Search PubMed⌕ Search

Biomedical subjects

N Sahyoun

Publications and source records attributed to N Sahyoun.

At least 55 records · Page 3Linked to original sources
PubMed1985

Cytoskeletal calmodulin-dependent protein kinase. Characterization, solubilization, and purification from rat brain.

Brain calmodulin-dependent protein kinase was isolated as the major cytoskeletal component of the post-synaptosomal fraction, accounting for about 50% of the total protein. The alpha- and beta-subunits of the enzyme were present in a ratio of about 6:1. The cytoskeletal preparation was also distinguished by the regular presence of tubulin and actin in approximately equimolar amounts to the beta-subunit. Electron microscopic morphology suggested that the enzyme formed a hierarchy of cytoskeletal complexes with an average diameter of 85 nm. These results indicate that cytoskeletal calmodulin-dependent protein kinase may have a structural as well as a catalytic role in neurons. The cytoskeletal enzyme could be solubilized in 8 M urea, displaying similar properties to its cytosolic counterpart. The postsynaptosomal cytoskeleton provides a simplified and well-defined model for the study of the protein-protein interactions involving calmodulin-dependent protein kinase.

Animals↗
PubMed1984

Protein kinase C: rapid enzyme purification and substrate-dependence of the diacylglycerol effect.

Protein kinase C has been purified by a rapid method resulting in a high-yield, stable enzyme preparation. The catalytic and regulatory properties of this enzyme preparation were characterized employing histone H1 and HMG8, a proteolytic fragment of H1. The enzyme had a lower Km for HMG8, and was stimulated more effectively by diacylglycerol and phorbol esters in the presence of this substrate. Furthermore, these activators markedly increased the Km for HMG8 but not for H1. Protein kinase C and cyclic AMP-dependent protein kinase phosphorylate serine residues which are located in different, single tryptic peptides from HMG8.

Animals↗
PubMed1984

Ca2+-calmodulin-dependent protein kinase in neuronal nuclei.

Ca2+-calmodulin-dependent protein kinase represents a major enzyme activity in neuronal nuclei. Most of the protein kinase activity is associated with the nuclear matrix but it can phosphorylate a broad range of substrates which derive from different nuclear compartments. Notable among these substrates is a Mr = 50,000 component of the nuclear matrix and a high mobility group chromatin protein which behaves like HMG17. The high level of activity, age- and tissue-specificity, and sensitivity to low concentrations of calmodulin suggest a role for this Ca2+-calmodulin-dependent protein kinase in regulating neuronal function at the level of the nucleus.

Age Factors↗
PubMed1984

Specificity of a phosphatase for phospholipid, Ca2+-dependent protein kinase-phosphorylated histone H1 resides in the catalytic subunit.

A protein phosphatase from liver which acts preferentially on histone phosphorylated with phospholipid, Ca2+-dependent protein kinase has been purified and the intrinsic specificity determined to reside in the catalytic subunit of the enzyme complex. Comparison with a preparation of pork heart protein phosphatase suggests that this specificity may be a general property of a class of protein phosphatases. Protein kinase C-phosphorylated histone H1 represents an improved substrate for phosphatase detection providing a five to tenfold greater sensitivity than other substrates including cAMP-dependent protein kinase phosphorylated H1.

Animals↗
PubMed1984

Ca2+/calmodulin-dependent protein kinases from the neuronal nuclear matrix and post-synaptic density are structurally related.

A major Ca2+/calmodulin-dependent protein kinase has been isolated in association with the neuronal nuclear matrix. Nuclear matrix preparations contain highly phosphorylated polypeptides with Mr values of 50,000 and 60,000. These polypeptides were further characterized by peptide and phospho peptide mapping, two-dimensional isoelectrofocusing/NaDodSO4/PAGE, and 125I-labeled calmodulin binding. The results indicate that the Mr 50,000 and 60,000 polypeptides of the nuclear matrix closely resemble the alpha and beta subunits, respectively, of the Ca2+/calmodulin-dependent protein kinase of the post-synaptic density. These findings indicate that similar protein kinases mediate the neuronal effects of Ca2+ at the cytosolic, synaptosomal, and nuclear levels.

