Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A mixed micellar assay for the binding of phorbol-esters to protein kinase C was developed to investigate the specificity and stoichiometry of phospholipid cofactor dependence and oligomeric state of protein kinase C (Ca2+/phospholipid-dependent enzyme) required for phorbol ester binding. [3H]Phorbol dibutyrate was bound to protein kinase C in the presence of Triton X-100 mixed micelles containing 20 mol % phosphatidylserine (PS) in a calcium-dependent manner with a Kd of 5 X 10(-9) M. The [3H]phorbol dibutyrate X protein kinase C . Triton X-100 . PS mixed micellar complex eluted on a Sephacryl S-200 molecular sieve at an Mr of approximately 200,000; this demonstrates that monomeric protein kinase C binds phorbol dibutyrate. This conclusion was supported by molecular sieve chromatography of a similar complex where Triton X-100 was replaced with beta-octylglucoside. Phorbol dibutyrate activation of protein kinase C in Triton X-100/PS mixed micelles occurred and was dependent on calcium. The PS dependence of both phorbol ester activation and binding to protein kinase C lagged initially and then was highly cooperative. The minimal mole per cent PS required was strongly dependent on the concentration of phorbol dibutyrate or phorbol myristic acetate employed. Even at the highest concentration of phorbol ester tested, a minimum of 3 mol % PS was required; this indicates that approximately four molecules of PS are required. [3H]Phorbol dibutyrate binding was independent of micelle number at 20 mol % PS. The phospholipid dependencies of phorbol ester binding and activation were similar, with PS being the most effective; anionic phospholipids (cardiolipin, phosphatidic acid, and phosphatidylglycerol were less effective, whereas phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin did not support binding or activation. sn-1,2-Dioleoylglycerol displaced [3H]phorbol dibutyrate quantitatively and competitively. The data are discussed in relation to a molecular model of protein kinase C activation.
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PMID:Phorbol ester binding and activation of protein kinase C on triton X-100 mixed micelles containing phosphatidylserine. 345 28

Sphingosine inhibited protein kinase C activity and phorbol dibutyrate binding. When the mechanism of inhibition of activity and phorbol dibutyrate binding was investigated in vitro using Triton X-100 mixed micellar methods, sphingosine inhibition was subject to surface dilution; 50% inhibition occurred when sphingosine was equimolar with sn-1,2-dioleoylglycerol (diC18:1) or 40% of the phosphatidylserine (PS) present. Sphingosine inhibition was modulated by Ca2+ and by the mole percent of diC18:1 and PS present. Sphingosine was a competitive inhibitor with respect to diC18:1, phorbol dibutyrate, and Ca2+. Increasing levels of PS markedly reduced inhibition by sphingosine. Since protein kinase C activity shows a cooperative dependence on PS, the kinetic analysis of competitive inhibition was only suggestive. Sphingosine inhibited phorbol dibutyrate binding to protein kinase C but did not cause protein kinase C to dissociate from the mixed micelle surface. Sphingosine addition to human platelets blocked thrombin and sn-1,2-dioctanoylglycerol-dependent phosphorylation of the 40-kDa (47 kDa) dalton protein. Moreover, sphingosine was subject to surface dilution in platelets. The mechanism of sphingosine inhibition is discussed in relation to a previously proposed model of protein kinase C activation. The possible physiological role of sphingosine as a negative effector of protein kinase C is suggested and a plausible cycle for its generation is presented. The potential physiological significance of sphingosine inhibition of protein kinase C is further established in accompanying papers on HL-60 cells (Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A., Bell, R. M., Kinkade, J. M., Jr. (1986) J. Biol. Chem. 261, 12010-12615) and human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). These results also suggest that sphingosine will be a useful inhibitor for investigating the function of protein kinase C in vitro and in living cells.
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PMID:Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets. 346 88

