scholarly journals The soluble ‘low-Km’ 5′-nucleotidase of rat kidney represents solubilized ecto-5′-nucleotidase

1991 ◽  
Vol 273 (2) ◽  
pp. 409-413 ◽  
Author(s):  
G Piec ◽  
M Le Hir
Keyword(s):  
Phospholipase C ◽  
High Speed ◽  
Catalytic Properties ◽  
Rat Kidney ◽  
Gel Filtration ◽  
Main Peak ◽  
Soluble Enzyme ◽  
Renal Brush Border Membranes ◽  
Triton X ◽  
Renal Brush Border

A soluble ‘low-Km’ 5′-nucleotidase has been described previously in several organs. It has been presumed to be of cytosolic origin and thus to play a role in the intracellular production of adenosine. Its catalytic properties are similar to those of the ecto-5′-nucleotidase of cell membranes. In the present study we compared molecular properties of the two enzymes in the kidney of the rat. The Mr of the main peak of soluble ‘low-Km‘ 5′-nucleotidase in gel-filtration chromatography was similar to that of the ecto-5′-nucleotidase solubilized by a phosphatidylinositol-specific phospholipase C from renal brush-border membranes. In phase-partition experiments using Triton X-114, the soluble enzyme appeared to be hydrophobic. Its hydrophobicity was decreased on treatment with a phosphatidylinositol-specific phospholipase C, suggesting that the soluble ‘low-Km’ 5′-nucleotidase contains the phosphatidylinositol anchor which is characteristic for the ecto-enzyme. An anti-ecto-5′-nucleotidase antiserum provoked an almost complete inhibition of the soluble enzyme. Immunoblotting using anti-ecto-5′-nucleotidase antiserum revealed in the high-speed supernatants a polypeptide with a similar Mr to the subunit of the ecto-5′-nucleotidase. The soluble ‘low-Km’ 5′-nucleotidase, like the ecto-5′-nucleotidase, bound specifically to concanavalin A. We conclude that the soluble ‘low-Km’ 5′-nucleotidase is not a cytosolic enzyme, but that it most probably originates from the solubilization of the ecto-5′-nucleotidase, and that it therefore cannot participate in the intracellular production of adenosine.

scholarly journals Incorporation of d-glucose-, l-alanine- and phosphate-transport systems from rat renal brush-border membranes into liposomes

1977 ◽  
Vol 168 (2) ◽  
pp. 311-314 ◽  
Author(s):  
R Kinne ◽  
R G Faust
Keyword(s):  
Brush Border ◽  
Rat Kidney ◽  
Protein Composition ◽  
Phosphate Transport ◽  
Transport Systems ◽  
Brush Border Membranes ◽  
Naturally Occurring ◽  
Renal Brush Border Membranes ◽  
Triton X ◽  
Renal Brush Border

An extract of soluble proteins was prepared from a rat kidney brush-border membranes by Triton X-100 solubilization followed by centrifugation for 1 h at 100000g. Its protein composition was markedly different from that of the brush-border membranes. Proteoliposomes were formed by co-sonication of the Triton X-100-free extract with a naturally occurring mixture of phospholipids extracted from rat kidney. These proteoliposomes were shown to contain Na+-stimulated D-glucose-, L-alanine- and phosphate-transport systems.

Solubilization of Adenylyl Cyclase from Human Myometrium in a αS-Coupled Form

2003 ◽  
Vol 23 (4) ◽  
pp. 175-186
Author(s):  
Ana M. Bajo ◽  
Juan C. Prieto ◽  
Pedro Valenzuela ◽  
Pilar Martinez ◽  
Luis G. Guijarro
Keyword(s):  
Adenylyl Cyclase ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Catalytic Subunit ◽  
Human Myometrium ◽  
Main Peak ◽  
Guanine Nucleotide Binding Protein ◽  
Chromatographic Profile ◽  
Guanine Nucleotide Binding ◽  
Triton X