Animals↗
PubMed1983

Histones H3 and H4 inhibit protein kinase C specifically.

The lysine-rich histone H1 is a preferred substrate for the Ca2+-phospholipid-dependent protein kinase (protein kinase C). Histones H3 and H4 are poor substrates but potent inhibitors of the enzyme. The inhibitory effect of H3 and H4 seems to result mainly from a decreased sensitivity of protein kinase C to stimulation by phosphatidylserine (PS). These observations suggest that site-specific phosphorylation of one histone type can be regulated by other histones.

Animals↗
PubMed1983

Cytosolic activator of adenylate cyclase: Reconstitution, characterization, and mechanism of action.

Rat liver and isolated hepatocytes contain high levels of a soluble adenylate cyclase stimulator, whereas rat erythrocytes lack this activity. Accordingly, a reconstitution system was developed with adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from erythrocyte ghosts and the soluble activator from liver cytosol. Pretreatment of erythrocyte ghosts with the cytosolic factor resulted in a 5- to 15-fold activation of adenylate cyclase in the presence or absence of NaF, 5'-guanylyl imidodiphosphate, or isoproterenol and GTP. The sequence of addition of the cytosolic component and the other activators was critical in determining the maximal activity of the enzyme. The cytosolic factor appears to be a heat-labile Mr 105,000 protein, which activates adenylate cyclase in a saturable reaction involving binding of the protein to the erythrocyte ghosts. This molecular interaction was accompanied by stabilization of a labile thiol group that was essential for catalytic activity. The cytosolic component also unmasks latent adenylate cyclase activity in human erythrocyte ghosts and in cytoskeletal preparations from rat erythrocyte ghosts. These observations suggest that the cytosolic activator may also occur as a native, peripheral membrane component of adenylate cyclase systems and may be required for the expression and stabilization of catalytic activity.

Adenylyl Cyclases↗
PubMed1983

A specific phosphoprotein phosphatase acts on histone H1 phosphorylated by protein kinase C.

A phosphohistone phosphatase from rat liver cytosol acts specifically on histone H1 that is phosphorylated with the Ca2+-phospholipid-dependent protein kinase (protein kinase C). The apparent Km for 32P-labeled H1 is 1 microM; other histones or cytosolic proteins phosphorylated with protein kinase C or with cyclic AMP-dependent protein kinase are poor substrates for the phosphatase. The enzyme has been partially purified by gel-permeation chromatography and by utilizing a high-performance liquid chromatography ion-exchange column. The physical properties of this enzyme include a Stokes radius of 5.0 nm, a sedimentation coefficient (s20,w) of 7.0 S, and a Mr of 150,000. The detection of protein kinase as well as the specific phosphohistone phosphatase in purified rat liver nuclei suggests a physiologic role for a histone H1 phosphorylation-dephosphorylation cycle mediated by protein kinase C and the corresponding phosphohistone phosphatase.

Animals↗
PubMed1982

Formation and identification of cytoskeletal components from liver cytosolic precursors.

Liver cytosol forms a macroscopic fibrillary network in the presence of low concentrations of MgCl2. This process represents the generation of 3- to 11-nm filaments from soluble precursors, involving selectively at least 12 major polypeptides. Similar polypeptides are enriched in the detergent-insoluble fraction from hepatocytes, suggesting that they may be important constituents of the native cytoskeleton. AcA 34 gel-permeation chromatography resolves the cytosol into three independently "polymerizing" peaks: A, B, and C. The formation of filaments follows biphasic kinetics in peaks B and C, whereas peak A lacks the slow phase. Filament formation in all three systems is inhibited by 1-15 mM inorganic phosphate, 10 mM NaF, or 10 mM sodium molybdate. The polymerization of peak C only is inhibited by 0.2-2 mM ATP. CaCl2 (1-100 microM) has no apparent regulatory effect. Two-dimensional polypeptide analysis and peptide mapping show that actin is a major component of peak C, while peaks A and B contain prominent polypeptides that may be related to intermediate filament subunits. In addition, all three systems contain two or three high molecular weight (greater than 170,000) polypeptides that may participate in modulating and extending the filament network. The filaments from peaks A and B are soluble in 8 M urea and reform on removal of the urea in the presence of 5 mM MgCl2. The polypeptide composition remains constant through three such cycles.

