UMLS:C0027960 - FACTA Search

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

We report here the isolation and deduced amino acid sequence of the flavoprotein, NADPH-cytochrome P450 (cytochrome c) reductase (EC 1.6.2.4), associated with the microsomal fraction of etiolated mung bean seedlings (Vigna radiata var. Berken). An 1150-fold purification of the plant reductase was achieved, and SDS/PAGE showed a predominant protein band with an apparent molecular mass of approximately 82 kDa. The purified plant NADPH-P450 reductase gave a positive reaction as a glycoprotein, exhibited a typical flavoprotein visible absorbance spectrum, and contained almost equimolar quantities of FAD and FMN per mole of enzyme. Specific antibodies revealed the presence of unique epitopes distinguishing the plant and mammalian flavoproteins as demonstrated by Western blot analyses and inhibition studies. Peptide fragments from the purified plant NADPH-P450 reductase were sequenced, and degenerate primers were used in PCR amplification reactions. Overlapping cDNA clones were sequenced, and the deduced amino acid sequence of the mung bean NADPH-P450 reductase was compared with equivalent enzymes from mammalian species. Although common flavin and NADPH-binding sites are recognizable, there is only approximately 38% amino acid sequence identity. Surprisingly, the purified mung bean NADPH-P450 reductase can substitute for purified rat NADPH-P450 reductase in the reconstitution of the mammalian P450-catalyzed 17 alpha-hydroxylation of pregnenolone or progesterone.
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PMID:Purification, characterization, and cDNA cloning of an NADPH-cytochrome P450 reductase from mung bean. 846 4

The flavoprotein domain of P450BM-3 (BMR), which is functionally analogous to eukaryotic NADPH-P450 oxidoreductases, contains both FAD and FMN. When BMR is titrated with NADPH or sodium dithionite under anaerobic conditions, addition of 2 electron equivalents per mole of BMR results in the reduction of the high potential flavin (FMN) without the accumulation of semiquinone intermediates. Additional sodium dithionite first produces some neutral, blue flavin semiquinone radical and, finally, fully reduced FADH2. During reduction with NADPH, an absorbance increase characteristic of the formation of a flavin-pyridine nucleotide charge-transfer complex was observed only during the addition of the second mole of NADPH per mole of BMR. On the basis of these results, we conclude that the midpoint reduction potential for the FMN semiquinone/FMNH2 couple is more positive than that for FMN/FMN semiquinone. The kinetics of reduction of BMR with NADPH were studied by stopped-flow spectrophotometry. With a 1:1 ratio of NADPH to BMR, the absorbance changes can be fit to five consecutive first order reactions with rate constants of 350 s-1, 130 s-1, 27 s-1, 2.3 s-1, and 0.05 s-1. These reactions are most probably the following: (a) complex formation between BMR and NADPH; (b) reduction of FAD with formation of the NADP(+)-FADH- charge-transfer complex; (c) transfer of the first electron from FADH- to FMN to form an anionic, red FMN semiquinone leaving the FAD as the neutral, blue semiquinone. Precise identification of intermediates beyond this point is difficult. In the presence of a 10-fold molar excess of NADPH, the absorbance changes and rate constants are somewhat different due to the formation of several additional reduced species of BMR. The rate of the first step increases, confirming that this is the formation of the NADPH-BMR complex. Our results indicate that the kinetic and thermodynamic control of the flavins in BMR is significantly different from that in microsomal P450 reductase. The low potential of the anionic FMN semiquinone can be utilized to reduce the P450 heme. When the anionic semiquinone becomes protonated, its potential becomes more positive and it is readily reduced to FMNH2, which is not capable of reducing P450.
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PMID:Equilibrium and transient state spectrophotometric studies of the mechanism of reduction of the flavoprotein domain of P450BM-3. 867 31

