ISOLATION AND CHARACTERIZATION OF THE MAJOR ANTIGEN OF RAT KIDNEY

1967 ◽  
Vol 45 (6) ◽  
pp. 781-789 ◽  
Author(s):  
Bao-Linh Dinh
Keyword(s):  
Rat Kidney ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Sedimentation Coefficient ◽  
Molecular Weights ◽  
Isolation And Characterization ◽  
Antigenic Properties ◽  
Major Antigen ◽  
Purified Antigen

The major antigen of rat kidney (KMA) was isolated by chromatography on DEAE-cellulose and Sephadex G-25 gel filtration. The sedimentation coefficient of the purified antigen was about 1.2 S and its electrophoretic mobility corresponded to that of α2-globulins. Its diffusion coefficient was estimated by the method of Allison and Humphrey as 11.4 × 10−7 cm2 sec−1. On gel filtration through Sephadex G-100, the purified preparation was resolved into two components with identical antigenic properties but of different molecular weights, estimated as 16,600 and 4,100. The concentrations of KMA were determined in different tissues, urine, and serum by immunodiffusion. The probable relationship between physical and immunochemical properties and the possible physio-pathological role of KMA are discussed.

scholarly journals α-Crystallin. The isolation and characterization of distinct macromolecular fractions

1971 ◽  
Vol 124 (2) ◽  
pp. 337-343 ◽  
Author(s):  
Abraham Spector ◽  
Lu-Ku Li ◽  
Robert C. Augusteyn ◽  
Arthur Schneider ◽  
Thomas Freund
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Chemical Difference ◽  
Molecular Weights ◽  
Isolation And Characterization ◽  
Different Populations ◽  
Molecular Weight Fractions

α-Crystallin was isolated from calf lens periphery by chromatography on DEAE-cellulose and gel filtration. Three distinct populations of macromolecules have been isolated with molecular weights in the ranges approx. 6×105−9×105, 0.9×106−4×106and greater than 10×106. The concentration of macromolecules at the molecular-weight limits of a population are very low. The members of the different populations do not appear to be in equilibrium with each other. Further, in those molecular-weight fractions investigated, no equilibrium between members of the same population was observed. The population of lowest molecular weight comprises 65–75% of the total material. The amino acid and subunit composition of the different-sized fractions appear very similar, if not identical. The only chemical difference observed between the fractions is the presence of significant amounts of sugar in the higher-molecular-weight fractions. Subunit molecular weights of approx. 19.5×103and 22.5×103were observed for all α-crystallin fractions.

Isolation of specific protease inhibitors from Neurospora crassa

1976 ◽  
Vol 54 (8) ◽  
pp. 699-703 ◽  
Author(s):  
Peter H. Yu ◽  
Maria R. Kula ◽  
Hsin Tsai
Keyword(s):  
Neurospora Crassa ◽  
Protease Inhibitors ◽  
Gel Filtration ◽  
Physiological Role ◽  
Exchange Chromatography ◽  
Proteinase K ◽  
Tryptophan Synthase ◽  
Molecular Weights ◽  
Specific Protease

Four natural protease inhibitors have been partially purified by heat treatment, ion-exchange chromatography and gel filtration from Neurospora crassa. The inhibitory activity has been estimated by measuring the inhibition of proteolysis of casein as well as by the protection of Neurospora tryptophan synthase from proteolytic inactivation. The inhibitors are all oligopeptides and possess molecular weights in the range 5000 – 24 000 and appear to be very specific to Neurospora proteases. They may be classified into two types. The first are specific to Neurospora alkaline protease and the second to acidic protease. None of them exhibited any effect on other proteases including trypsin, chymotrypsin, papain, pepsin, thermolysin, subtilisin and proteinase K. The possible physiological role of these inhibitors is discussed.

scholarly journals Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues

1978 ◽  
Vol 175 (3) ◽  
pp. 1051-1067 ◽  
Author(s):  
K K Mäkinen ◽  
P L Mäkinen
Keyword(s):  
Substrate Inhibition ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Gel Permeation Chromatography ◽  
Ph Optimum ◽  
Preparative Scale ◽  
Molecular Weights ◽  
Gel Permeation ◽  
Lysine Residues ◽  
Enzyme I