Actins↗
PubMed1981

Tissue glutathione as a cyst(e)ine reservoir during fasting and refeeding of rats.

Previous reports suggest that liver glutathione serves as a cyst(e)ine reservoir in both starved rats and rats fed a cyst(e)ine-deficient diet. We investigated whether glutathione concentrations in other tissues also decrease and whether component amino acids of glutathione increase under such conditions. Tissue glutathione and free amino acid concentrations were measured in six groups of growing rats (120-150 g; four animals/group) during fasting and refeeding regimens. All animals were fed a rat stock diet ad libitum for 5 days. Two groups were than fasted for 24 or 48 hours, respectively. Other groups were fasted for 48 hours and then refed for 10, 24 or 48 hours respectively the stock diet. Significant changes (P less than or equal to 0.05) in liver and intestine glutathione concentrations were noted with 24 and 48-hour fasting, and a 24-hour refeeding was necessary to return concentrations to prefasting values. Muscle glutathione concentrations decreased slightly with fasting, but remained low with refeeding. Erythrocyte glutathione concentrations were independent of dietary treatment. Liver cysteine and glycine concentrations increased after 24 hours' fasting, but glutamate levels were not affected. The data indicate that liver and intestine glutathione serve as cyst(e)ine reservoirs during fasting. Since erythrocyte and muscle glutathione levels did not decrease, levels in these tissues cannot be used to measure cyst(e)ine status in premature infants fed parenterally.

Animals↗
PubMed1978

Insulin and epidermal growth factor-urogastrone: affinity crosslinking to specific binding sites in rat liver membranes.

Both insulin and human epidermal growth factor-urogastrone (EGF/URO) can be covalently linked to specific rat liver membrane binding sites by glutaraldehyde coupling followed by sodium borohydride reduction to yield affinity-labeled membrane constituents sufficiently stable for solubilization and further analysis by various techniques. Solubilization of membranes covalently labeled with (125)I-labeled insulin yields a component with chromatographic properties identical to those of a soluble insulin receptor characterized in previous studies. A second soluble insulin-binding component that is not revealed by the affinity-labeling method and that has not yet been reported can also be detected. Membranes similarly labeled with (125)I-labeled EGF/URO yield one major and two minor ligand-specific soluble (Triton X-100) affinity-labeled components, as detected by chromatography on Sepharose 6B. Further analysis of the EGF/URO-labeled components by affinity chromatography on concanavalin A-Sepharose, by disc gel electrophoresis, and by enzymatic digestion suggests that the major specific binding component for EGF/URO in liver membranes is a glycoprotein subunit of approximately 100,000 daltons that possesses a 20,000-dalton portion inaccessible to proteolytic cleavage when the subunit is anchored in the membrane. The affinity labeling approach described should prove of use for the study of other polypeptide receptors that, like the EGF/URO receptor, lose their ligand recognition property subsequent to membrane solubilization.

Affinity Labels↗
PubMed1978

Properties of the interaction of fluoride- and guanylyl-5'-imidodiphosphate-regulatory proteins with adenylate cyclase.