Cytosolic and microsomal protein kinase preparations from cultured chicken osteoblasts were found to phosphorylate up to six major proteins with Mrs 66, 58, 50, 36, 32, and 22 kDa in chicken bone extract. Use of heparin led to the conclusion that these proteins were predominantly phosphorylated by factor-independent protein kinase (FIPK) present both in microsomal and cytosolic preparations. It was confirmed that microsomal preparation contained predominantly FIPK, whereas cytosolic preparation contained additional kinases, that can phosphorylate the bone proteins. Use of purified chicken bone osteopontin (OPN) (58 kDa) and recombinant OPN led to the same conclusions. The identify of the protein kinases was clearly established by using a series of synthetic peptide substrates. Quantitative analysis utilizing pure protein kinases and purified chicken bone OPN, recombinant mouse OPN, and bovine bone OPN and BSP led to introduction of approximately 9 moles of phosphate/mole of OPN and 6.6 moles phosphate/mole bovine bone sialoprotein (BSP) by casein kinase II. cGMP-dependent protein kinase and protein kinase C both introduced 0.5-1.2 moles phosphate/mole of OPN and BSP, whereas cAMP-dependent protein kinase led to no significant phosphorylation of OPN or BSP. Consistent with the above results, sites of phosphorylation identified for OPN (metabolically labeled) and BSP (labeled by casein kinase II) revealed that predominant phosphorylated sites have recognition sequences for FIPK.
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PMID:Protein kinases of cultured chicken osteoblasts that phosphorylate extracellular bone proteins. 908 59

Nine investigated chlorobromine-, bromine-, and dibromine-ifosfamide analogues including 3 racemates and 6 enantiomers, caused about 10-fold increase in in vitro cytotoxic activity, similar to reference standards ifosfamide and cyclophosphamide in HeLa (KB) human tumor cell culture systems with the addition of rat liver microsomal preparations (ED50 = 0.11 - 0.27 x 10(-3) mole/l) as compared to microsomally non induced samples. The chlorobromine-analogues (ED50 = 0.11 - 0.20 x 10(-3) mole/l) demonstrated the highest cytotoxicity in comparison with bromine-, or dibromine-analogues (SAR). Their levorotatory (-)-(S)- enantiomers (ED50 = 0.11 : 0.21 : 0.24 x 10(-3) mole/l) appear to be more active than their dextrorotatory (+)-(R)-antipodes (ED50 = 0.20 : 0.26 : 0.27 x 10(-3) mole/l, respectively) (ESAR). The stereodifferentiated enhancement of their in vitro cytotoxicity, correlated with the decreasing of in vivo L1210 antileukemic effect following phenobarbital metabolic induction in terms of a whole--strong antitumor activity, indicate that their cytostatic activity depends enantioselectively on the mixed function oxidases-system activity, and presumably on the efficacy of the rate of drug metabolic transformation to their cytostatically active metabolites/intermediates.
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PMID:Enantioselective cytotoxic activity of bromine-substituted analogues of ifosfamide. A microsomal implication. 943 59

Small angle X-ray diffraction was used to examine arterial smooth muscle cell (SMC) plasma membranes isolated from control and cholesterol-fed (2%) atherosclerotic rabbits. A microsomal membrane enriched with plasma membrane obtained from animals fed cholesterol for up to 13 weeks showed a progressive elevation in the membrane unesterified (free) cholesterol:phospholipid (C/PL) mole ratio. Beyond 9 weeks of cholesterol feeding, X-ray diffraction patterns demonstrated a lateral immiscible cholesterol domain at 37 degrees C with a unit cell periodicity of 34 A coexisting within the liquid crystalline lipid bilayer. On warming, the immiscible cholesterol domain disappeared, and on cooling it reappeared, indicating that the immiscible cholesterol domain was fully reversible. These effects were reproduced in a model C/PL binary lipid system. In rabbits fed cholesterol for less than 9 weeks, lesser increases in membrane C/PL mole ratio were observed. X-ray diffraction analysis demonstrated an increase in membrane bilayer width that correlated with the C/PL mole ratio. This effect was also reproduced in a C/PL binary lipid system. Taken together, these findings demonstrate that in vivo, feeding of cholesterol causes cholesterol-phospholipid interactions in the membrane bilayer that alter bilayer structure and organization. This interaction results in an increase in bilayer width peaking at a saturating membrane cholesterol concentration, beyond which lateral phase separation occurs resulting in the formation of separate cholesterol bilayer domains. These alterations in structure and organization in SMC plasma membranes may have significance in phenotypic modulation or aortic SMC during early atherogenesis.
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PMID:Physical effects of cholesterol on arterial smooth muscle membranes: evidence of immiscible cholesterol domains and alterations in bilayer width during atherogenesis. 961 Jul 60

