scholarly journals Enzymic degradation of keratosulphates

1970 ◽  
Vol 119 (1) ◽  
pp. 39-47 ◽  
Author(s):  
Michiko Nishida-Fukuda ◽  
Fujio Egami
Keyword(s):  
Column Chromatography ◽  
Liver Extract ◽  
Concerted Action ◽  
Sephadex Column ◽  
Marine Gastropod ◽  
Multienzyme System ◽  
Shark Cartilage

1. A multienzyme system capable of degrading keratosulphates to yield galactose, N-acetylglucosamine and sulphate was found in the liver extract of a marine gastropod, Charonia lampas. 2. During the degradation, neither oligosaccharides nor sulphated sugars were produced. 3. It is suggested that the degradation could be attributed to the concerted action of β-galactosidase, β-N-acetylglucosaminidase and a sulphatase (sulphohydrolase), tentatively designated keratosulphatase. 4. Two forms of keratosulphatase (I and II) were separated by DEAE-Sephadex column chromatography. Both forms could release all the sulphate from keratosulphates and neither appeared to be identical with glycosulphatase or chondrosulphatase, both of which are also present in Charonia lampas. 5. β-Galactosidase and β-N-acetylglucosaminidase could degrade keratopolysulphate to a greater extent in the presence of keratosulphatase than in its absence. 6. It is suggested that keratosulphate was first desulphated by the action of keratosulphatase, and the desulphated polymer was then degraded to galactose and N-acetylglucosamine by the action of β-galactosidase and β-N-acetylglucosaminidase. 7. β-Galactosidase alone released a small amount of galactose from shark cartilage keratopolysulphate, but β-N-acetylglucosaminidase alone did not release N-acetylglucosamine. This indicates that unsulphated galactose residues occupy all the non-reducing terminal positions in keratopolysulphate chains.

scholarly journals Synthesis of adult-type hemoglobin in human erythremia cell line

Blood ◽  
1984 ◽  
Vol 64 (1) ◽  
pp. 314-317 ◽  
Author(s):  
M Kaku ◽  
K Yagawa ◽  
K Nakamura ◽  
H Okano
Keyword(s):  
Cell Line ◽  
Isoelectric Focusing ◽  
Column Chromatography ◽  
Cytosine Arabinoside ◽  
Adult Type ◽  
Sephadex Column ◽  
Cell Lysate ◽  
Globin Chains ◽  
Exposed Cell ◽  
Carboxy Methyl

Abstract KMOE -2/05 cells, derived from a patient with acute erythremia, became benzidine-positive after the addition of cytosine arabinoside (CA). Radioimmunoassays using antihuman hemoglobin antibodies revealed an elevated amount of hemoglobin in the CA-exposed cells over that in the control cells (without CA). Isoelectric focusing of the CA-exposed cell lysate formed benzidine-positive foci in the positions of human adult Hb (HbA) and human fetal Hb (HbF). To determine the types of globin synthesized in the CA-exposed cells, globin chains internally labeled with [3H] leucine were purified by carboxy-methyl (CM)-Sephadex column chromatography, immunoadsorption by Sepharose-coupled antihuman Hb antibodies and Sephadex G-100. The labeled globin chains were finally separated by CM-cellulose chromatography in urea. Two distinct peaks of radioactivity were shown in exactly the same fractions as carrier human globin alpha- and beta-chains. These observations indicate that these KMOE -2/05 cells synthesize HbA.

