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K Meflah

Publications and source records attributed to K Meflah.

85 records · Page 5Linked to original sources
PubMed1989

Differences in the release of 5'-nucleotidase and alkaline phosphatase from plasma membrane of several cell types by PI-PLC.

1. We have compared the effect of phosphatidyl inositol specific phospholipase C (PI-PLC) on the attachment of both 5'-nucleotidase and alkaline phosphatase to the liver plasma membrane from different species. 2. Our results demonstrate differences in the susceptibilities of both enzymes to PI-PLC treatment in relation to their origin. 3. These results were confirmed by immunoblotting using polyclonal anti-5'-nucleotidase antibodies. 4. In addition, in a single animal, susceptibility of both enzymes to PI-PLC treatment is different from one tissue to another. 5. The different percentages of released enzymes could be explained either by a polymorphism in the anchoring of these proteins at the cell surface membrane, or by a different steric hindrance or environment at the cleavage site itself.

5'-Nucleotidase↗
PubMed1988

Purification of bovine liver cytosolic 5'-nucleotidase. Kinetic and structural studies as compared to the membrane isoenzyme.

Cytosolic 5'-nucleotidase from bovine liver has been purified to homogeneity. Two affinity chromatographies on concanavalin A and 5'AMP-Sepharose columns result in a 12,000-fold purification. The sequential elution of glycoproteins from the concanavalin-A-Sepharose column with methyl alpha-D-glucoside and methyl alpha-D-mannoside greatly increases the degree of purification of the enzyme. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate shows two subunits having apparent molecular masses of 65 kDa and 57 kDa respectively, while only one band at 70 kDa is observed in the case of the membrane-bound 5'-nucleotidase. Both the Stokes radii, measured by gel exclusion HPLC, and the sedimentation coefficient, determined by density gradient ultracentrifugation, indicate that the cytosolic enzyme is a heterodimer of about 130 kDa. This contrasts with the membrane-bound 5'-nucleotidase which is a homodimer of 140 kDa. Moreover, the antibodies raised against the membrane 5'-nucleotidase inhibited the cytosolic form indicating that a common antigenic determinant(s) exists between the two isoenzymes. However, structural differences are revealed by immunoblotting. In the same way, the effect of lectins suggests that differences in the structure of the carbohydrate chains exist between the two isoenzymes. The purified cytosolic enzyme has lower affinity for the nucleotides than does the membrane enzyme. In addition, while ADP, [alpha,beta-CH2]ADP and ATP were strong competitive inhibitors of the membrane enzyme, ADP and ATP activate the cytosolic form and [alpha,beta-CH2]ADP has no effect. Moreover, two pH optima at 7.5 and 9.5 are observed in the cytosolic enzyme while only one at 7.5 occurred in the membrane form. Finally the exogenous cations, MgCl2 and MnCl2, are necessary for the maximal activity of the cytosolic but not of the membrane 5'-nucleotidase. All these observations indicate that the two isoenzymes are different.

5'-Nucleotidase↗
PubMed1988

The carbohydrate moiety of mineral-bound proteins from fetal enamel: a basis for enamelins heterogeneity.

Enamelins were prepared from the soft enamel of bovine fetuses. They were purified on synthetic hydroxyapatite and separated in two fractions by affinity chromatography on a ConA-ultrogel column. The two fractions were different with respect to their electrophoretic behavior, stainability, amino acid composition, phosphorylation, and glycosylation. The ConA-binding fraction, consisting of three molecular species with apparent molecular weights of 33, 37, and 45 kD, contained organic phosphorus and high levels of sugars. The Gal/Man ratio suggested a biantennary structure. The ConA-unbound fraction contained two major molecular species with molecular weights of 70 and 56 kD, and represented 70% of the total enamelin preparation. The amino acid composition of this fraction showed a higher level of alanine and a lower level of proline when compared with that of total enamelins. Its sugar composition was unusual, being principally constituted of N-acetyl galactosamine and N-acetyl glucosamine.

Amino Acids↗
PubMed1986

Chemical modification of essential carboxyl group and histidine residue in the plasma-membrane 5'-nucleotidase.

