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)

Treatment of relaxed skinned rabbit psoas muscle fibers with 0.1 mM N-phenylmaleimide (NPM) for 1 h locks all of the crossbridges in a weakly-binding state resembling that of the myosin.ATP crossbridge. Under these conditions, NPM reacts mainly with myosin heavy chain (Barnett et al. (1992) Biophys. J. 61, 358-367). Here the specific sites for that reaction are explored. Small bundles of rabbit psoas muscle fibers were treated with Triton X-100 to make the fiber sarcolemmas permeable. The bundles were treated with 0.1 mM [14C]NPM for 1 h, and homogenized for SDS-PAGE. 43 +/- 2.2% of the muscle fiber protein ran in the myosin heavy chain band, the same as for untreated fibers. An alkylating stoichiometry of 2.2 +/- 0.33 moles NPM per mole myosin heavy chain was determined. Exhaustive trypsin digestion followed by two-dimensional thin-layer chromatography and reverse-phase HPLC revealed two major sites on myosin heavy chain for NPM binding. The sites contained about the same amount of linked NPM, suggesting that the reaction stoichiometry of each site under the conditions studied is approx. 1 mol NPM/mol myosin heavy chain. Comparison of the labeled tryptic peptides with NPM-reacted synthetic SH1 and SH2 tryptic peptides and analysis of the treated fiber bundles' ATPase activity suggested that the sites for NPM reaction on myosin heavy chain when it locks crossbridges in a weakly-binding state are Cys-697 (SH2) and Cys-707 (SH1).
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PMID:The site and stoichiometry of the N-phenylmaleimide reaction with myosin when weakly-binding crossbridges are formed in skinned rabbit psoas fibers. 749 34

A trifluoromethyl ketone analogue of arachidonic acid in which the COOH group is replaced with COCF3 (AACOCF3) was prepared and found to be a tight- and slow-binding inhibitor of the 85-kDa cytosolic human phospholipase A2 (cPLA2). Enzyme inhibition was observed when AACOCF3 was tested in assays using either phospholipid vesicles or phospholipid/Triton X-100 mixed micelles. The fact that the inhibition developed over several minutes in both assays establishes that AACOCF3 inhibits by direct binding to the enzyme rather than by decreasing the fraction of enzyme bound to the substrate interface. From the measured values of the inhibitor association and dissociation rate constants, an upper limit of the equilibrium dissociation constant for the Ca(2+).AACOCF3.PLA2 complex of 5 x 10(-5) mole fraction was obtained. Thus, detectable inhibition of cPLA2 by AACOCF3 occurs when this compound is present in the assay at a level of one inhibitor per several thousand substrates. Arachidonic acid analogues in which the COOH group is replaced by COCH3, CH(OH)CF3, CHO, or CONH2 did not detectably inhibit the cPLA2. The arachidonyl ketones AACOCF2CF3 and AACOCF2Cl were found by 19F NMR to be less hydrated than AACOCF3 in phospholipid/Triton X-100 mixed micelles, and compared to AACOCF3 these compounds are also weaker inhibitors of cPLA2. In keeping with the fact that cPLA2 displays substrate specificity for arachidonyl-containing phospholipids, the arachidic acid analogue C19H39COCF3 is a considerably less potent inhibitor compared to AACOCF3.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Slow- and tight-binding inhibitors of the 85-kDa human phospholipase A2. 801 13