STUDIES ON PLASMINOGEN: V. PURIFICATION OF BOVINE AND HUMAN PLASMINOGENS BY SEPHADEX CHROMATOGRAPHY

1966 ◽  
Vol 44 (4) ◽  
pp. 475-485 ◽  
Author(s):  
John Y. S. Chan ◽  
Edwin T. Mertz
Keyword(s):  
Human Serum ◽  
Column Chromatography ◽  
Continuous Flow ◽  
Sephadex Column ◽  
Molecular Weights ◽  
Starch Gel ◽  
Electrophoresis Method ◽  
Human Plasminogen ◽  
Specific Activities

Purified euglobulins prepared from bovine and human serum were fractionated by Sephadex column chromatography at pH 3.5. The products obtained were purified further by phosphate precipitation (bovine) or lysine solubilization (human) to yield final products with activities comparable to those prepared by the continuous-flow electrophoresis method. Plasminogen from Cohn fraction III-4 was also purified. The method yields preparations from serum which contain essentially no altered plasminogen. The specific activities, the starch-gel patterns, the molecular weights, and the recoveries of the purified bovine and human plasminogen preparations are reported and discussed.

scholarly journals Separation of Cr(III)-Hexacyanoferrate(II)-Complexes by Sephadex Column Chromatography

1968 ◽  
Vol 41 (10) ◽  
pp. 2542-2542 ◽  
Author(s):  
Yoshio Matsumoto ◽  
Michiko Shirai ◽  
Hiroko Saito
Keyword(s):  
Column Chromatography ◽  
Sephadex Column

METHODOLOGICAL PROBLEMS IN COMPETITIVE PROTEIN-BINDING TECHNIQUES; THE USE OF SEPHADEX COLUMN CHROMATOGRAPHY TO SEPARATE STEROIDS

1970 ◽  
Vol 65 (1_Suppl) ◽  
pp. S37-S60 ◽  
Author(s):  
Beverley E. Pearson Murphy
Keyword(s):  
Sample Preparation ◽  
Protein Binding ◽  
Column Chromatography ◽  
Plasma Protein ◽  
Binding Proteins ◽  
Sephadex Column ◽  
Tissue Protein ◽  
Competitive Protein Binding ◽  
Methodological Problems

ABSTRACT The problems to be investigated in setting up a competitive protein-binding (CPB) assay have been considered: the choice of plasma protein, tissue protein or antibody as the assay protein; the separation of bound and unbound fractions; sample preparation with respect to the removal or destruction of interfering binding-proteins and the separation of competing analogues. Factors giving rise to blank values have been discussed as well as problems encountered in running CPB assays routinely.

EFFECT OF TRICHLORACETIC ACID ON BASOPHILS AND ON CORTICOTROPHIN CONTENT OF HUMAN PITUITARY GLANDS: EXTRACTION OF LOW MOLECULAR WEIGHT CORTICOTROPHIN

1963 ◽  
Vol 42 (1) ◽  
pp. 69-84 ◽  
Author(s):  
A. R. Currie ◽  
Beryl M. A. Davies
Keyword(s):  
Molecular Weight ◽  
Column Chromatography ◽  
Periodic Acid ◽  
Low Molecular Weight ◽  
Posterior Lobe ◽  
Human Pituitary ◽  
Trichloracetic Acid ◽  
Simple Method ◽  
Sephadex Column ◽  
Pituitary Glands

ABSTRACT Experiments are reported which show that 2.5% trichloracetic acid (TCA) causes marked loss of periodic acid-Schiff staining of the basophil granules of the pituitary gland and that there is extraction of some ACTH into the TCA. There is, however, no direct correlation between the basophil changes and the extraction of the hormone from the gland. The vesiculate chromophobe, the colloid and the cytoplasmic granules of the acidophils are not apparently affected by TCA treatment. Little is known about the chemical state of storage of ACTH in the human adenohypophysis. The amino acid structure and sequence of human ACTH has recently been proposed but there is as yet no chemical basis for a histochemical technique specific for ACTH. The 2.5% TCA-extractable ACTH, which represents about 30% of the total ACTH activity of batches of 'old' pituitary glands, has been characterized by ultrafiltration, Sephadex column chromatography and ultracentrifugation as a single component of minimum molecular weight about 3200. This is a simple method for extracting low molecular weight ACTH-extraction in 2.5% TCA (or water acidified to pH 5.0) for 1 hour at 4° C followed by ultrafiltration and Sephadex column chromatography. About 68% of the original activity in the TCA extract is recovered in the final product. Extraction was equally efficient whether 'pieces' or homogenates were used and in the presence or absence of posterior lobe tissue. Autolysis results in a decrease of both residual gland and TCA-extractable hormone.