Two arylamidases (I and II) were purified from human erythrocytes by a procedure that comprised removal of haemoglobin from disrupted cells with CM-Sephadex D-50, followed by treatment of the haemoglobin-free preparation subsequently with DEAE-cellulose, gel-permeation chromatography on Sephadex G-200, gradient solubilization on Celite, isoelectric focusing in a pH gradient from 4 to 6, gel-permeation chromatography on Sephadex G-100 (superfine), and finally affinity chromatography on Sepharose 4B covalently coupled to L-arginine. In preparative-scale purifications, enzymes I and II were separated at the second gel-permeation chromatography. Enzyme II was obtained as a homogeneous protein, as shown by several criteria. Enzyme I hydrolysed, with decreasing rates, the L-amino acid 2-naphtylamides of lysine, arginine, alanine, methionine, phenylalanine and leucine, and the reactions were slightly inhibited by 0.2 M-NaCl. Enzyme II hydrolysed most rapidly the corresponding derivatives of arginine, leucine, valine, methionine, proline and alanine, in that order, and the hydrolyses were strongly dependent on Cl-. The hydrolysis of these substrates proceeded rapidly at physiological Cl- concentration (0.15 M). The molecular weights (by gel filtration) of enzymes I and II were 85 000 and 52 500 respectively. The pH optimum was approx. 7.2 for both enzymes. The isoelectric point of enzyme II was approx. 4.8. Enzyme I was activated by Co2+, which did not affect enzyme II to any noticeable extent. The kinetics of reactions catalysed by enzyme I were characterized by strong substrate inhibition, but enzyme II was not inhibited by high substrate concentrations. The Cl- activated enzyme II also showed endopeptidase activity in hydrolysing bradykinin.

scholarly journals Isolation of palmitoyl-CoA hydrolases from human blood platelets

1981 ◽  
Vol 199 (3) ◽  
pp. 639-647 ◽  
Author(s):  
R K Berge ◽  
L E Hagen ◽  
M Farstad
Keyword(s):  
Human Blood ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Cytosol Fraction ◽  
Molecular Weights ◽  
Apparent Homogeneity ◽  
Coa Hydrolase ◽  
Human Blood Platelets

The palmitoyl-CoA hydrolase activity, which in human blood platelets is mainly localized in the cytosol fraction [Berge, Vollset & Farstad (1980) Scand. J. Clin. Lab. Invest. 40, 271--279], was found to be extremely labile. Inclusion of glycerol or palmitoyl-CoA stabilized the activity during preparation. Gel-filtration studies revealed multiple forms of the enzyme with molecular weights corresponding to about 70 000, 40 000 and 24 000. The relative recovery of the mol.wt.-70 000 form was increased by the presence of 20% (v/v) glycerol or 10 microM-palmitoyl-CoA. The three enzyme forms are probably unrelated, since they were not interconvertible. The three different species of palmitoyl-CoA hydrolase were purified by DEAE-cellulose and hydroxyapatite chromatography, isoelectric focusing and high-pressure liquid chromatography (h.p.l.c.) to apparent homogeneity. The three enzymes had isoelectric points (pI) of 7.0, 6.1 and 4.9. The corresponding molecular weights were 27 000--33 000, 66 000--72 000 and 45 000--49 000, calculated from h.p.l.c. and Ultrogel AcA-44 chromatography. The apparently purified enzymes were unstable, as most of the activity was lost during purification. The enzyme with an apparent molecular weight of 45 000--49 000 was split into fractions with molecular weights of less than 10 000 by re-chromatography on h.p.l.c. concomitantly with a loss of activity. The stimulation of the activity by the presence of serum albumin seems to depend on the availability of palmitoyl-CoA, as has been reported for other palmitoyl-CoA hydrolases. [Berge & Farstad (1979) Eur. J. Biochem. 96, 393--401].

Studies on the purification of Entamoeba histolytica antigens by gel filtration. II. The antigenic properties of the isolated fractions

1970 ◽  
Vol 16 (6) ◽  
pp. 493-498 ◽  
Author(s):  
Z. Ali Khan ◽  
E. Meerovitch
Keyword(s):  
Entamoeba Histolytica ◽  
Gel Filtration ◽  
Water Soluble ◽  
The Other ◽  
Soluble Extract ◽  
Serological Tests ◽  
Molecular Weights ◽  
Antigenic Properties ◽  
Water Soluble Extract

Partially purified fractions of the water-soluble extract of Entamoeba histolytica (DKB strain) obtained by chromatography in Sephadex G-200 (F1, F2, and F4) and G-100 (F3a, F3b, F3c, and F3d) gels were used in various serological tests and their reactivities compared with the whole 1:40 antigen. The main haemagglutinating (HA) and complement-fixing (CF) activities were confined to fractions F1 and F2, which had molecular weights of 650 000 and 229 000, respectively. The 4 to 6 μg/ml of protein contained in these fractions at the optimum dilution gave antibody liters comparable to those for the whole antigen, which had about 77 μg/ml of protein. The other antigen fractions (F4, F3a, F3b, F3c, and F3d) showed very little activity. Fraction F1 had two main precipitin bands (1 and 2), which showed reaction of identity with two of the bands in fraction F2. The other fractions which showed some HA and CF reactivity had trace amounts of these antigens. It is presumed that the antigens specific for precipitin bands 1 and 2 are the main CF and HA antigens.

