PURIFICATION OF "INHIBIN" FROM HUMAN OVARIAN FOLLICULAR FLUID

1979 ◽  
Vol 90 (1) ◽  
pp. 157-166 ◽  
Author(s):  
S. Chari ◽  
C. R. N. Hopkinson ◽  
E. Daume ◽  
G. Sturm
Keyword(s):  
Follicular Fluid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Male Rats ◽  
Acetone Powder ◽  
Gel Chromatography ◽  
Apparent Homogeneity ◽  
Ovarian Follicular Fluid

ABSTRACT Following the earlier demonstration of inhibin-like activity in human ovarian follicular fluid a method for its purification to apparent homogeneity is described. The fluid was converted to acetone powder and subjected sequentially to ammonium sulphate fractionation, gel chromatography on Sephadex G-200, continuous gradient ion-exchange chromatography on DEAE-cellulose, first with a pH gradient from 8.0 to 4.0 and then with a NaCl gradient to 1 m at pH 5.2. The active fraction from this step was subjected to gel filtration on Sephadex G-100 and finally passed through an Amicon Centriflo membrane CF-25 (cut off point: 25 000 m.w.). The ultrafiltrate was homogeneous by SDS-polyacrylamide gel electrophoresis, had a molecular weight of the order of 23 000 and was capable of suppressing serum gonadotrophin levels in the castrated male rats in as low a dose as 25 μg/rat.

scholarly journals Isolation and characterization of a T lymphocyte-derived differentiation inducing factor for the myeloid leukemic cell line HL- 60

Blood ◽  
1984 ◽  
Vol 63 (3) ◽  
pp. 510-517 ◽  
Author(s):  
IL Olsson ◽  
MG Sarngadharan ◽  
TR Breitman ◽  
RC Gallo
Keyword(s):  
Cell Line ◽  
T Lymphocyte ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Periodate Oxidation ◽  
Morphological Characteristics ◽  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Isolation And Characterization ◽  
Sds Page

Abstract Mitogen-stimulated mononuclear blood cells produce differentiation inducing factors (DIFs) for the promyelocytic cell line HL-60. We report that DIF is produced constitutively by a malignant T lymphocyte line HUT-102. DIF was purified 7,000-fold from HUT-102 conditioned media by utilizing ion-exchange chromatography with DEAE-Sepharose, gel chromatography, Blue-Sepharose chromatography, and preparative SDS- polyacrylamide gel electrophoresis (SDS-PAGE). The final preparation is susceptible to protease treatment, has a molecular weight of 46,000, as determined by SDS-PAGE and approximately 55,000 by gel filtration, has an isoelectric point of approximately 5.2, does not adhere to lectin- Sepharose and is resistant to periodate oxidation, and is free of colony-stimulating factor. DIF induced maturation of HL-60 into phagocytizing nitro blue tetrazolium reducing cells with the morphological characteristics of myelomonocytic or monocyte-like cells. An activity, co-chromatographing with DIF, acts synergistically with retinoic acid to induce maturation not only of HL-60, but also of the monoblast-like cell line U-937 (measured as percentage of cells reducing NBT).

scholarly journals Mouse Uterine Alkaline Phosphatase: Improved Purification by Affinity Chromatography and Further Characterization of the Enzyme

1980 ◽  
Vol 33 (3) ◽  
pp. 279 ◽  
Author(s):  
RN Murdoch ◽  
Louise E Buxton ◽  
DJ Kay
Keyword(s):  
Alkaline Phosphatase ◽  
Affinity Chromatography ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Anion Exchange Chromatography ◽  
Pregnant Mouse ◽  
Exchange Chromatography ◽  
Enzyme Preparations

An improved procedure for the purification of alkaline phosphatase from about 10 g of day 7 pregnant mouse uterine tissue is described. Following homogenization, the procedure involved solubilization and extraction with 0�8% (v/v) Triton X-lOO and 20% (v/v) n-butanol, ammonium sulfate precipitation, concanavalin A-Sepharose 4B affinity chromatography, DEAE-cellulose anion-exchange chromatography and Sephacryl S200 gel filtration. On subjecting 2162-fold purified enzyme preparations to polyacrylamide-gel electrophoresis, a single band of protein coincident with the zone of enzyme activity and having an apparent molecular weight of 205 OOO� lOOOO was identified. Affinity chromatography yielded the largest increase in purity of any step in the procedure and established the glycoprotein nature of the uterine enzyme.