Adenylyl cyclase (AC) was extracted from human myometrium with either non-ionic (Lubrol-PX or Triton X-100) or zwitterionic (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, CHAPS) detergents. The soluble enzyme was stimulated by forskolin, a hydrophobic activator, in the presence of Mg2+ indicating that the catalytic subunit had not been damaged after solubilization. The enzyme was also activated by 5′-guanylyl imidodiphosphate (Gpp(NH)p) showing that the catalytic unit was not separated from stimulatory guanine nucleotide binding protein (Gs) during the extraction. Both activators showed different effects on the stimulatory efficacy and potency of AC activity solobulized with detergents. Gel filtration of Lubrol-PX and CHAPS extracts over a Sepharose CL-2B column partially resolved AC and its complexes. The chromatographic profile for Lubrolsolubilized AC presented a main peak of about 200 kDa whereas CHAPS-solubilized AC showed a dominant peak of about 1100 kDa. The heterodisperse peaks obtained revealed that the catalytic AC subunit was not separated from Gs proteins after gel filtration, and that AC could be associated with other cellular proteins. When Lubrol extract was submitted to anionic-exchange chromatography, the enzyme was purified about 7.5 fold (enzymatic activity of 48.1 pmol/min/mg of protein). The catalytic subunit was co-eluted with both AC-activating proteins Gαs large (52.2 kDa) and Gαs small (48.7 kDa). This is the first demonstration of the stable physical association of AC with both αs subunits of G proteins in human myometrium.

An ATP-inhibited soluble 5'-nucleotidase of rat kidney

1988 ◽  
Vol 254 (2) ◽  
pp. F191-F195
Author(s):  
M. Le Hir ◽  
U. C. Dubach
Keyword(s):  
High Speed ◽  
Catalytic Properties ◽  
Divalent Cations ◽  
Rat Kidney ◽  
Maximal Activity ◽  
Rate Of Hydrolysis ◽  
Membrane Bound ◽  
Ph Range ◽  
Hydrolysis Of

Hydrolysis of 5'-AMP by 5'-nucleotidase is a possible source of adenosine in the kidney. A renal membrane-bound ecto-5'-nucleotidase has been previously described. The present study deals with the catalytic properties of a 5'-AMP phosphohydrolase partially purified from high-speed supernatants of rat kidney homogenates. It exhibits phosphatase activity toward 5'-AMP, 5'-IMP, and 5'-GMP, but not toward 2'- and 3'-AMP and corresponds therefore to a 5'-nucleotidase. The hydrolysis of 5'-AMP by the soluble 5'-nucleotidase requires divalent cations. Maximal activity is reached with 10 microM of either Mn2+ or Co2+, whereas half-maximal activity is obtained with approximately 400 microM Mg2+. The soluble 5'-nucleotidase exhibits Michaelis-Menten kinetics with a Km of 9.5 microM for 5'-AMP. In the presence of 1 mM of free Mg2+, physiological concentrations of ATP provoke an increase of the Km for 5'-AMP and a decrease of Vmax. An increase of the pH of 0.4 units in the pH range 6.4-7.4 roughly doubles the rate of hydrolysis of 5'-AMP. The effects of ATP and of the pH are compatible with a role of the renal soluble 5'-nucleotidase in the hydrolysis of 5'-AMP and in the production of adenosine during hypoxia.

scholarly journals Isolation of a subpopulation of glycoprotein IIb-III from platelet membranes that is bound to membrane actin.

1985 ◽  
Vol 100 (2) ◽  
pp. 652-657 ◽  
Author(s):  
R G Painter ◽  
K N Prodouz ◽  
W Gaarde
Keyword(s):  
High Speed ◽  
High Efficiency ◽  
Gel Filtration ◽  
Insoluble Fraction ◽  
Human Platelets ◽  
Independent Manner ◽  
Platelet Surface ◽  
Sds Page ◽  
Triton X

Triton X-100-insoluble residues, or skeletons, of plasma membrane-rich vesicles obtained from unstimulated human platelets were isolated by high speed centrifugation. About 10-15% of the total surface iodinatable glycoproteins IIb and III (GPIIb and GPIII, respectively) co-isolated with the insoluble fraction. After sonication and centrifugation the solubilized material was further purified by affinity chromatography on Lens culinaris lectin-Sepharose. SDS PAGE analysis of this material revealed the presence of at least three major proteins, which were shown to be GPIIb, GPIII, and membrane actin, as judged by their electrophoretic properties and on the basis of immunological criteria. Antibodies directed against platelet surface glycoproteins and antibodies directed against rabbit actin were able to immunoprecipitate all three proteins, which indicates that they were noncovalently associated with one another. Gel filtration of the Lens lectin-purified Triton-insoluble complex on Ultrogel AcA 22 showed that greater than 85% of the total surface GPIIb and III was associated with an actin-rich peak that eluted in the void volume. In contrast, the form of GPIIb-III present in the Triton-soluble membrane fraction behaved as monomeric species when chromatographed under identical conditions. Finally, the GPIIb-III membrane actin complex bound with high efficiency to rabbit f-actin in vitro in a Ca++-independent manner, whereas the monomeric forms found in the Triton-soluble fraction did not bind to actin. These results indicate that two forms of GPIIb and III exist: one that binds directly to endogenous membrane actin and one that does not.