The mechanism of activation of adenylate cyclase by guanylyl-5'-imidodiphosphate [Gpp(NH)p] and NaF has been investigated by studying the reconstitution of Gpp(NH)p and NaF sensitivity of an enzyme rendered insensitive to these agents by differential detergent extraction of a particulate brain enzyme. Such reconstitution can be achieved by the addition of macromolecular regulatory factors from membranes of various tissues. Trypsin digestion and thermal inactivation provide evidence for the existence of two distinct regulatory functions, one capable of restoring the Gpp(NH)p response and another the NaF response. The regulatory protein(s) seem to interact with their respective activators in an easily reversible, divalent cation-independent reaction. This appears to be followed by a high-affinity interaction between the catalytic and regulatory components of adenylate cyclase in a slow, temperature-dependent, divalent cation-dependent process tha produces the persistently activated state of the enzyme. The enzyme activation can be reversed by methods that separate catalytic from regulatory components and the resulting enzyme activity can be restimulated by the reconstitution technique.

Adenylyl Cyclases↗
PubMed1977

Topographic separation of adenylate cyclase and hormone receptors in the plasma membrane of toad erythrocyte ghosts.

Brief sonication of whole erythrocyte plasma membranes (ghosts) from toads at 4 degrees does not inactivate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] or destroy the receptor binding properties of hydroxybenzylpindolol or insulin. The hormonal (but not the fluoride-induced) stimulation of this enzyme is, however, lost. Fractionation of the small, resealed membrane fragments (vesicles) on discontinuous sucrose gradients results in the separation of vesicle populations differing grossly in size and protein composition. In addition, the distribution of the beta-adrenergic receptor, an insulin binding site, and adenylate cyclase among these vesicles fractions differs. The pattern of distribution of these functional structures can be altered differentially by manipulations of the ghosts before sonication. For example, brief preincubation with isoproterenol leads to a change in the relative distribution of beta-receptor (but not adenylate cyclase) among the various vesicle fractions; this effect is not obtained with beta-receptor antagonists, which block the isoproterenol effect. Exposure of the ghosts to different temperatures, changes in the divalent cation composition of the medium, or the addition of ATP also leads to changes in the distribution of surface markers of the subsequently formed vesicles. The results indicate gross asymmetries in the distribution of protein components within the plane of the membrane and raise important questions regarding the manner whereby functionally related and coupled components, such as hormone receptors and adenylate cyclase, interact.

Adenosine Triphosphate↗
PubMed1976

Structure and function of cholera toxin and hormone receptors.

The enterotoxin from Vibrio cholerae is a protein of 100,000 mol wt which stimulates adenylate cyclase activity ubiquitously. The binding of biologically active 125I-labeled choleragen to cell membranes is of extraordinary affinity and specificity. The binding may be restricted to membrane-bound ganglioside GM1. This ganglioside can be inserted into membranes from exogenous sources, and the increased toxin binding in such cells can be reflected by an increased sensitivity to the biological effects of the toxin. Features of the toxin-activated adenylate cyclase, including conversion of the enzyne to a GTP-sensitive state, and the increased sensitivity of activation by hormones, suggest analogies between the basic mechanism of action of choleragen and the events following binding of hormones to their receptors. The action of the toxin is probably not mediated through intermediary cytoplasmic events, suggesting that its effects are entirely due to processes involving the plasma membrane. The kinetics of activation of adenylate cyclase in erythrocytes from various species as well as in rat adipocytes suggest a direct interaction between toxin and the cyclase enzyme which is difficult to reconcile with catalytic mechanisms of adenylate cyclase activation. Direct evidence for this can be obtained from the comigration of toxin radioactivity with adenylate cyclase activity when toxin-activated membranes are dissolved in detergents and chromatographed on gel filtration columns. Agarose derivatives containing the "active" subunit of the toxin can specifically absorb adenylate cyclase activity, and specific antibodies against the choleragen can be used for selective immunoprecipitation of adenylate cyclase activity from detergent-solubilized preparations of activated membranes. It is proposed that toxin action involves the initial formation of an inactive toxin-ganglioside complex which subsequently migrates and is somehow transformed into an active species which involves relocation within the two-dimensional structure of the membrane with direct perturbation of adenylate cyclase molecules (virtually irreversibly). These studies suggest new insights into the normal mechanisms by which hormone receptors modify membrane functions.

Adenylyl Cyclases↗