scholarly journals Characterization of catabolin, the major product of pig synovial tissue that induces resorption of cartilage proteoglycan in vitro

1981 ◽  
Vol 199 (3) ◽  
pp. 705-714 ◽  
Author(s):  
Jeremy Saklatvala
Keyword(s):  
Synovial Tissue ◽  
Culture Medium ◽  
Gel Filtration ◽  
Septal Cartilage ◽  
Major Product ◽  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Connective Tissues ◽  
Dialysis Tubing

1. Pig synovium in organ culture produces material which induces living cartilage to resorb its proteoglycan in vitro. 2. The bioassay for this material was to measure glycosaminoglycan released from explants of bovine nasal-septal cartilage cultured for 8 days. The performance of the assay was greatly improved by adding cortisol succinate (0.1mug/ml). This decreased the release of glycosaminoglycan from unstimulated cartilage without inhibiting its response to catabolic factors from the synovium. 3. By using this improved assay it was shown that 90% of the active materials in synovial culture medium were retained by dialysis membrane. 4. An active protein was partially purified from synovial culture medium by (NH4)2SO4 precipitation, ion-exchange chromatography, gel filtration and preparative isoelectric focusing. 5. This protein, called catabolin, had mol.wt. 17000 and pI4.6. 6. Synovial culture medium concentrated in dialysis tubing was subjected to gel chromatography and found to contain one major active component, which was eluted at the same position as the partially purified catabolin. 7. The synovial culture medium was not inactivated by heating (70°C for 10min), nor were diluted preparations of partially purified catabolin, but concentrated crude preparations were thermolabile. 8. These results suggest that catabolin is the major substance produced by the synovial tissue in culture which induces resorption of proteoglycan of living cartilage in vitro. 9. Other cultured soft connective tissues produced catabolin-like activity. The example of sclera is shown, and production was inhibited by cortisol succinate (0.1mug/ml). 10. It is suggested that catabolin may be a general product of soft connective tissues in culture, and its physiological function may be to induce resorption of connective-tissue matrix after injury.

scholarly journals The isolation and partial characterization of low-molecular-weight phosphorylated component of the non-histone proteins of mouse nuclei

1978 ◽  
Vol 175 (1) ◽  
pp. 35-46 ◽  
Author(s):  
A J MacGillivray ◽  
C Johnston ◽  
R MacFarlane ◽  
D Rickwood
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Low Molecular Weight ◽  
Gel Chromatography ◽  
Liver Nuclei ◽  
Acidic Proteins

After labelling of mouse liver nuclei with [gamma-32P]ATP in vitro, 10-20% of the radioactivity incorporated into the saline-soluble nuclear and HAP2 chromatin fractions was located in a low-molecular-weight component (component 10) with pI near 4.5 in urea. By using combinations of ion-exchange chromatography, preparative thin-layer isoelectric focusing and gel filtration, this component was isolated from both nuclear fractions. Recovery from the saline-soluble fraction was poor under conditions that allow endogenous phosphatases to be active. Component 10 was shown to be a phosphoprotein on the basis of enzyme-digestion experiments and the detection of phosphoserine and phosphothreonine. The 32P radioactivity did not appear to be associated with phosphorylated basic amino acids. Its molecular weight was determined by gel chromatography and electrophoresis in sodium dodecyl sulphate/polyacrylamide gels as approx. 10000, and tryptic digestion of the reduced carboxymethylated protein in urea yielded two 32P-labelled peptides. It has not been possible as yet to assign a function to component 10, though its similarity to other low-molecular-weight acidic proteins is discussed.

Co-purification of bone resorbing activity and adenylate cyclase stimulating activity from human tumours associated with the humoral hypercalcaemia of malignancy

1987 ◽  
Vol 114 (1) ◽  
pp. 18-26 ◽  
Author(s):  
Chohei Shigeno ◽  
Itsuo Yamamoto ◽  
Shegiharu Dokoh ◽  
Megumu Hino ◽  
Jun Aoki ◽  
...  
Keyword(s):  
Bone Resorption ◽  
High Performance ◽  
Basic Protein ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Protein Factor ◽  
Human Tumours ◽  
Acid Stable ◽  
Bone Resorbing Activity

Abstract. We have partially purified a tumour factor capable of stimulating both bone resorption in vitro and cAMP accumulation in osteoblastic ROS 17/2 cells from three human tumours associated with humoral hypercalcaemia of malignancy. Purification of tumour factor by sequential acid urea extraction, gel filtration and cation-exchange chromatography, reverse-phase high performance liquid chromatography followed by analytical isoelectric focussing provided a basic protein (pI > 9.3) with a molecular weight of approximately 13 000 as a major component of the final preparation which retained both the two bioactivities. Bone resorbing activity and cAMP-increasing activity in purified factor correlated with each other. cAMP-increasing activity of the factor was heat- and acid-stable, but sensitive to alkaline ambient pH. Treatment with trypsin destroyed cAMP-increasing activity of the factor. Synthetic parathyroid hormone (PTH) antagonist, human PTH-(3– 34) completely inhibited the cAMP-increasing activity of the factor. The results suggest that this protein factor, having its effects on both osteoclastic and osteoblastic functions, may be involved in development of enhanced bone resorption in some patients with humoral hypercalcaemia of malignancy.