The hydrophobic, membrane-binding domain of purified human erythrocyte acetylcholinesterase was labeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. The radiolabel was incorporated when the enzyme was prepared in detergent-free aggregates, in detergent micelles, or in phospholipid liposomes, but the highest percentage of labeling occurred in the detergent-free aggregates. Papain digestion of the enzyme released the hydrophobic domain, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate or gel exclusion chromatography demonstrated that the label was localized exclusively in the cleaved hydrophobic domain fragment. This fragment was purified in a three-step procedure. Digestion was conducted with papain attached to Sepharose CL-4B, and the supernatant was adsorbed to acridinium affinity resin to remove the hydrophilic enzyme fragment. The nonretained fragment associated with Triton X-100 micelles was then chromatographed on Sepharose CL-6B, and finally detergent was removed by chromatography on Sephadex LH-60 in an ethanol-formic acid solvent. The fragment exhibited an apparent molecular weight of 3100 on the Sephadex LH-60 column when compared with peptide standards. However, amino acid analysis of the purified fragment revealed only 1 mol each of histidine and glycine per mole of fragment in contrast to the 25-30 mole of amino acids expected on the basis of the molecular weight estimate. This result suggests a novel non-amino acid structure for the hydrophobic domain of human erythrocyte acetylcholinesterase.
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PMID:Selective radiolabeling and isolation of the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase. 352 70

Human KB cells produce two immunologically cross-reactive folate-binding proteins: a particulate cell-associated protein which is solubilized by Triton X-100, and a soluble protein which is released into their growth medium. This compartmentation of these two folate-binding proteins provides a convenient system for studies of their biochemical relationship. The two folate-binding proteins behave similarly to the purified particulate and soluble folate-binding proteins of human milk in analysis by radioactive folate binding, Sephacryl S-200 gel filtration profiles, polyacrylamide gel electrophoresis in either Triton X-100 or sodium dodecyl sulfate, and in Triton X-100 binding based on sucrose density gradient ultracentrifugation in H2O and D2O. The two folate-binding proteins were endogenously labeled by pulsing methionine-starved KB cells with [35S]methionine, and each protein was purified to apparent homogeneity by affinity chromatography at different times during the chase with nonradioactive methionine. The time course of the changes in specific activity (moles of [35S]methionine per mole of folate-binding protein) revealed a more rapid initial rate of synthesis and an earlier maximum in specific activity for the cell-associated folate-binding protein than for the soluble folate-binding protein released into the growth medium. Differences in the levels and specific activities of the two folate-binding proteins of cells exposed to cycloheximide compared with simultaneous controls after pulsing with [35S]methionine suggest that, whereas the cell-associated folate-binding protein is probably produced by de novo protein synthesis, the soluble folate-binding protein seems to be produced from a cellular pool of an already synthesized protein. These results combined with the immunologic cross-reactivity of the two folate-binding proteins strongly suggest a precursor-product relationship between them.
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PMID:The interrelationship of the soluble and membrane-associated folate-binding proteins in human KB cells. 353 8

Trehalase (alpha, alpha-trehalase, EC 3.2.1.28) was solubilized from the brush border membrane of pig kidney cortex by Triton X-100 and sodium deoxycholate in the presence of inhibitors of proteolytic enzymes. The kidney enzyme was purified 3060-fold using gel filtration, ion exchange chromatography, Con A-Sepharose chromatography, phenyl-Sepharose CL-4B hydrophobic interaction chromatography, Tris-Sepharose 6B affinity chromatography, and hydroxylapatite chromatography. Tris-Sepharose 6B was utilized to absorb contaminant proteins. Purity was estimated as 99% or greater, based on amino-terminal amino acid analysis. The purified enzyme had a specific activity of 278 units/mg protein, showed one major band after silver staining, and had an estimated molecular weight of 80,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was a glycoprotein and contained 2 mol of glucosamine per mole of trehalase. Kidney trehalase was inhibited by Tris, HgCl2, and phlorizin with Ki values of 3.8 mM, 11 microM, and 2.4 mM, respectively. Inclusion of Cl- in the reaction mixture protected the enzyme from inactivation by HgCl2. The apparent Km for trehalose was calculated to be 2.1 mM. Kidney trehalase was highly specific for trehalose and exhibited an optimal pH of 5.9. The isoelectric point was between pH 4.7 and 4.4, as measured by chromatofocusing.
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PMID:Purification and properties of detergent-solubilized pig kidney trehalase. 359 57