The effect of detergents on the overall catalytic turnover by secreted phospholipase A2 (PLA2) on codispersions of the substrate phospholipid is characterized. The overall rate of interfacial catalytic turnover depends on the effective substrate "concentration" (mole fraction) that the bound enzyme "sees" at the interface. Therefore, besides the intrinsic catalytic turnover rate determined by the Michaelis-Menten cycle in the interface [Berg et al. (1991) Biochemistry 30, 7283], two other interfacial processes significantly alter the overall effective rate of hydrolysis: first, the fraction of the total enzyme at the interface; second, the rate of replenishment of the substrate. At low mole fractions (< 0.3), bile salts promote the binding of pig pancreatic PLA2 to zwitterionic vesicles, and the rate of hydrolysis increases with the fraction of the enzyme in the interface. At higher (> 0.3) mole fractions of the detergent, the bilayer is disrupted, and the rate of hydrolysis decreases by more than a factor of 10. The detergent-dependent decrease in the rate of hydrolysis of the sn-2-oxyphospholipids is much larger than that of sn-2-thiophospholipid, and therefore the element effect (O/S ratio) decreases from about 10 in bilayers to less than 2 in mixed micelles. This loss of the element effect in mixed micelles shows that the chemical step is no longer rate-limiting during the hydrolysis of mixed micelles formed by the disruption of vesicles by the detergent. Such effects were observed with phospholipase A2 from several sources acting on substrates dispersed in a variety of detergents including bile salts, 2-deoxylysophosphatidylcholine, and Triton X-100.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The chemical step is not rate-limiting during the hydrolysis by phospholipase A2 of mixed micelles of phospholipid and detergent. 834 32

Although tyrosylprotein sulfation has been implicated in the processing of several secretory proteins, nothing is known about the regulation of the enzyme responsible for this event. When poly(Glu6, Ala3, Tyr1) (EAY; M(r) 47,000) was employed as sulfate acceptor, the tyrosylprotein sulfotransferase (TPST) from Golgi membranes of submandibular salivary gland was used to study the effect of various lipids on the expression of its activity. The TPST activity in the Golgi membrane was 38 pmol (mg of protein)-1 (30 min)-1. Approximately 90% of the total activity present in Golgi membranes was extracted by NaCl and Triton X-100 treatment. The Km values of solubilized TPST for EAY and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) were 0.04 and 0.25 microM, respectively. Among the various lipids tested, sphingosine showed maximum inhibition of TPST activity followed by sphingomyelin and phosphatidylcholine (PC). Of the two sphingosine analogs tested, threosphinganine was as effective as sphingosine in TPST inhibition, while erythrosphinganine had little effect. In contrast, the acidic phospholipids phosphatidylinositol (PI) and phosphatidylserine (PS) and oleic acid showed slight stimulation. Half-maximal inhibition of TPST was obtained at 150 microM sphingosine (6 mol % when expressed as mole percent of sphingosine to total phospholipids plus Triton X-100). The inhibition was competitive with respect to EAY and uncompetitive with respect to PAPS. The inhibition caused by sphingosine could be reversed by PI, PS, and oleic acid but not by PC and sphingomyelin. Sphingosine inhibition of TPST activity was also observed in the enzyme isolated from several other tissues such as liver, lung, heart, and cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibition of tyrosylprotein sulfotransferase by sphingosine and its reversal by acidic phospholipids. 842 47

The mixing behaviour of plant oils (ricebran, saffola and clove) with water in presence of amphiphiles (Triton X-100, Tween-60, Aerosol OT, Igepal, Na-oleate, ethanol and cinnamic alcohol) in various ternary and quaternary combinations has been studied. The phase behaviour at different mass proportions and temperature has been investigated in the absence and presence of additives such as NaCl, glucose, urea and cholesterol. Of all the combinations studied, those with ethanol plus sodium oleate as amphiphile have shown maximum extent of single phase microemulsion formation. The presence of urea in the aqueous medium has further increased the monophasic extent whereas NaCl has decreased it. Cholesterol in oil and glucose in water have apparently shown inert effects. The effects of the additives on the formation of biphasic or triphasic formulations, on the other hand, have been found to be distinct and well-dependent on [H2O]/[amphiphile] mole ratio and temperature. Spectral measurements of I3- in the aqueous micropool in microemulsion of clove oil/(ethanol + Na-oleate)/water have shown the microenvironment to be physicochemically different from bulk water.
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PMID:Biological microemulsions V: mutual mixing of oils, amphiphiles and water in ternary and quaternary combinations. 882 91