Purification and characterization of prostate-specific antigen (PSA) complexed to alpha 1-antichymotrypsin: potential reference material for international standardization of PSA immunoassays

1995 ◽  
Vol 41 (9) ◽  
pp. 1273-1282 ◽  
Author(s):  
Z Chen ◽  
A Prestigiacomo ◽  
T A Stamey
Keyword(s):  
Molecular Mass ◽  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Molar Ratio ◽  
Free Psa ◽  
Apparent Homogeneity ◽  
International Standardization

Abstract We describe for the first time a protocol to purify to apparent homogeneity an in vitro-prepared complex of prostate-specific antigen (PSA) and alpha 1-antichymotrypsin (ACT) by using a combination of gel filtration and ion-exchange chromatography. The purity of the PSA-ACT complex was confirmed by gel electrophoresis and Western blot. The PSA-ACT complex was stable in the pH range 6.0 to 7.8; it was also stable in various matrices, temperatures, and high concentrations of salt. Purification of the PSA-ACT complex was highly reproducible. An absorptivity of 0.99 L x g-1 x cm-1 at 280 nm was assigned to the PSA-ACT complex, based on amino acid analysis. Because PSA and ACT bind in a 1:1 molar ratio, we determined the molecular mass of the PSA-ACT complex as the mass encoded by the cDNA of ACT (plus 26% carbohydrate) plus the molecular mass of PSA (28,430 Da), which totals 89,280 Da. Using this material, we made two common calibrators, one of 100% PSA-ACT complex and one of 90% PSA-ACT complex plus 10% free PSA by volume (90:10 calibrator). Substitution of these calibrators for the manufacturers' calibrators in nine commercial immunoassays substantially reduced differences between immunoassays, especially for serum PSA values between 4 and 10 micrograms/L. The 90:10 calibrator is recommended as a universal calibrator for international standardization of PSA immunoassays.

THE UPTAKE OF TESTOSTERONE AND ZINC IN VITRO BY THE HUMAN BENIGN HYPERTROPHIC PROSTATE

1973 ◽  
Vol 58 (3) ◽  
pp. 405-419 ◽  
Author(s):  
M. JOAN REED ◽  
S. R. STITCH
Keyword(s):  
Ion Exchange ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Zinc Binding ◽  
Supernatant Fraction ◽  
Low Molecular Weight ◽  
Molecular Weight Peak ◽  
Steroid Binding

SUMMARY The uptake of 65Zn and [1,2-3H]testosterone by minced tissue of human benign hypertrophic prostates and the subcellular distribution of radioactivity were examined. The nature of steroid and 65Zn binding by the cytosol (105000 g supernatant) fraction was investigated by gel filtration, ion-exchange chromatography and electrophoresis. It was found that steroid binding after incubation at 4°C was specific. One or two regions of steroid binding were observed after gel filtration of the cytosol using Sephadex G-200, depending upon incubation conditions. Binding of 65Zn was found in the low molecular weight peak after G-200 gel filtration. Equimolar CdCl2 and 65ZnCl2 were incubated with [1,2-3H]testosterone and minced tissue and the cytosol was subjected to gel filtration. Compared with control values, without CdCl2, reduction of 65Zn binding by about 50% occurred, while binding of 3H-labelled steroid was unaffected. Electrophoresis and ion-exchange chromatography showed that 65Zn and 3H-labelled steroid were bound to different proteins. A sample of the zinc-binding protein was prepared by ion-exchange chromatography and the homogeneity was checked by electrophoresis.

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).

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 Evidence for N-linked glycosylation in Toxoplasma gondii

1993 ◽  
Vol 291 (3) ◽  
pp. 713-721 ◽  
Author(s):  
M Odenthal-Schnittler ◽  
S Tomavo ◽  
D Becker ◽  
J F Dubremetz ◽  
R T Schwarz
Keyword(s):  
Toxoplasma Gondii ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Surface Glycoprotein ◽  
Common Precursor ◽  
The Common ◽  
Oligosaccharide Structures