The phospholipid, sn-1,2-diacylglycerol, and calcium dependencies of rat brain protein kinase C were investigated with a mixed micellar assay (Hannun, Y., Loomis, C., and Bell, R.M. (1985) J. Biol. Chem. 260, 10039-10043). Protein kinase C activity was independent of the number of Triton X-100, phosphatidylserine (PS), and sn-1,2-dioleoylglycerol (diC18:1) mixed micelles. Activation was strongly dependent on the mole per cent of PS and diC18:1. Activity of protein kinase C was dependent on PS, diC18:1, and calcium in mixed micelles prepared from detergents other than Triton X-100. This is consistent with the micelle providing an inert surface into which the lipid cofactors partition. Molecular sieve chromatography provided direct evidence for the homogeneity of Triton X-100, PS, and diC18:1 mixed micelles. Mixing studies and surface dilution studies indicated that PS and diC18:1 rapidly equilibrate among the mixed micelles. At saturating calcium, the diC18:1 dependence was strongly dependent on the mole per cent PS present. At 10 mol % PS, 0.25 mol % diC18:1 gave maximal activity whereas 6 mol % PS and 6 mol % diC18:1 did not give maximal activity. diC18:1 dependencies were hyperbolic at all PS levels tested. The data support the conclusion that a single molecule of diC18:1/micelle is sufficient to activate monomeric protein kinase C. The mole per cent PS required for maximal activation was reduced markedly as the mole per cent diC18:1 increased. Under all conditions tested, the PS dependence of protein kinase C activation lagged until greater than 3 mol % PS was present. Then activation occurred in a cooperative manner with Hill numbers near 4. These data indicate that 4 or more molecules of PS are required to activate monomeric protein kinase C. PS was the most effective of all the phospholipids tested in the mixed micelle assay. diC18:1 was found to modulate the amount of calcium required for maximal activity. As the level of Ca2+ increased, the mole per cent PS required reached a limiting value of 3 mol %. A number of sn-1,2-diacylglycerols containing short chain fatty acids activated protein kinase C in a saturable manner in mixed micelles. The data are discussed in relation to a model for protein kinase activation.
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PMID:Protein kinase C activation in mixed micelles. Mechanistic implications of phospholipid, diacylglycerol, and calcium interdependencies. 371 Oct 83

The plasma membrane of bovine aortic endothelium was isolated, characterized, and found to contain at least four membrane-associated cytoskeletal proteins. Exposure of the plasma membranes to salt media (up to 1M KCl) resulted in the release of 30% of the total plasma membrane-associated proteins and extraction with 1% Triton X-100, 60%. At least four heavily glycosylated bands (185-, 165-, 150-, and 130,000 mol-wt) were evident. The Triton-insoluble pellet fraction contained several major polypeptides (30-, 43-, 58-, and 240,000 mol-wt), two of which were identified by immunoblotting as cytoplasmic actin (43,000 mol-st) and vimentin (58,000 mol-wt). Strikingly, vimentin and a 240,000 mol-wt polypeptide were routinely present in approximately a mole ratio of 4:1 in more than 60% of the plasma membrane preparations. We also report the presence of a 2.1-like and a 4.1-like protein associated with plasma membranes. The 2.1-like protein demonstrated similar solubilities and apparent molecular weight (210,000) as erythroid protein 2.1. Likewise, the endothelial 4.1-like protein exhibited similar solubilities and apparent molecular weight as erythroid protein 4.1. Immunofluorescence staining of fixed and permeabilized cultures with anti-2.1 antibodies showed a fibrillar pattern. In contrast, cells stained with anti-protein 4.1 were brightly fluorescent, bearing both a diffuse and punctate pattern. This paper presents several novel observations pertaining to the composition of bovine aortic endothelial cell plasma membranes, namely: the presence of two erythroid-like cytoskeletal polypeptides; the presence of vimentin and a 240,000 mol-wt polypeptide in a 4:1 mole ratio in more than 60% of the plasma membrane preparations and the co-elution in a 4:1 mol ratio with a protein perturbant; and the inability to release actin from the plasma membrane preparations, suggesting the association of actin with other molecules in the plasma membrane preparation.
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PMID:Isolation of bovine aortic endothelial cell plasma membranes: identification of membrane-associated cytoskeletal proteins. 373 85