The thermal inactivation of horse spleen ferritin was studied over a range of temperatures (36-52 degrees C) in 0.1 M acetate buffer (pH 4.2) as a decrease of its peroxidase activity during tetramethylbenzidine (TMB) oxidation by hydrogen peroxide. The activation energy of this process was 163.3 kJ/mole. Thermodynamic activation parameters for the loss of peroxidase activity of ferritin were calculated. The influence of various detergents on ferritin-dependent oxidation of TMB, ortho-tolidine, and ortho-phenylenediamine (PDA) by hydrogen peroxide was studied in 0.1 M phosphate buffer (pH 6.0) at 20 degrees C. Relatively high concentrations of charged detergents (SDS and cetyltrimethylammonium bromide) decreased the peroxidase activity of ferritin with all three amines, whereas moderate concentrations of the nonionic detergent Triton X-100 did not influence oxidation of these substrates. Increase of dimethylformamide concentration in 0.02 M acetate buffer (pH 4.2) from 5 to 40% strongly decreased the rate of TMB and PDA oxidation by hydrogen peroxide or cumene hydroperoxide. Decrease in the activity of thermally inactivated ferritin with TMB as substrate, reduction of alpha-helical content of the protein at 40-50 degrees C, an inactivating effect of charged surfactants and organic co-solvent on the peroxidase activity of ferritin indicate a very important role of the apoprotein in peroxidase function. A possible mechanism of apoferritin participation in peroxidase catalysis is discussed.
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PMID:Role of the apoprotein in the catalytic peroxidase-like function of ferritin. 948 74

We report here the isolation and partial characterization of a flavoprotein, NADPH-cytochrome P450 (cytochrome c) reductase. The enzyme is a part of steroid 11 alpha-hydroxylating system and is associated with the microsomal fraction of the fungus Rhizopus nigricans. Fungal reductase was solubilized from microsomal membranes with Triton X-100 and purified to apparent homogeneity by affinity and high-performance ion-exchange chromatography. A 350-fold purification of the enzyme with specific activity of 37 mumol cytochrome c reduced/min/mg protein was achieved. A single protein band was obtained on SDS-PAGE analysis with an apparent molecular weight of 79 kDa. Purified reductase contained approximately equimolar quantities of flavin adenine dinucleotide and flavin mononucleotide per mole of the enzyme. Upon induction of the steroid hydroxylating system with progesterone the activity of microsomal NADPH-cytochrome c (P450) reductase increased 10-fold. This is in good correlation with the increase in content of fungal cytochrome P450. Purified fungal flavoprotein was active in a reconstituted system with cytochrome P450 C21 from adrenal gland but could not replace adrenodoxin reductase in the mitochondrial steroid 11 beta-hydroxylating system. We were able to confirm the role of the enzyme by reconstituting steroid 11 alpha-hydroxylating activity from the separated components NADPH-cytochrome P450 reductase and cytochrome P450, partly purified from fungal microsomes.
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PMID:Purification and characterization of NADPH-cytochrome P450 reductase from filamentous fungus Rhizopus nigricans. 973 72