Purification and characterization of the extracellular α-amylase activity of the yeast Schwanniomyces alluvius

1987 ◽  
Vol 65 (10) ◽  
pp. 899-908 ◽  
Author(s):  
F. Moranelli ◽  
M. Yaguchi ◽  
G. B. Calleja ◽  
A. Nasim
Keyword(s):  
Amino Acid ◽  
Amylase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Proteinase K ◽  
Ph Optimum ◽  
Sds Page

The extracellular α-amylase activity of the yeast Schwanniomyces alluvius has been purified by anion-exchange chromatography on DEAE-cellulose and gel-filtration chromatography on Sephadex G-100. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) and N-terminal amino acid analysis of the purified sample indicated that the enzyme preparation was homogeneous. The enzyme is a glycoprotein having a molecular mass of 52 kilodaltons (kDa) estimated by SDS–PAGE and 39 kDa by gel filtration on Sephadex G-100. Chromatofocusing shows that it is an acidic protein. It is resistant to trypsin but sensitive to proteinase K. Its activity is inhibited by the divalent cation chelators EDTA and EGTA and it is insensitive to sulfhydryl-blocking agents. Exogenous divalent cations are inhibitory as are high concentrations of monovalent salts. The enzyme has a pH optimum between 3.75 and 5.5 and displays maximum stability in the pH range of 4.0–7.0. Under the conditions tested, the activity is maximal between 45 and 50 °C and is very thermolabile. Analysis of its amino acid composition supports its acidic nature.

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 Platelet-derived neutrophil adherence-inhibiting factor in humans

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2368-2373
Author(s):  
K Iwabuchi ◽  
T Yamashita
Keyword(s):  
Type Iv Collagen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Human Platelets ◽  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Adsorption Sites ◽  
Type Iv ◽  
Neutrophil Adherence

The effect of constituents of human platelets on leukocyte adherence was examined. Adherence-inhibiting factors (AIFs), which strongly inhibited neutrophil adherence to glass, were present in both cytosol and granule fractions of human platelets. On the Superose 6 gel chromatography (Pharmacia LKB Biotechnology, Uppsala, Sweden), the granular AIF was eluted as a single active peak (2,600 Kd), whereas cytosolic AIFs were eluted at two different positions (2,600 and 480 Kd). When platelets were stimulated by thrombin, granular AIF was released extracellularly without releasing a cytosolic marker. Using DE32 anion exchange chromatography and Superose 6 gel filtration, granular AIF was completely purified. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis analysis suggests that granular AIF consists of two subunits with molecular masses of approximately 340 and 190 Kd. Purified granular AIF inhibited human neutrophil adherence to glass, plastic, and type IV collagen-coated plastic, whereas it did not affect monocyte adherence. These results suggest that granular AIF inhibits neutrophil adherence not only via nonspecific adsorption sites, but also via type IV collagen receptors.

Lectins from bulbs of the Chinese daffodil Narcissus tazetta (family Amaryllidaceae)

2000 ◽  
Vol 78 (4) ◽  
pp. 463-468 ◽  
Author(s):  
L SM Ooi ◽  
T B Ng ◽  
Yunqi Geng ◽  
V EC Ooi
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Syncytium Formation ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Lung Cells ◽  
Narcissus Tazetta ◽  
Mannose Binding ◽  
Fetal Bovine

The isolation of three lectins with similar N-terminal amino acid sequences from the bulbs of the Chinese daffodil Narcissus tazetta was achieved. The isolation protocol involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on mannose-agarose, and fast protein liquid chromatography-gel filtration on Superose 12. The lectins were all adsorbed on mannose-agarose and demonstrated a single band with a molecular weight of 13 kDa in SDS-polyacrylamide gel electrophoresis and a single 26 kDa peak in gel filtration, indicating that they were mannose-binding, dimeric proteins. The lectins differed in hemagglutinating activity, with the magnitude of the activity correlating with the ionic strength of the buffer required to elute the lectin from the DEAE-cellulose column. The bulb lectin did not exert potent cytotoxicity against cancer cell lines or fetal bovine lung cells but inhibited syncytium formation in, and reinstated viability of, fetal bovine lung cells infected with bovine immunodeficiency virus.Key words: lectins, daffodil bulbs, chromatography.

scholarly journals Platelet-derived neutrophil adherence-inhibiting factor in humans

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2368-2373 ◽  
Author(s):  
K Iwabuchi ◽  
T Yamashita
Keyword(s):  
Type Iv Collagen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Human Platelets ◽  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Adsorption Sites ◽  
Type Iv ◽  
Neutrophil Adherence

Abstract The effect of constituents of human platelets on leukocyte adherence was examined. Adherence-inhibiting factors (AIFs), which strongly inhibited neutrophil adherence to glass, were present in both cytosol and granule fractions of human platelets. On the Superose 6 gel chromatography (Pharmacia LKB Biotechnology, Uppsala, Sweden), the granular AIF was eluted as a single active peak (2,600 Kd), whereas cytosolic AIFs were eluted at two different positions (2,600 and 480 Kd). When platelets were stimulated by thrombin, granular AIF was released extracellularly without releasing a cytosolic marker. Using DE32 anion exchange chromatography and Superose 6 gel filtration, granular AIF was completely purified. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis analysis suggests that granular AIF consists of two subunits with molecular masses of approximately 340 and 190 Kd. Purified granular AIF inhibited human neutrophil adherence to glass, plastic, and type IV collagen-coated plastic, whereas it did not affect monocyte adherence. These results suggest that granular AIF inhibits neutrophil adherence not only via nonspecific adsorption sites, but also via type IV collagen receptors.

