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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Some of the unusual molecular and catalytic properties of a high molecular weight dihydro-orotate dehydrogenase (DHOD) from Neurospora crassa have been determined. Comparison of the properties of this enzyme with the properties of the soluble biosynthetic enzyme of prokaryotes has revealed several important differences. The fungal enzyme is located in a mitochondrial membrane in a position consistent with linkage with the respiratory chain through ubiquinone (Miller, R. W.: Arch. Biochem, Biophys. 146, 256-270 (1971)). Release of the enzyme from the membrane results in a solubilized protein complex containing bound lipids and inactive hydrophobic proteins. Non-specific protein aggregation is minimized during purification by Triton-X-100 and phospholipase treatments. The catalytically active enzyme has an apparent molecular weight of 210 000. In contrast to soluble DHOD preparations the high molecular weight enzyme has no endogenous dihydro-orotate oxidase (EC 1.3.3.1) activity and is relatively insensitive to inactivation by sulfhydryl-reactive reagents in the presence of dihydro-orotate (DHO). The enzyme activity is highly sensitive to conditions causing oxidation of flavin mononucleotide (FMN). The activity cannot be restored by cysteine or other means. FMN is present in all purified preparations in a bound, non-fluorescent (reduced) form until dihydro-orotic acid is removed or oxidized. Catalytic efficiency of the purified enzyme was 12 000 mol DHO oxidized per minute per mole FMN. This high turnover rate is due in part to the small flavin content of the purified enzyme, equivalent to 1 mol FMN per 120 000 g of catalytically active protein. Iron was detected in the purified enzyme by atomic absorption spectroscopy but labile sulfide was absent. Thenoyltrifluoroacetone, an iron chelator, only partially inhibited DHO oxidation regardless of electron acceptor. Fatty acids interact with a hydrophobic site of the enzyme in non-competitive fashion but under certain conditions appear to significantly alter the Km for ubiquinone. Orotate, by comparison, is a purely competitive inhibitor. Both types of inhibitor may function to regulate the biosynthesis of orotate in vivo. Superoxide anion is not produced in significant quantities by the DHO-reduced enzyme unless both ubiquinone and a suitable single electron carrier such as phenazine methosulfate are present. DHOD has been proposed as a source of superoxide anion in mammalian mitochondria (Forman, H. J. & Kennedy, J. A.: J. Biol. Chem. 250, 4322-4326 (1975)).
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PMID:A high molecular weight dihydro-orotate dehydrogenase of Neurospora crassa. Purification and properties of the enzyme. 13 Jan 99

The interaction of bee venom melittin with erythrocyte membrane ghosts has been investigated by means of fluorescence quenching of membrane tryptophan residues, fluorescence polarization and ESR spectroscopy. It has been revealed that melittin induces the disorders in lipid-protein matrix both in the hydrophobic core of bilayer and at the polar/non-polar interface of melittin complexed with erythrocyte membranes. The peptide has been found to act most efficiently at the concentration of the order of 10(-10) mol/mg membrane protein. The apparent distance separating the membrane tryptophan and bound 1-anilino-8-naphthalenesulphonate (ANS) molecules is decreased upon melittin binding, which results in a significant increase of the maximum energy transfer efficiency. Significant changes in the fluorescence anisotropy of both 1,6-diphenyl-1,3,5-hexatriene and 1-anilino-8-naphthalenesulphonate bound to erythrocyte ghosts, which have been observed in the presence of melittin and crude venom, indicate membrane lipid bilayer rigidization. The effect of crude honey bee venom has been found to be of similar magnitude as the effect of pure melittin at the concentration of 10(-10) mol/mg membrane protein. Using two lipophilic spin labels, methyl 5-doxylpalmitate and 16-doxylstearic acid, we found that melittin at its increasing concentrations induces a well marked rigidization in the deeper regions of lipid bilayer, whereas the effect of rigidization near the membrane surface maximizes at the melittin concentration of 10(-10) mol/mg (10(-4) mol melittin per mole of membrane phospholipid). The decrease in the ratio hw/hs of maleimide and the rise in relative rotational correlation time (tau c) of iodacetamid spin label, indicate that melittin effectively immobilizes membrane proteins in the plane of the lipid bilayer. We conclude that melittin-induced rigidization of the lipid bilayer may induce a reorganization of lipid assemblies as well as the rearrangements in membrane protein pattern and consequently the alterations in lipid-protein interactions. Thus, the interaction of melittin with erythrocyte membranes is supposed to produce local conformational changes in membranes, which are discussed in the connection with their significance during the synergistic action of melittin and phospholipase of bee venom on red blood cells.
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PMID:Melittin-induced alterations in dynamic properties of human red blood cell membranes. 131 7

