-hydroxy-kynurenine and of total ommochromes were determined independently at different stages during transformation of the larva towards the pupa. While the levels of non-protein nitrogen and of total free amino acids remain nearly constant, there is a three- and twentyfold increase of tryptophan and 3-hydroxy-kynurenine, respectively. It is proposed, that ommochrome formation serves in the removal of tryptophan liberated by proteolysis.

LYTIC ENZYMES OF SORANGIUM SP.: ACTION OF THE α- AND β-LYTIC PROTEASES ON TWO BACTERIAL MUCOPEPTIDES

1965 ◽  
Vol 43 (12) ◽  
pp. 1971-1983 ◽  
Author(s):  
C. S. Tsai ◽  
D. R. Whitaker ◽  
L. Jurášek ◽  
D. C. Gillespie
Keyword(s):  
Amino Acids ◽  
Glutamic Acid ◽  
Hydrolysis Product ◽  
Electrophoretic Pattern ◽  
Titratable Acidity ◽  
Exchange Chromatography ◽  
Molar Ratio ◽  
Gas Liquid Chromatography ◽  
Muramic Acid ◽  
Lytic Enzymes

The α- and β-lytic proteases hydrolyzed mucopeptides from the walls of Arthrohacter globiformis and Micrococcus lysodeikticus cells. The Arthrobacter mucopeptide was the more readily degraded substrate; the β-enzyme was the more active enzyme. Hydrolysates of Micrococcus mucopeptide showed the simpler electrophoretic pattern of ninhydrin-positive components.Analyses for C-terminal amino acids (after hydrazinolysis) and for dinitrophenyl amino acids (after dinitrophenylation) showed that the hydrolyses of Micrococcus mucopeptide were accompanied by increases in (a) C-terminal glycine and, to a lesser extent, in (b) C-terminal alanine, and by increases in (c) free ε-amino groups of lysine residues and in (d) N-terminal alanine residues. A brief hydrolysis with the β-enzyme gave slightly to moderately greater increases in a, b, and c, and a much greater increase in d, than a prolonged hydrolysis with the α-enzyme at a higher enzyme concentration.The soluble peptides hydrolyzed from Micrococcus mucopeptide by each enzyme were isolated by ion-exchange chromatography. Their composition, and that of the main hydrolysis product of the α-enzyme (an insoluble gel), was compared with that of untreated mucopeptide. The amino acids of the four products remained in approximately the same molar ratio relative to glutamic acid: Ala2.2, Gly1.0, Lys1.0, Glu1.0. N-Acetyl muramic acid and N-acetyl glucosamine were estimated by gas–liquid chromatography. Relative to glutamic acid, the molar ratio of each amino sugar was about 3.0 in the mucopeptide, 5.0 in the gel, and 0.6 in the soluble peptides. Half the glycyl residues of the mucopeptide and virtually all the glycyl residues of the other three products were analyzed as C-terminal glycine. Subtractive Edman analyses showed little change in the Ala:Glu ratio of the mucopeptide and of the gel but a substantial decrease in the ratios for the soluble peptides. The titratable acidity of the mucopeptide and the gel was consistent with these analyses.It is concluded that cross-linkages from the C-terminus of the peptide chains of the mucopeptide are hydrolyzed by both enzymes and that the linkage between N-acetyl muramic acid and the alanyl residue at the N-terminus of the peptide chains is hydrolyzed rapidly by the β-enzyme and slowly by the α-enzyme.

Purification and characterization of tonin

1976 ◽  
Vol 54 (9) ◽  
pp. 788-795 ◽  
Author(s):  
S. Demassieux ◽  
R. Boucher ◽  
C. Grisé ◽  
J. Genest
Keyword(s):  
Analytical Ultracentrifugation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Potassium Phosphate ◽  
Kinetic Studies ◽  
Exchange Chromatography ◽  
Sedimentation Equilibrium ◽  
Angiotensin I ◽  
Ph Optimum ◽  
Ammonium Sulphate Precipitation

Tonin was purified from rat submaxillary glands by differential centrifugation, ammonium sulphate precipitation, gel filtration on Sephadex G150, and by ion-exchange chromatography on DEAE-cellulose, phospho-cellulose, SP-Sephadex C25, and SP-Sephadex C50. Purified tonin was shown to be homogeneous by analytical electrophoresis and by analytical ultracentrifugation analysis. Purified tonin was very stable when stored in buffers of low pH values or when incubated at high temperatures in neutral solutions. The molecular weight estimated by sedimentation equilibrium was 28 700. The pH optimum was near 6.8 in a 0.1 M potassium phosphate buffer. The Michaelis–Menten constant for tonin using angiotensin I as substrate was about 4 × 10−5 M. Tonin activity was strongly inhibited by plasma. Kinetic studies using angiotensin I as substrate showed that the inhibition of tonin by plasma was of the non-competitive type.