Purification and characterization of a β-glucosidase from solid-state cultures ofHumicola griseavar.thermoidea

1996 ◽  
Vol 42 (1) ◽  
pp. 1-5 ◽  
Author(s):  
Edivaldo Ximenes Ferreira Filho
Keyword(s):  
Solid State ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Performance Liquid Chromatographic ◽  
Exchange Chromatography ◽  
Glucosidase Activity ◽  
Apparent Homogeneity ◽  
Sds Page

The thermophilic fungus Humicola grisea var. thermoidea produced β-glucosidase activity when grown in a solid-state culture on wheat bran as carbon source. A β-glucosidase was purified to apparent homogeneity by ultrafiltration, gel filtration chromatography on Sephacryl S-100, and ion-exchange chromatography on S-Sepharose, as judged by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) on a 12.5% (w/v) slab gel. The enzyme had a molecular mass of 82 and 156 kDa, as estimated by SDS–PAGE and gel filtration on a high performance liquid chromatographic column, respectively, suggesting that the native enzyme may consist of two identical subunits. The purified enzyme was thermostable at 60 °C for 1 h with a half-life of 15 min at 65 °C, and displayed optimum activity at 60 °C and a pH range of 4.0–4.5. The Kmand Vmaxvalues for p-nitrophenyl β-D-glucopyranoside were determined to be 0.316 mM and 0.459 IU∙mL−1, respectively. D-Glucose, D-gluconic acid lactone, Hg2+, Cu2+, and Mn2+inhibited β-glucosidase activity. The enzyme activity was competitively inhibited by D-glucose (Ki = 0.6 mM). The purified enzyme was very active against cellobiose and p-nitrophenyl β-D-glucopyranoside.Key words: Humicola, β-glucosidase, purification, characterization.

Purification and characterization of a murine cellular retinoic acid-binding protein

1988 ◽  
Vol 66 (7) ◽  
pp. 750-757 ◽  
Author(s):  
John Stuart Bailey ◽  
Chi-Hung Siu
Keyword(s):  
Retinoic Acid ◽  
Ion Exchange ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Retinol Binding Protein ◽  
Amino Terminal ◽  
Acid Binding Protein

A cellular retinoic acid-binding protein from 1-day-old mouse pups has been purified to homogeneity. The isolation procedure included gel filtration on Sephadex G-75, ion exchange chromatography on DEAE cellulose, and chromatofocusing on PBE 9–4 ion exchange resin. The chromatofocusing step was most useful in removing the major contaminants, which were otherwise difficult to remove. The binding protein was finally subjected to two cycles of high performance liquid chromatography on a DEAE-5PW column to achieve homogeneity. The protein has an isoelectric point of 4.75 and consists of a single polypeptide, migrating with an apparent Mr of 14 600 in SDS – polyacrylamide gel electrophoresis. Amino-terminal sequence analysis showed that the mouse cellular retinoic acid-binding protein has a high percentage of amino acid identity with other retinoid-binding proteins, However, it is immunologically distinct from the cellular retinol-binding protein.

scholarly journals Purification and chemical characterization of human hexosaminidases A and B

1976 ◽  
Vol 159 (3) ◽  
pp. 535-539 ◽  
Author(s):  
J E S. Lee ◽  
A Yoshida
Keyword(s):  
Column Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Deae Cellulose Column ◽  
Hexosaminidase A ◽  
Cellulose Column

N-Acetyl-β-hexosaminidases A and B were purified to homogeneity from human placenta. In the initial step of purification, the enzymes were adsorbed on concanavalin A-Sepharose 4B and eluted from the column with α-methyl D-mannosides. Subsequent purification steps included DEAE-cellulose column chromatography, QAE-Sephadex [diethyl-(2-hydroxypropyl)aminoethyl-Sephadex] column chromatography, Sephadex G-200 gel filtration and preparative disc polyacrylamide-gel electrophoresis, followed by another QAE-Sephadex chromatography for the hexosaminidase A preparation, and DEAE-cellulose column chromatography, calcium phosphate gel chromatography, Sephadex G-200 gel filtration, QAE-Sephadex chromatography and CM-cellulose chromatography for the hexosaminidase B preparation. The purified preparations, particularly hexosaminidase A, had significantly higher specific enzyme activities than previously reported. The preparations moved on polyacrylamide-gel electrophoresis as single protein bands, which also stained for enzyme activity. Sedimentation-equilibrium centrifugation indicated homogenous dispersion of the enzymes, and the molecular weight was estimated as about 110000 for both enzymes. Complete amino acid and carbohydrate compositions of the two isoenzymes were determined, and, in contrast with previous suggestions, no sialic acid was found in the enzymes.

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