Purification and Kinetic Studies on a Circulating Anticoagulant in a Suspected Case of Lupus Erythematosus*

1965 ◽  
Vol 14 (01/02) ◽  
pp. 088-115 ◽  
Author(s):  
E. T Yin ◽  
L. W Gaston
Keyword(s):  
Lupus Erythematosus ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Sedimentation Coefficient ◽  
Kinetic Studies ◽  
Suspected Case ◽  
Active Inhibitor ◽  
Heat Stable ◽  
Species Specific ◽  
Active Lupus

Summary1. A circulating anticoagulant in a suspected case of lupus erythematosus has been highly purified by a combination of Sephadex gel filtration and DEAE cellulose chromatography.2. The inhibitor is a gamma globulin with a sedimentation coefficient of 6.6 Svedberg units.3. For its anticoagulant action, the lupus inhibitor requires a co-factor which is present both in the lupus and normal blood.4. The cofactor is located in the gamma globulins fraction which is relatively heat stable, but less than the inhibitor, at 56° C.5. The active lupus inhibitor (inhibitor + cofactor) is not species specific against human prothrombin.6. Working with highly purified systems, the active inhibitor is found to inhibit prothrombin conversion by formed prothrombin activator. It does not appear to inhibit the formation of prothrombin activator nor does it affect purified prothrombin.

ISOLATION OF LH, FSH AND TSH FROM HUMAN PITUITARIES AFTER THE REMOVAL OF HGH

1974 ◽  
Vol 77 (3) ◽  
pp. 485-497 ◽  
Author(s):  
P. A. Torjesen ◽  
T. Sand ◽  
N. Norman ◽  
O. Trygstad ◽  
I. Foss
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Disc Electrophoresis ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Molecular Weights ◽  
Pituitary Glands

ABSTRACT Highly purified human LH, FSH and TSH were isolated from batches of 300 frozen pituitary glands (200 g) by pH, acetone and ethanol fractionation, Sephadex gel filtration, ion-exchange chromatography on DEAE-cellulose and CM-Sephadex, and preparative polyacrylamide-gel electrophoresis. Sodium dodecyl-sulphate (SDS) polyacrylamide gel electrophoresis was used in order to check the purity, the identity and the molecular weight of the purified LH, FSH and TSH. This procedure showed that the hormone preparations consisted of two subunits with molecular weights of: LH: 21 300 and 17 900, FSH: 22 100 and 18 300 and TSH: 20 800 and 16 400. The purity of the hormone preparations was also evaluated by analytical disc electrophoresis at pH 8.9. The purified hormone preparations had radioimmunological activity as follows: LH: 20 000 IU/mg, FSH: 16 500 IU/mg and TSH: 5 IU/mg. All preparations had high biological potency.

scholarly journals Isolation and characterization of two sialoproteins present only in bone calcified matrix

1985 ◽  
Vol 232 (3) ◽  
pp. 715-724 ◽  
Author(s):  
A Franzén ◽  
D Heinegård
Keyword(s):  
Sialic Acid ◽  
Core Protein ◽  
Deae Cellulose ◽  
Sedimentation Coefficient ◽  
Dry Weight ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bovine Bone ◽  
Guanidinium Chloride ◽  
The Core ◽  
Isolation And Characterization