scholarly journals Soluble low-Km 5′-nucleotidase from electric-ray (Torpedo marmorata) electric organ and bovine cerebral cortex is derived from the glycosyl-phosphatidylinositol-anchored ectoenzyme by phospholipase C cleavage

1992 ◽  
Vol 284 (3) ◽  
pp. 621-624 ◽  
Author(s):  
M Vogel ◽  
H Kowalewski ◽  
H Zimmermann ◽  
N M Hooper ◽  
A J Turner
Keyword(s):  
Cerebral Cortex ◽  
Phospholipase C ◽  
High Speed ◽  
Electric Organ ◽  
Bovine Brain ◽  
Soluble Enzyme ◽  
Torpedo Marmorata ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Membrane Bound

Soluble and membrane-bound low-Km 5′-nucleotidase was isolated from high-speed supernatants and membrane fractions derived from the electric organ of the electric ray (Torpedo marmorata) or from bovine brain cerebral cortex. Purification of both enzymes included chromatography on concanavalin A-Sepharose and AMP-Sepharose. The contribution to the total of soluble enzyme activity was lower in electric organ (1.6%) than in bovine cerebral cortex (27.9%). Membrane-bound and soluble forms have very similar Km values for AMP and are inhibited by micromolar concentrations of ATP. Both forms cross-react with, and are inhibited by, an antibody against the membrane-bound surface-located (ecto-) 5′-nucleotidase from electric organ. The HNK-1 carbohydrate epitope is present on both forms of the Torpedo enzyme, but is entirely absent from bovine cerebral-cortex 5′-nucleotidase. An antibody specific for the inositol 1,2-(cyclic)monophosphate that is formed on phospholipase C cleavage of an intact glycosyl-phosphatidylinositol (GPI) anchor binds to the soluble, but not to the membrane-bound, form of the enzyme from both sources. Our results suggest that soluble low-Km 5′-nucleotidase in both electric organ and bovine brain is derived from the membrane-bound GPI-anchored form of the enzyme by the action of a phospholipase C and is not a soluble cytoplasmic enzyme.

Conversion of an apparent 100 kDa folate binding protein from human milk, choroid plexus and semen to a 25 kDa molecular species by phosphatidylinositol-specific phospholipase C

1992 ◽  
Vol 12 (2) ◽  
pp. 87-93 ◽  
Author(s):  
Steen Ingemann Hansen ◽  
Jan Holm
Keyword(s):  
Human Milk ◽  
Choroid Plexus ◽  
Phospholipase C ◽  
Molecular Species ◽  
Binding Protein ◽  
Gel Filtration ◽  
Molecular Size ◽  
Membrane Anchor ◽  
Folate Binding Protein ◽  
Triton X

Gel filtration studies in the presence of Triton X-100 showed that treatment with phosphatidylinositol-specific phospholipase C reduced the apparent molecular size of the 100 kDa folate binding protein from human milk, choroid plexus and semen to 25 kDa. Cleavage of a hydrophobic glycosly phosphatidylinositol domain (a membrane anchor) inserting the protein into Triton X-100 micelles could account for this phenomenon.

Binding of Human Platelet Glycoprotein lb and Actin to Fragments of Actin-Binding Protein

1992 ◽  
Vol 67 (02) ◽  
pp. 252-257 ◽  
Author(s):  
Anne M Aakhus ◽  
J Michael Wilkinson ◽  
Nils Olav Solum
Keyword(s):  
Actin Filaments ◽  
High Speed ◽  
Binding Protein ◽  
Protein A ◽  
Actin Binding ◽  
Platelet Glycoprotein ◽  
Actin Binding Protein ◽  
Crossed Immunoelectrophoresis ◽  
Triton X ◽  
Calpain Inhibitors