A phosphatidylinositol 4-kinase (Ptdlns 4-kinase, M(r) approximately 95,000) from the membranes of the electric organ of Torpedo californica was purified to apparent homogeneity. The Michaelis constant for ATP (KM = 280 +/- 60 microM at 20 degrees C) and the inhibition constant for adenosine (Ki = 0.4 mM at 20 degrees C) qualify the electrocyte Ptdlns 4-kinase as a type III kinase. The Ptdlns 4-kinase phosphorylates preferentially exogenous Ptdlns, added in the form of mixed Ptdlns/Triton X-100 micelles, whereas endogenously bound Ptdlns in the membrane fragments of electrocytes is a very poor substrate. It is important that the enzyme and the substrate Ptdlns are situated in different lipid bilayers. The catalytic turnover constant for exogenous Ptdlns is k = 55.3 +/- 6 min-1 at 20 degrees C and the molar Triton X-100/Ptdlns ratio of 16:1. For the substrate Ptdlns in the 'micellar solvent' Triton X-100, steady state kinetics were analysed in terms of the mole fraction X = n(Ptdlns)/[n(Ptdlns) + n(Triton X)] yielding the characteristic Michaelis mole fraction XM = 0.019 +/- 0.005 at 20 degrees C. The activity of the enzyme was enhanced about 5-fold in the presence of Triton X-114, yielding k = 277 +/- 30 min-1 at 20 degrees C. Triton X-114 has a shorter head-group, indicating that the vicinity of the Ptdlns head group in the mixed micelles should not be screened by bulky neighbours. The inhibition of the enzyme activity by Ca2+ is highly cooperative yielding the Hill inhibition constant Ki = 0.47 +/- 0.1 mM and the Hill coefficient h = 3.6 +/- 0.5. The enthalpy of activation is 100 +/- 10 kJ/mol between 0 degree C and 20 degrees C. Although the Ptdlns 4-kinase can be affinity-chromatographically copurified with the nicotinic acetylcholine (AcCho) receptor, suggesting tight association between the two proteins. AcCho does not affect the activity of the Ptdlns 4-kinase in the presence of the AcCho receptor.
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PMID:Phosphatidylinositol 4-kinase of Torpedo californica electrocytes: physico-chemical characterization and regulation by calcium and vicinal molecules of phosphatidylinositol. 985 9

The interactions, at sublytic concentration, of Triton X-100 and sodium cholate with sonicated and extruded liposomes of egg and soya lecithins were considered to analyze the integrity and/or the barrier efficiency of liposomal membranes. Results are discussed in terms of surfactant partition between the aqueous and the lipid phases and of the release of a fluorescent hydrophilic probe. Phospholipid nature and liposome size influence detergent partition, whereas the content release is mainly affected by the surfactant mole fraction in the bilayer, and by the liposome size.
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PMID:Surfactant-induced leakage from liposomes: a comparison among different lecithin vesicles. 1038 52

The formation and the photophysical properties of the europium-thenoyltrifluoroacetone (TTA) trioctylphosphine oxide (TOPO)-Triton X-100 chelate were investigated. When the medium is buffered with acetate, there is a strong competition between acetate and TTA for coordination with europium ions. When TOPO is added into the solution, the Eu-TTA-TOPO ternary chelate forms more easily, probably because the coligand acts as a synergic agent and would favour the formation of the enol form of TTA. Although the stoichiometric composition of the chelate is expected to be Eu(TTA)3(TOPO)2, the Eu-TTA and the Eu-TOPO mole ratios may be within 2-3 and 1-2, respectively, depending on the composition of the solution. However, the fluorescent properties of the chelate seem to be mainly dominated by its actual concentration into the solution rather than by its composition. Time resolution of europium emission spectra in the microsecond range has shown that energy transfer occurs from the TTA ligand to the 5D1 level of europium. Then, the emitting 5D0 level is populated through non-radiative deactivation of 5D1. The observed lifetimes of the 5D1 and 5D0 states are 1.25 and 860 micros, respectively. The overall fluorescence quantum yield of the chelate, measured by the photothermal method, is found to be 0.22. On the basis of the time-resolved photothermal experiments, the fluorescence quantum yield of the 5D0 state is expected to be > 0.8 and the energy transfer efficiency < 0.28.
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PMID:Investigation of chelate formation, intramolecular energy transfer and luminescence efficiency and lifetimes in the Eu-thenoyltrifluoroacetone-trioctylphosphine oxide-Triton X-100 system using absorbance, fluorescence and photothermal measurements. 1079 45


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