In this paper we report experiments demonstrating the presence of N-linked oligosaccharide structures in Toxoplasma gondii tachyzoites, providing the first direct biochemical evidence that this sporozoan parasite is capable of synthesizing N-linked glycans. The tachyzoite surface glycoprotein gp23 was metabolically labelled with [3H]glucosamine and [3H]mannose. Gel-filtration chromatography on Bio-Gel P4 columns produced four radiolabelled N-linked glycopeptides which were sensitive to peptidase-N-glycanase F, but resistant to endoglycosidases H and F. Using chemical analysis and exoglycosidase digestions followed by Dionex-high-pH anion-exchange chromatography and size fractionation on Bio-Gel P4 we show that gp23 has N-linked glycans in the hybrid- or complex-type structure composed of N-acetylgalactosamine, N-acetylglucosamine and mannose and devoid of sialic acid and fucose residues. In addition, the sensitivity of glycopeptides from glycoprotein extracts to endoglycosidases H and F revealed the in vivo synthesis of oligomannose-type structures by T. gondii tachyzoites. We have extended these findings by demonstrating the ability of T. gondii microsomes to synthesize in vitro a glucosylated lipid-bound high-mannose structure (Glc3Man9GlcNAc2) that is assumed to be identical with the common precursor for N-glycosylation in eukaryotes.

scholarly journals A Novel Hemagglutinin with Antiproliferative Activity against Tumor Cells from the Hallucinogenic MushroomBoletus speciosus

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Jian Sun ◽  
Tzi-Bun Ng ◽  
Hexiang Wang ◽  
Guoqing Zhang
Keyword(s):  
Cation Exchange ◽  
Antiproliferative Activity ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Lymphocytic Leukemia ◽  
Hemagglutinating Activity ◽  
Cation Exchange Chromatography ◽  
Type Ab

Little was known about bioactive compounds from the hallucinogenic mushroomBoletus speciosus. In the present study, a hemagglutinin (BSH,B. speciosushemagglutinin) was isolated from its fruiting bodies and enzymatic properties were also tested. The chromatographic procedure utilized comprised anion exchange chromatography on Q-Sepharose, cation exchange chromatography on CM-Cellulose, cation exchange chromatography on SP-Sepharose, and gel filtration by FPLC on Superdex 75. The hemagglutinin was a homodimer which was estimated to be approximately 31 kDa in size. The activity of BSH was stable up to 60°C, while there was a precipitous drop in activity when the temperature was elevated to 70°C. BSH retained 25% hemagglutinating activity when exposed to 100 mM NaOH and 25 mM HCl. The activity was potently inhibited by 1.25 mM Hg2+and slightly inhibited by Fe2+, Ca2+, and Pb2+. None of the sugars tested showed inhibition towards BSH. Its hemagglutinating activity towards human erythrocytes type A, type B, and type AB was higher than type O. The hemagglutinin showed antiproliferative activity towards hepatoma Hep G2 cells and mouse lymphocytic leukemia cells (L1210)in vitro, with IC50of 4.7 μM and 7.0 μM, respectively. It also exhibited HIV-1 reverse transcriptase inhibitory activity with an IC50of 7.1 μM.

scholarly journals Characterization of proteins from human synovium and mononuclear leucocytes that induce resorption of cartilage proteoglycan in vitro

1983 ◽  
Vol 209 (2) ◽  
pp. 337-344 ◽  
Author(s):  
J Saklatvala ◽  
S J Sarsfield ◽  
L M C Pilsworth
Keyword(s):  
Articular Cartilage ◽  
Synovial Tissue ◽  
Gel Filtration ◽  
Human Articular Cartilage ◽  
Nasal Cartilage ◽  
Cartilage Proteoglycan ◽  
Mononuclear Leucocytes ◽  
Culture Supernatants

Both human synovial tissue in culture and lectin-stimulated mononuclear leucocytes produced a protein that induced proteoglycan resorption in explants of bovine nasal cartilage and human articular cartilage. On gel filtration the protein had Mr 16000-20000 and on isoelectric focusing its pI was 5.2-5.3. The protein corresponded to catabolin, which has previously been identified as a product of cultured porcine synovial tissue and mononuclear leucocytes. The action of partially purified human catabolin was not inhibited by cortisol, although the activity of the leucocyte supernatants from which it had been isolated was inhibited. For this reason it is not possible to be sure that the active factor detected in the bioassay of the crude leucocyte culture supernatants is in fact catabolin.

Isolation of Fasciola hepatica haemoglobin

Parasitology ◽  
1995 ◽  
Vol 111 (2) ◽  
pp. 209-215 ◽  
Author(s):  
S. McGonigle ◽  
J. P. Dalton
Keyword(s):  
Fasciola Hepatica ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequence Analysis ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Haemoglobin Molecule

SUMMARYA haemoprotein released in vitro by adult Fasciola hepatica was purified by gel filtration chromatography on Sephacryl S-200 and ion-exchange chromatography on DEAE-Sepharose. The molecule, with an apparent molecular weight of > 200 kDa, contains a haem group and has absorption spectra characteristics similar to haemoglobins. N-terminal amino acid sequence analysis revealed no similarity between the F. hepatica haemoglobin and other vertebrate or invertebrate haemoglobins. Antibodies to the haemoglobin molecule can be detected in the sera of F. hepatica-infected bovines as early as 1 week after infection.

Sign in / Sign up

Export Citation Format

Share Document