SummaryActin-binding protein (ABP) is degraded into fragments of 190 and 90 kDa by calpain. A monoclonal antibody (MAb TI10) against the 90 kDa fragment of ABP coprecipitated with the glycoprotein lb (GP lb) peak observed on crossed immunoelectrophoresis of Triton X-100 extracts of platelets prepared without calpain inhibitors. MAb PM6/317 against the 190 kDa fragment was not coprecipitated with the GP lb peak under such conditions. The 90 kDa fragment was adsorbed on protein A agarose from extracts that had been preincubated with antibodies to GP lb. This supports the idea that the GP Ib-ABP interaction resides in the 90 kDa region of ABP. GP lb was sedimented with the Triton-insoluble actin filaments in trace amounts only, and only after high speed centrifugation (100,000 × g, 3 h). Both the 190 kDa and the 90 kDa fragments of ABP were sedimented with the Triton-insoluble actin filaments.

scholarly journals Production of Porous Ceramic Materials from Spent Fluorescent Lamps

2021 ◽  
Vol 11 (13) ◽  
pp. 6056
Author(s):  
Egle Rosson ◽  
Acacio Rincón Rincón Romero ◽  
Denis Badocco ◽  
Federico Zorzi ◽  
Paolo Sgarbossa ◽  
...  
Keyword(s):  
Rare Earth ◽  
Rare Earth Elements ◽  
High Speed ◽  
Porous Ceramic ◽  
Ceramic Materials ◽  
Porous Ceramics ◽  
Fluorescent Lamps ◽  
Naoh Solution ◽  
Commercial Glass ◽  
Triton X

Spent fluorescent lamps (SFL) are classified as hazardous materials in the European Waste Catalogue, which includes residues from various hi-tech devices. The most common end-of-life treatment of SFL consists in the recovery of rare earth elements from the phosphor powders, with associated problems in the management of the glass residues, which are usually landfilled. This study involves the manufacturing of porous ceramics from both the coarse glass-rich fraction and the phosphor-enriched fraction of spent fluorescent lamps. These porous materials, realizing the immobilization of Rare Earth Elements (REEs) within a glass matrix, are suggested for application in buildings as thermal and acoustic insulators. The proposed process is characterized by: (i) alkaline activation (2.5 M or 1 M NaOH aqueous solution); (ii) pre-curing at 75 °C; (iii) the addition of a surfactant (Triton X-100) for foaming at high-speed stirring; (iv) curing at 45 °C; (v) viscous flow sintering at 700 °C. All the final porous ceramics present a limited metal leaching and, in particular, the coarse glass fraction activated with 2.5 M NaOH solution leads to materials comparable to commercial glass foams in terms of mechanical properties.

scholarly journals Characterization of glycoconjugates of human gastrointestinal mucosa by lectins. II. Lectin binding to the isolated glycoproteins of normal and malignant gastric mucosa.

1984 ◽  
Vol 32 (7) ◽  
pp. 690-696 ◽  
Author(s):  
J Fischer ◽  
G Uhlenbruck ◽  
P J Klein ◽  
M Vierbuchen ◽  
R Fischer
Keyword(s):  
Molecular Weight ◽  
Gastric Mucosa ◽  
High Molecular Weight ◽  
Lectin Binding ◽  
Gel Filtration ◽  
Molar Ratio ◽  
Gastric Cancer Cells ◽  
Tissue Sections ◽  
Gradient Gel Electrophoresis ◽  
Triton X

Using affinity chromatography on HPA-, PNA-, Con A, and WGA-agarose columns only a part (10-30%) of the high molecular weight mucous glycoproteins could be isolated from the Triton X-100 solubilized components of normal as well as carcinomatous gastric mucosa. The main part of the mucus was not bound by the lectins, which corresponds to our earlier lectin histochemical observations on paraffin-embedded tissue sections. The lectin-bound mucous glycoproteins had a relatively lower molecular weight, ranging from about 250-1,000 kilodaltons, as indicated by polyacrylamide gradient gel electrophoresis and by gel filtration on Biogel A 1.5 m column. In gas chromatographic analysis the molar ratio of aminohexoses to galactose was found to be much higher (3:1) in the lectin-bound mucous substances than in the whole high molecular weight mucus (1:1). This finding indicates that lectins have a higher affinity to the hexosamine rich components of mucus, which may be special forms of mucous glycoprotein molecules or the incompletely glycosylated core and backbone regions of the oligosaccharide chains of mucus. Extremely high hexosamine values (10:1) were found in the PNA isolated mucus of gastric adenocarcinoma. Since it is known that PNA binds to the terminal disaccharide, beta-galactose-(1-3)-N-acetylgalactosamine, which is localized at the reducing end of the oligosaccharide chains of mucus, it is highly probable that the elongation of the oligosaccharide side chains is disturbed in gastric cancer cells.

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