Two different sialoproteins were isolated from the mineralized matrix of bovine bone by using extraction with guanidinium chloride first without and then with EDTA. The sialoproteins were purified by chromatography on DEAE-cellulose eluted with a sodium acetate gradient in 7 M-urea, pH 6. Two sialoproteins (I and II) were then separated by chromatography on DEAE-cellulose eluted with a sodium chloride gradient in 7 M-urea, pH 4. The ratio between recovered sialoprotein I and II was 1:5. The chemical analysis of the two sialoproteins showed that they differed. Both, however, had very high contents of aspartic acid/asparagine and glutamic acid/glutamine though they differed markedly in contents of leucine and glycine. Both sialoproteins contained phosphate, sialoprotein I more than sialoprotein II. Content of sialic acid was substantially higher in the more prominent sialoprotein II (13.4% of dry weight) than in sialoprotein I (4.8% of dry weight). The peptide patterns produced by trypsin digests of [125I]iodinated sialoproteins I and II showed both structural similarities and structural differences. Sialoprotein II, being the major component, was characterized further. Its molecular mass was 57300 Da determined by sedimentation-equilibrium centrifugation in 6 M-guanidinium chloride, and its sedimentation coefficient (S0(20),w) was 2.53 S. Upon rotary shadowing, sialoprotein II appeared as an extended rod, having a core with an average length of 40 nm. Two types of oligosaccharides, N-glycosidically and O-glycosidically linked to the core protein, were isolated from sialoprotein II. Contents of mannose and sialic acid in the O-linked oligosaccharide were surprisingly high. Antibodies against sialoprotein II were raised in rabbits and an enzyme-linked immunosorbent assay was developed. Antigenicity of sialoprotein II was not affected by reduction and alkylation, was only partially lost upon trypsin digestion and was completely lost upon fragmentation of the core protein by alkaline-borohydride treatment, indicating that all antigenic sites were located in the protein portion. Sialoprotein I expectedly showed only partial immunological cross-reactivity with sialoprotein II. The quantity of sialoprotein II in bone extracts was found to be about 1.5 mg/g wet wt. of bone, but the protein was not detected in extracts of a number of other bovine tissues i.e. aorta, cartilage, dentine, kidney, liver, muscle, sclera, skin and tendon.

scholarly journals Amino acid activation in mammalian brain. Purification and characterization of tryptophan-activating enzyme from buffalo brain

1973 ◽  
Vol 135 (2) ◽  
pp. 367-373 ◽  
Author(s):  
C.-C. Liu ◽  
C.-H. Chung ◽  
M.-L. Lee
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Mammalian Brain ◽  
Acid Activation ◽  
High Concentration ◽  
Molecular Weights ◽  
Fractionation Column ◽  
Quaternary Structures

l-Tryptophan-activating enzyme [l-tryptophan–tRNA ligase (AMP), EC 6.1.1.2] of water-buffalo brain was purified to near homogeneity by heat and pH treatments, ammonium sulphate fractionation, column chromatography on DEAE-cellulose, hydroxyapatite and Amberlite CG-50, and gel filtration on Sephadex G-200. The purified enzyme catalyses tryptophanyl-tRNA formation with yeast tRNA, but not with Escherichia coli tRNA. The enzyme exhibits multiple peaks of activity in Sephadex gel filtration with molecular weights corresponding to 155000, 105000 and 50000. However, only one peak of activity with molecular weight of 155000 can be detected when the enzyme is subjected to gel filtration at high concentration. Disc gel electrophoresis in the presence of sodium dodecyl sulphate reveals a single band with molecular weight of 55000. The activity of the enzyme is concentration dependent. Different Km and Vmax. values are obtained at different enzyme concentrations. These data suggest that this enzyme may exist in different quaternary structures, each with its own kinetic constants. The enzyme activity is inhibited by p-chloromercuribenzoate, and is not protected by the presence of the substrates, l-tryptophan, Mg2+, ATP, in any combination.

Two types of xylanases of alkalophilic Bacillus sp. No. C-125

1985 ◽  
Vol 31 (6) ◽  
pp. 538-542 ◽  
Author(s):  
H. Honda ◽  
T. Kudo ◽  
Y. Ikura ◽  
K. Horikoshi
Keyword(s):  
Ammonium Sulfate ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Bacillus Sp ◽  
Ammonium Sulfate Precipitation ◽  
Molecular Weights ◽  
Protein Molecules ◽  
Activity Curve ◽  
Alkalophilic Bacillus ◽  
Hydrolysis Of

One alkalophilic Bacillus sp. strain C-125 (FERM No. 7344) was isolated from soil. From this organism, two types of xylanases, designated xylanase A and xylanase N, were purified by an ammonium sulfate precipitation followed by Biogel P-30 gel filtration, DEAE-cellulose chromatography, and Sephadex G-75 gel filtration. The molecular weights of xylanase A and N were estimated as 43 000 and 16 000, respectively. Immunological experiments indicated that xylanase A and xylanase N were entirely different protein molecules. Xylanase N was most active at pH 6.0–7.0, but xylanase A had a very broad pH activity curve (pH 6–10) and was still active even at pH 12.0. The maximum hydrolysis of xylan by the enzymes was about 25%. Both enzymes split xylan and yielded xylobiose and higher oligosaccharides but could hydrolyze neither xylobiose nor xylotriose. Trans xylosidation activities were detected in both enzymes.

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