The interaction of the nonionic detergent Triton X-100 with phospholipid bilayers of liposomes made of egg yolk phosphatidylcholine was studied through the behavior of several physical properties. The dielectric permittivity spectra between 30 kHz and 13 MHz, the viscosity, the density, and the d.c. conductivity (1 kHz) of aqueous liposomes suspensions at various mole ratios were measured at 22 degrees C. For detergent-to-phospholipid ratios lower than 3, a dielectric relaxation process of characteristic frequency of about 50 kHz was recorded. This process does not appear for the liposomes in water, and becomes smaller and smaller for detergent-to-phospholipid ratios higher than 3. The viscosity of these suspensions showed a biphasic behavior, being remarkably increased by the detergent for concentration ratios lower than 3. The measured d.c. conductivity of these samples showed no relation with this process, being slightly increased when the detergent content is increased. As a conclusion of these results a well defined concentration range appears where the phospholipid organization changes forming highly asymmetrical structures.
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PMID:Effect of Triton X-100 on the physical properties of liposomes. 379 May 61

Purified human placental insulin receptors were incorporated into small unilamellar phospholipid vesicles by the addition of n-octyl beta-glucopyranoside solubilized phospholipids, followed by removal of the detergent on a Sephadex G-50 gel filtration column and extensive dialysis. The vesicles have an average diameter of 142 +/- 24 nm by Sephacryl S-1000 gel filtration chromatography and 119 +/- 20 nm by transmission electron microscopy. These vesicles are impermeant to small molecules as indicated by their ability to retain [gamma-32P]ATP, which could be released by the addition of 0.05% Triton X-100. Detergent permeabilization or freeze-thawing of the insulin receptor containing vesicles in the presence of 125I-insulin indicated that approximately 75% of the insulin binding sites were oriented right side out (extravesicularly). Sucrose gradient centrifugation of insulin receptors incorporated at various protein to phospholipid mole ratios demonstrated that the insulin receptors were inserted into the phospholipid bilayer structure in a concentration-dependent manner. Addition of [gamma-32P]ATP to the insulin receptor containing vesicles was relatively ineffective in promoting the autophosphorylation of the beta subunit in the absence or presence of insulin. Permeabilization of the vesicles with low detergent concentrations, however, stimulated the beta-subunit autophosphorylation approximately 2-fold in the absence and 10-fold in the presence of insulin. Insulin-stimulated beta-subunit autophosphorylation was also observed under conditions such that 94% of those vesicles containing insulin receptors had a single receptor per vesicle, suggesting that the initial beta-subunit autophosphorylating activity is intramolecular. Phospho amino acid analysis of the vesicle-incorporated insulin receptors demonstrated that the basal and insulin-stimulated beta-subunit autophosphorylation occurs exclusively on tyrosine residues. It is concluded that when purified insulin receptors are incorporated into a phospholipid bilayer, they insert into the vesicles primarily in the same orientation as occurs in the plasma membrane of intact cells and retain insulin binding as well as insulin-stimulated beta-subunit autophosphorylating activities.
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PMID:Incorporation of the purified human placental insulin receptor into phospholipid vesicles. 408 39

Oxidation of NADH by rat erythrocyte plasma membrane was stimulated by about 50-fold on addition of decavanadate, but not other forms of vanadate like orthovanadate, metavanadate aad vanadyl sulphate. The vanadate-stimulated activity was observed only in phosphate buffer while other buffers like Tris, acetate, borate and Hepes were ineffective. Oxygen was consumed during the oxidation of NADH and the products were found to be NAD+ and hydrogen peroxide. The reaction had a stoichiometry of one mole of oxygen consumption and one mole of H2O2 production for every mole of NADH that was oxidized. Superoxide dismutase and manganous inhibited the activity indicating the involvement of superoxide anions. Electron spin resonance in the presence of a spin trap, 5, 5'-dimethyl pyrroline N-oxide, indicated the presence of superoxide radicals. Electron spin resonance studies also showed the appearance of VIV species by reduction of VV of decavanadate indicating thereby participation of vanadate in the redox reaction. Under the conditions of the assay, vanadate did not stimulate lipid peroxidation in erythrocyte membranes. Extracts from lipid-free preparations of the erythrocyte membrane showed full activity. This ruled out the possibility of oxygen uptake through lipid peroxidation. The vanadate-stimulated NADH oxidation activity could be partially solubilized by treating erythrocyte membranes either with Triton X-100 or sodium cholate. Partially purified enzyme obtained by extraction with cholate and fractionation by ammonium sulphate and DEAE-Sephadex was found to be unstable.
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PMID:A vanadate-stimulated NADH oxidase in erythrocyte membrane generates hydrogen peroxide. 608 22


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