The effect of myelin basic protein (MBP) on the activity of phospholipase A2 (PLA2, EC 3.1.1.4) against monolayers of dilauroylphosphatidylcholine (dlPC) or dilauroylphosphatidic acid (dlPA) containing different proportions of sulfatide (Sulf) and galactocerebroside (GalCer) was investigated. MBP was introduced into the interface by direct spreading as an initial constitutive component of the lipid-protein film or by adsorption and penetration from the subphase into the preformed lipid monolayers. The effect of MBP on PLA2 activity depends on the type of phospholipid and on the proportion of MBP at the interface. At a low mole fraction of MBP, homogeneously mixed lipid-protein monolayers are formed, and the PLA2 activity against dlPC is only slightly modified while the degradation of dlPA is markedly inhibited. This is probably due to favorable charge-charge interactions between dlPA and MBP that interfere with the enzyme action. The PLA2 activity against either phospholipid is increased when the mole fraction of MBP exceeds the proportion at which immiscible surface domains are formed. GalCer has little effect on the modulation by MBP of the phospholipase activity. The effect of Sulf depends on its proportions in relation to MBP. The individual effects of both components balance each other, and a finely tuned modulation is regulated by the interactions of MBP with Sulf or with the phospholipid.
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PMID:Concerted modulation by myelin basic protein and sulfatide of the activity of phospholipase A2 against phospholipid monolayers. 137 78

More than 100 amphiphilic phosphoesters, possible tetrahedral transition-state analogues capable of coordinating to the calcium ion at the active site of phospholipase A2, were designed, synthesized, and tested as inhibitors for the hydrolysis of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol vesicles in the scooting mode. This assay system permits the study of structurally diverse inhibitors with phospholipase A2S from different sources, and it is not perturbed by factors that change the quality of the interface. As a prototype, 1-hexadecyl-3-trifluoroethylglycero-2-phosphomethanol (MJ33) was investigated in detail. Only the (S)-(+) analogue of MJ33 is inhibitory, and it is as effective as the sn-2 phosphonate or the sn-2 amide analogues of sn-3 phospholipids. The inhibitory potencies of the various phosphoesters depended strongly on the stereochemical and structural features, and the mole fractions of inhibitors required for 50% inhibition, X1(50), ranged from more than 1 to less than 0.001 mole fraction. The affinity of certain inhibitors for enzymes from different sources differed by more than 200-fold. The inhibitors protected the catalytic site residue His-48 from alkylation in the presence of calcium but not barium as expected if the formation of the EI complex is supported only by calcium. The equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface was correlated with the XI(50) values, which were different if the inhibition was monitored in the pseudo-zero-order or the first-order region of the progress curve. These results show that the inhibitors described here interfered only with the catalytic turnover by phospholipase A2's bound to the interface, their binding to the enzyme occurred through calcium, and the inhibitors did not have any effect on the dissociation of the enzyme bound to the interface.
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PMID:Active-site-directed specific competitive inhibitors of phospholipase A2: novel transition-state analogues. 193 54

Small bilayer particles form spontaneously from gel-state long-chain phospholipids such as dipalmitoylphosphatidylcholine and 0.2 mol fraction short-chain lecithins (e.g., diheptanoyl-phosphatidylcholine). When the particles are incubated at temperatures greater than the Tm of the long-chain phosphatidylcholine (PC), the particles rapidly fuse (from 90-A to greater than or equal to 5000-A radius); this transition is reversible. A possible explanation for this behavior involves patching or phase separation of the short-chain component within the gel-state particle and randomization of both lipid species above Tm. Differential scanning calorimetry, 1H T1 values of proteodiheptanoyl-PC in diheptanoyl-PC-d26/dipalmitoyl-PC-d62 matrices of varying deuterium content, solid-state 2H NMR spectroscopy as a function of temperature, and fluorescence pyrene excimer-to-monomer ratios as a function of mole fraction diheptanoyl-PC provide evidence that such phase separation must occur. These results are used to construct a phase diagram for the diheptanoyl-PC/dipalmitoyl-PC system, to propose detailed geometric models for the different lipid particles involved, and to understand phospholipase kinetics toward the different aggregates.
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PMID:Phase separation in short-chain lecithin/gel-state long-chain lecithin aggregates. 226 48