An investigation, using specific chemical reagents, of the amino acids involved in the catalytic activity of the purified 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) from bovine liver plasma membranes, was carried out. The enzyme was irreversibly inactivated by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The inhibition kinetics were of the first-order type and decreased partially in the presence of nucleotides and divalent cations. These results indicate for the first time that a carboxyl group is essential for the catalytic process of 5'-nucleotidase. Moreover, chemical modification by diethylpyrocarbonate also produced inactivation of the enzyme and showed a differential spectrum with a peak at 240 nm characteristic of N-carbethoxyhistidine residues. This inactivation was efficiently released upon decarbethoxylation by hydroxylamine only when the extent of inactivation, due to low concentration of diethylpyrocarbonate, was limited. The time-dependent inactivation followed first-order kinetics and nucleotides afforded significant protection against diethylpyrocarbonate modification. The results indicate the involvement of the histidine residue in catalysis.

5'-Nucleotidase↗
PubMed1985

Structural differences between plasma-membrane 5'-nucleotidase in different cell types as evidenced by antibodies.

Antibodies raised against bovine 5'-nucleotidase inhibit this enzyme as well as 5'-nucleotidase from other bovine tissues, showing common structure(s) between these proteins. However, an IgG fraction directed against the glucidic moiety of the liver enzyme did not cross-react with the enzyme from lymphocyte or caudate nuclei, a clear indication that within the same species the 5'-nucleotidase differs from one cell type to another. In addition, immunoblots after electrophoresis show that the previous antibodies recognize 5'-nucleotidase from human, mouse or chicken origin. However, only human 5'-nucleotidase activity can be inhibited by the antibodies. Thus at least three groups of antigenic determinants must exist on the 5'-nucleotidase: one related to the glucidic moiety of the glycoprotein whose binding inhibits the enzyme activity, another related to the catalytic site, as its binding also led to enzyme inhibition, and a last one of structural nature. It seems that the third group of determinant is common to many species, whereas the second one is more restricted.

5'-Nucleotidase↗
PubMed1984

Evidence for a metalloprotein structure of plasma membrane 5'-nucleotidase.

To point out the metalloprotein structure of bovine liver plasma membrane 5'-nucleotidase, we studied the inhibition mechanism of the purified enzyme by EDTA: this apparently non-competitive inhibition seems to be dependent on EDTA concentration, pH, temperature and incubation time. When the restoration of activity was assayed by addition of divalent cations or by gel filtration, the inhibition became progressively irreversible with time. Incubation of the enzyme with [14C]EDTA allowed us to observe, after gel filtration as well as after sucrose gradient ultracentrifugation, that the chelating agent is bound to 5'-nucleotidase.

5'-Nucleotidase↗
PubMed1984

5'-Nucleotidase from bovine caudate nucleus synaptic plasma membranes: specificity for substrates and cations; study of the carbohydrate moiety by glycosidases.

We studied 5'-nucleotidase in preparations of synaptic plasma membranes from bovine caudate nucleus. The best substrates for this membrane-bound enzyme were purine nucleotides, particularly 5'AMP. Effects of metal cations and chelating agents suggest that 5'-nucleotidase is a metalloprotein. Optimal conditions for solubilization of the 5'-nucleotidase were found by using a low concentration of the zwitterionic detergent sulfobetaine 14. In contrast, another membrane-bound enzyme, acetylcholinesterase, was not solubilized under these conditions, but only in the presence of Triton X-100. The effects of lectins (concanavalin A, Lens culinaris agglutinin, wheat germ agglutinin, and Limulus polyphemus agglutinin) showed that both enzymes are glycoproteins. Sequential hydrolysis with specific glycosidases produced modifications of the effect of lectins on these enzymes. The results suggest the presence of a complex-type glycosylation, with a fucose residue on the internal N-acetyl-D-glucosamine of the pentasaccharide core.

5'-Nucleotidase↗
PubMed1983

Purification and properties of bovine liver plasma membrane 5' nucleotidase.