Properties of 1-phosphofructokinase from Pseudomonas piutida

1977 ◽  
Vol 23 (6) ◽  
pp. 721-725 ◽  
Author(s):  
Sookie S. Bang ◽  
Paul Baumann ◽  
Mark H. Sawyer
Keyword(s):  
Pseudomonas Putida ◽  
Adenosine Triphosphate ◽  
Column Chromatography ◽  
Inhibitory Activity ◽  
Reaction Rate ◽  
Adenosine Monophosphate ◽  
The Other ◽  
Kinetic Properties ◽  
Sephadex Column ◽  
Sigmoidal Kinetics

The 1-phosphofructokinase (1-PFK, EC 2.7.1.56) from Pseudomonas putida was partially purified by a combination of (NH4)2SO4 fractionation and DEAE-Sephadex column chromatography. In its kinetic properties, this enzyme resembled the 1-PFK's from other bacteria. With the substrates fructose-1-phosphate (F-1-P) and adenosine triphosphate (ATP) Michaelis–Menten kinetics were observed, the Km for one substrate being unaffected by a variation in the concentration of the other substrate. At pH 8.0, the Km values for F-1-P and ATP were 1.64 × 10−4 M and 4.08 × 10−4 M, respectively. At fixed concentrations of F-1-P and ATP, an increase in the Mg2+ resulted in sigmoidal kinetics. Activity was inhibited by ATP when the ratio of ATP:Mg2+ was greater than 0.5 suggesting that ATP:2 Mg2+ was the substrate and free ATP was inhibitory. Activity of 1-PFK was stimulated by K+ and to a lesser extent by NH4+ and Na+. The reaction rate was unaffected by 2 mM K2HPO4, pyruvate, phosphoenolpyruvate, adenosine monophosphate, adenosine 3′,5′-cyclic monophosphate, fructose-6-phosphate, glucose-6-phosphate, 6-phosphogluconate, 2-keto-3-deoxy-6-phosphogluconate, or citrate. The results indicated that the 1-PFK from P. putida was not allosterically regulated by a number of metabolites which may play an important role in the catabolism of D-fructose.

THE INDUCTION OF NAD KINASE BY DDT IN TRIATOMA INFESTANS

1967 ◽  
Vol 45 (5) ◽  
pp. 619-626 ◽  
Author(s):  
M. Agosin ◽  
Jovita Ilivicky ◽  
S. Litvak
Keyword(s):  
Column Chromatography ◽  
De Novo ◽  
Deae Cellulose ◽  
Triatoma Infestans ◽  
Protective Mechanism ◽  
Nad Kinase ◽  
De Novo Synthesis ◽  
Enzyme Protein ◽  
Sephadex Column ◽  
Immunochemical Techniques

The NAD kinase (EC 2.7.1.23) from Triatoma infestans has been purified and a specific antiserum against it prepared. Immunochemical techniques have shown that the increase in the levels of NAD kinase in nymphs of T. infestans is accompanied by an increase in the amount of enzyme protein. The enzyme is labeled after injection of 14C-labeled leucine in both induced and non-induced insects, but labeling is greater in the former, which further supports the concept that a de novo synthesis of enzyme protein occurs during induction by DDT. The enzyme is heterogeneous by DEAE-cellulose and DEAE-Sephadex column chromatography, but the antiserum does not distinguish this heterogeneity. NAD kinase induction may correspond to a protective mechanism of the insects by increasing the availability of coenzymes required for DDT detoxication.

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