We have studied the interaction of divalent and trivalent with a potent phospholipase A(2) neurotoxin, crotoxin, from Crotalus durissus terrificus venom. The pharmacological action of crotoxin requires dissociation of its catalytic subunit (component B) and of its non-enzymatic chaperone subunit (component A), then the binding of the phospholipase subunit to target sites on cellular membranes and finally phospholipid hydrolysis. In this report, we show that the phospholipase A(2) activity of crotoxin and of component B required Ca2+ and that other divalent cations (Sr2+, Cd2+ and Ba2+) and trivalent lanthanide ions are inhibitors. The lowest phospholipase A(2) activity was observed in the presence of Ba2+, which proved to be a competitive inhibitor of Ca2+. The binding of divalent cations and trivalent lanthanide ions to crotoxin and to its subunits has been examined by equilibrium dialysis and by spectrofluorimetric methods. We found that crotoxin binds two divalent cations per mole with different affinities; the site presenting the highest affinity (K(d) in the mM range) in involved in the activation (or inhibition) of the phospholipase A(2) activity and must therefore be located on component B, the other site (K(d) higher than 10 mM) is probably localized on component A and does not play any role in the catalytic activity of crotoxin. We also observed that crotoxin component B binds to vesicular and micellar phospholipids, even in the absence of divalent cations. The affinity of this interaction either does not change or else increases by an order of magnitude in the presence of divalent cations.
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PMID:Binding of divalent and trivalent cations with crotoxin and with its phospholipase and its non-catalytic subunits: effects on enzymatic activity and on the interaction of phospholipase component with phospholipids. 259 66

A phospholipase A2 was isolated from the venom of the mexican beaded lizard (Heloderma horridum horridum) by phenyl-Sepharose chromatography followed by Sephadex G-75 gel filtration and two additional steps on ion exchange resins (DE-32 cellulose). The affinity chromatographic method (PC-Sepharose 4B) reported for the isolation of other phospholipases [Rock, Ch. O., & Snyder, F. (1975) J. Biol. Chem. 250, 2564-2566; King, T. P., Alagon, A. C., Kwan, J., Sobotka, A. K., & Lichteinstein, L. M. (1983) Mol. Immunol. 20, 297-308; King, T. P., Kochoumian, L., & Joslyn, A. (1984) Arch. Biochem. Biophys. 230, 1-12] was uneffective for the separation of this enzyme. The monomeric form of the Heloderma phospholipase has an apparent Mr of 18 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 19 060 as calculated from amino acid analysis. It also contains on the order of 7% carbohydrates per mole of enzyme. The N-terminal amino acid sequence was shown to be very different from that of phospholipases isolated from mammalian pancreas and crotalids and elapids snake venoms. The first 39 amino acid residues at the N-terminal region have 56% homology with bee venom phospholipase but differ from the bee phospholipase in that its isoelectric point is acidic (pI = 4.5), instead of basic, and it has approximately 50 amino acid residues more in the molecule. The specificity of the enzyme is mainly A2 type with possible residual B-type activity. The enzymatic activity is Ca2+-dependent. Half-cystine alignment of the Heloderma phospholipase sequence with those of other known phospholipases shows the lack of an octadecapeptide at the N-terminal region, the existence of an extra hexapeptide at positions 42-47, and an exact correspondence of Heloderma Gly-12, Gly-14, His-36, and Asp-37 with Gly-30, Gly-32, His-48, and Asp-49 from other phospholipases shown to be important for Ca2+ binding (( Dijkstra, B. W., Drenth, J., Kalk, K. H., & Vandermaalen, P. J. (1978) J. Mol. Biol. 124, 53-60 )).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Biochemical characterization of the phospholipase A2 purified from the venom of the Mexican beaded lizard (Heloderma horridum horridum Wiegmann). 308 12