scholarly journals The isolation and properties of a second glycoprotein (LGP-II) from the articular lubricating fraction from bovine synovial fluid

1979 ◽  
Vol 179 (3) ◽  
pp. 465-471 ◽  
Author(s):  
D A Swann ◽  
G Mintz
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Synovial Fluid ◽  
Bovine Serum ◽  
Gel Permeation Chromatography ◽  
Sedimentation Equilibrium ◽  
Major Constituent ◽  
Precipitin Line ◽  
Acetylneuraminic Acid ◽  
Gel Permeation

A high-molecular-weight glycoprotein (LGP-I) was shown [Swann, Sotman, Dixon & Brooks (1977) Biochem. J. 161, 473–485] to be the major constituent in the articular lubricating fraction from bovine synovial fluid. In addition to the LGP-I component, a second glycoprotein (LGP-II) was also present. After fractionation of bovine synovial fluid by sequential sedimentation in CsCl density gradients, the LGP-I and LGP-II components were separated by gel-permeation chromatography. The LGP-II component was then purified by chromatography on DEAE Bio-Gel A and Bio-Gel P-150. The molecular weight of the LGP-II component was 48,800 calculated from sedimentation-equilibrium measurements. Amino acids represented 53% (w/w) and carbohydrate constituents 36% (w/w) of the molecule. Glutamic acid and lysine (144 and 100 residues/1000 residues) were the major amino acids. Glucosamine, mannose, galactose and N-acetylneuraminic acid [representing 8.0, 6.6, 9.5 and 11.9% (w/w) respectively] were the only carbohydrate constituents detected. Immunodiffusion analysis showed that LGP-II component did not form a detectable precipitin line with antiserum to bovine serum. It appears likely, therefore, that this glycoprotein is synthesized by the joint tissues and is not derived from serum.

Separation of Heparin into Subtractions by DEAE-Cellulose Chromatography

1979 ◽  
Vol 42 (05) ◽  
pp. 1452-1459 ◽  
Author(s):  
Robert H Yue ◽  
Toby Starr ◽  
Menard M Gertler
Keyword(s):  
High Activity ◽  
Uronic Acid ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Net Charge ◽  
Uronic Acid Content ◽  
Successful Separation ◽  
Salt Gradient ◽  
Structure And Activity ◽  
Low Activity

SummaryCommercial porcine heparin can be separated into three distinct subtractions by using DEAE-cellulose chromatography and a stepped salt gradient. Gram quantities of heparin can be fractionated by this technique. All three heparin subtractions can accelerate the inhibition of thrombin by antithrombin III with different efficiency. The specific activities of the high activity heparin, intermediate activity heparin and low activity heparin are 228 units/mg, 142 units/mg and 95 units/mg, respectively. Both the uronic acid content and the quantity of N-SO4 for all three heparin subfractions have been evaluated. The high activity heparin has the lowest uronic acid and N-SO4 content. The successful separation of commercial heparin into three distinct subfractions by means of ion-exchange chromatography suggests that the net charge on these three heparin components will serve as a model system in the elucidation of the structure and activity relationship to the biological function of heparin.

Post-proline endopeptidase, further characterization of the enzyme from pig kidneys

1984 ◽  
Vol 49 (8) ◽  
pp. 1846-1853 ◽  
Author(s):  
Karel Hauzer ◽  
Tomislav Barth ◽  
Linda Servítová ◽  
Karel Jošt
Keyword(s):  
Amino Acids ◽  
Aspartic Acid ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Relative Molecular Mass ◽  
Enzyme Molecule ◽  
Thiol Compounds ◽  
Modified Method ◽  
N Terminus

A post-proline endopeptidase (EC 3.4.21.26) was isolated from pig kidneys using a modified method described earlier. The enzyme was further purified by ion exchange chromatography on DEAE-Sephacel. The final product contained about 95% of post-proline endopeptidase. The enzyme molecule consisted of one peptide chain with a relative molecular mass of 65 600 to 70 000, containing a large proportion of acidic and alifatic amino acids (glutamic acid, aspartic acid and leucine) and the N-terminus was formed by aspartic acid or asparagine. In order to prevent losses of enzyme activity, thiol compounds has to be added.

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