5'-Nucleotidase from bovine liver plasma membranes has been extracted by the zwitterionic detergent sulfobetaine 14, and purified to apparent homogeneity. Two affinity chromatographies on concanavalin-A-Ultrogel and 5' AMP-Sepharose 4B followed by AcA-54-Ultrogel filtration resulted in a purification of 16000 times relative to the homogenate. Sodium dodecyl sulphate gel electrophoresis indicates that the apparent molecular weight of the subunit is 70000. Cross-linking of the native enzyme with dimethylpimelimidate followed by gel electrophoresis shows a band with an apparent molecular weight of 140000 indicating that the enzyme is a dimer. 5'-Nucleotidase is a glycoprotein and its activity is inhibited to different degrees by various lectins, indicating a direct interaction with the enzyme. The purified enzyme shows a sevenfold greater affinity for AMP than the membrane-bound enzyme. The optimum activity of the purified enzyme occurs at pH 7.5 while the membrane-bound enzyme showed a wide range of pH optimum (7.5-8.3). An Arrhenius plot of the membrane-bound enzyme shows a break at 28 degrees C, which disappears in the purified enzyme. The enzyme was inhibited by EDTA, and this inhibition was reversed by divalent cations. This, as well as other evidence, indicates that the enzyme contains a highly bound metal cation, perhaps Mn2+ or Mg2+.

5'-Nucleotidase↗
PubMedDate not supplied

Delineation of liver necrosis using double contrast-enhanced MRI.

The purpose of this study was to demonstrate the potential usefulness of the combination of gadolinium and dysprosium to enhance the different between normal and necrotic liver tissue. Small regions of acute necrosis were induced by injecting 200-300 microliters of 95% alcohol into the liver of 26 rats. MRI was performed 24 hours after necrosis induction, before and immediately after injection of one or both contrast agents. Using a mixed T1/T2-weighted sequence, the signal intensity of (SI) of the normal liver was reduced by 70%, whereas the necrotic regions had more than a 50% increase in SI after double contrast. The region that was enhanced corresponded largely with the region of necrosis as observed postmortem. The lesion size, when identified, was largely underestimated using either of the agents along, albeit using the common pulse sequences. The double contrast effect of simultaneous administration of gadolinium and dysprosium allows accurate delineation of liver necrosis.

Animals↗
PubMedDate not supplied

Induction of antigen presentation by macrophages after phagocytosis of tumour apoptotic cells.

Due to their resistance to classical chemotherapies, most human colorectal cancers have a high incidence and a poor prognosis. Immunotherapy using interleukin 2 (IL2) has provided disappointing results in the treatment of these cancers. Recently, however, we have demonstrated that a treatment combining a cell-differentiating agent, sodium butyrate (NaBut) with IL2 resulted in a remission of established peritoneal colorectal carcinomatosis in rats. Separately, neither NaBut nor IL2 treatment cured these tumour-bearing rats. NaBut is known to induce cell differentiation and subsequent apoptosis in epithelial cells, while IL2 stimulates the immune cells capable of participating in tumour rejection. We postulated that the significant therapeutic effect of NaBut/IL2 treatment could be attributed to a NaBut-induced increase in the immunogenicity of the cancer cells. We report here that NaBut induced an apoptotic process in rat colon tumour cells in vivo and in vitro. We observed, in an efficient cure, colocalization of apoptotic bodies and monocytes/macrophages at the periphery of the tumour. We propose that these apoptotic bodies are phagocytosed in vivo by the macrophages. We also showed in vitro that a subpopulation of macrophages involved in the phagocytic clearance of apoptotic cells expresses cell surface molecules associated with antigen presentation and stimulates the proliferation of naive splenocytes. Our data suggest that therapies that recruit massive induction of the apoptotic process in tumour cells could favour tumour antigen presentation via their specific phagocytosis by antigen-presenting cells (APCs). We propose that the development of specific therapies that stimulate both tumour cell apoptosis and the immune system could offer new opportunities in anti-cancer treatments of poorly immunogenic cancer cells.

Animals↗