The cholesterol to phospholipid mole ratio (C/PL) of human platelets was increased 1.3-fold or maintained at a normal value by incubating platelets with sonicated dispersions of cholesterol and phosphatidylcholine (PC) (C/PL = 3 or 1, respectively). Thrombin-induced mobilization of [3H]arachidonic acid from prelabeled phospholipids and subsequent formation of labeled cyclo-oxygenase and lipoxygenase products were increased in cholesterol-enriched platelets as a function of thrombin concentration. Elevated platelet cholesterol content affected thrombin-induced changes in platelet phospholipids: (a) hydrolysis of PC was more sensitive to thrombin and was markedly enhanced over a wide range of thrombin concentrations (0.1-2 units/ml); (b) hydrolysis of phosphatidylinositol (PI) was increased at thrombin concentrations greater than or equal to 0.2 unit/ml. Increased metabolism of [3H]arachidonic acid in stimulated cholesterol-enriched platelets was due to loss of [3H]arachidonate from PC at 0.1 unit/ml of thrombin. At higher thrombin concentrations (0.2-2 units/ml) it reflected enhanced hydrolysis of predominantly PC, but also PI. We conclude that cholesterol, possibly through its effect on platelet lipid organization, influences arachidonic acid metabolism in stimulated plates by promoting enhanced activity of platelet phospholipase(s) for liberation of arachidonic acid.
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PMID:Effect of membrane cholesterol on phospholipid metabolism in thrombin-stimulated platelets. Enhanced activation of platelet phospholipase(s) for liberation of arachidonic acid. 708 8

The kinetic parameters for the steady-state rate of hydrolysis of egg phosphatidylcholine in multilamellar vesicles by bee venom phospholipase A2 are measured in the presence of 27 alkanols and several organic solvents. In general, small nonpolar solutes like enflurane, tetrahydrofuran, benzene, chloroform and diethylether do not promote the hydrolysis of multilamellar vesicles. The rate of hydrolysis shows a biphasic dependence upon the alkanol concentration for all higher (C5-C9) alcohols examined, i.e., an optimal rate of hydrolysis is observed at a characteristic concentration for each alcohol. The alkanol to lipid mole ratio (D/L ratio) in the bilayer at the peak activating concentration of an alkanol was computed from its bilayer/water partition coefficient. The branched chain alcohols induce peak activation of hydrolysis at lower D/L ratios in the bilayer than the corresponding straight chain analogs. Similarly, the longer chain n-alkanols at peak activating concentration have a lower D/L ratio than the corresponding lower alcohols. Both the Km and Vm for phosphatidylcholine increase as a function of the chain length of the activating alcohol. These kinetic parameters also depend upon the position of the substituents on the activating alcohols. Both the D/L ratio and Vm for an alcohol are found to change with the cross-sectional area of the activating alcohol across its long axis: alcohols with a more asymmetric cross-section exhibit higher Vm and a lower D/L ratio. Such correlations of Vm and D/L ratio with the molecular parameters of the alkanols are interpreted to suggest that the accessibility of the substrate molecule in the bilayer to the phospholipase is modulated by the free space introduced by the alkanols in the bilayer. Effect of tetradecane derivatives and A2C (a membrane fluidizing agent) on the phase transition characteristics of DPPC bilayers, and their susceptibility to phospholipase A2 from bee venom and pig pancreas is also reported. These solutes cause a broadening of the transition profile and reduce the size of the cooperative unit and the enthalpy of transition. These effects depend upon the mole fraction of a solute in the bilayer; however, equal concentrations of these solutes do not induce equal response. Susceptibility of the modified bilayers to phospholipase A2 depends not only upon the structure of the solute but also upon the source of the enzyme. The data show that the activity of the membrane-bound enzyme is modulated to different extents by different solutes, and the bilayer perturbing ability of these solutes may be related to the asymmetry of their cross-sectional area and to the free space introduced by the alkanols in a bilayer.
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PMID:Intrinsic differences in the perturbing ability of alkanols in bilayer: action of phospholipase A2 on the alkanol-modified phospholipid bilayer. 719 Oct 9

Mitochondria obtained from hyperthyroid rabbit liver contained an approximately 4-fold greater amount of free fatty acids (FFA) than preparations from the control animals. The temperature dependence of FFA accumulation (29--38 degrees C) showed that the activation energy of mitochondrial lipid hydrolysis for control and experimental animals was 7 kcal/mole and 29 kcal/mole, respectively. The mitochondria were kept at 3 degrees C for 48 h. During this period there was an accumulation of fatty acids. The rate of the process in the mitochondria of hyperthyroid animals was several times than that in the control ones. This indicates the activation of mitochondrial phospholipase by the liver in hyperthyrosis. The increased mitochondrial phospholipase activity seen in hyperthyrosis is assumed to be determined by changes in the physical properties of lipid membranes.
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PMID:[Liver mitochondrial phospholipase activation in hyperthyroid rabbits]. 722 49


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