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

Acyl-CoA cholesterol acyltransferase (ACAT) (EC 2.3.1.26) in the yolk sac membrane of chicken eggs plays an important role in the transport of lipids, which serve as both structural components and as an energy source during embryogenesis. ACAT from the yolk sac membrane of chicken eggs 16 d after fertilization has higher activity and better stability than its mammalian liver counterpart. During our study of the avian enzyme, ACAT was found to be activated up to twofold during storage at 4 degrees C. The activation was investigated, and data suggest that redistribution of cholesterol within microsomal vesicles leads to the increase. Methyl-beta-cyclodextrin (MbetaCD) increases activation an additional twofold, possibly by facilitating the movement of cholesterol within microsomal fragments and allowing redistribution of cholesterol in lipid bilayers to a greater extent. Treatment of microsomes with MbetaCD removes cholesterol from the membranes. Controlled amounts of cholesterol can be restored to the membranes by mixing them with cholesterol-phosphatidylcholine liposomes in the presence of MbetaCD. Under these conditions, the plot of ACAT vs. cholesterol mole fraction in the liposomes is sigmoidal. The finding that MbetaCD can enhance cholesterol transfer between liposomes and microsomes and reduce the limitation of slow movement of nonpolar molecules in aqueous media should make cyclodextrins more useful in in vitro studies of apolar molecule transport between membrane vesicles.
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PMID:Activation of acyl-CoA cholesterol acyltransferase: redistribution in microsomal fragments of cholesterol and its facilitated movement by methyl-beta-cyclodextrin. 1023 Jul 20

The recent discovery that sterol carrier protein-2 (SCP-2) binds long chain++ (LCFA-CoA) with high affinity (A. Frolov et al., J. Biol. Chem. 271 (1997) 31878-31884) suggests new possible functions of this protein in LCFA-CoA metabolism. The purpose of the present investigation was to determine whether SCP-2 differentially modulated microsomal LCFA-CoA transacylation to cholesteryl esters, triacylglycerols, and phospholipids in vitro. Microsomal acyl-CoA:cholesterol acyltransferase (ACAT) activity measured with liposomal membrane cholesterol donors depended on substrate LCFA-CoA level, mol% cholesterol in the liposomal membrane, and total amount of liposomal cholesterol. As compared to basal activity without liposomes, microsomal ACAT was inhibited 30-50% in the presence of cholesterol poor (1.4 mol%) liposomes. In contrast, cholesterol rich (>25 mol%) liposomes stimulated ACAT up to 6.4-fold compared to basal activity without liposomes and nearly 10-fold as compared to cholesterol pool (1.4 mol%) liposomes. Increasing oleoyl-CoA reversed the inhibition of microsomal ACAT by cholesterol poor (1.4 mol%) liposomes, but did not further stimulate ACAT in the presence of cholesterol rich (35 mol%) liposomes. In contrast, high (100 microM) oleoyl-CoA inhibited ACAT nearly 3-fold. This inhibition was reversed by LCFA-CoA binding proteins, bovine serum albumin (BSA) and SCP-2. SCP-2 was 10-fold more effective (mole for mole) than BSA in reversing LCFA-CoA inhibited microsomal ACAT. Concomitantly, under conditions in which SCP-2 stimulated ACAT it equally enhanced transacylation of oleoyl-CoA into phospholipids, and 5.2-fold enhanced oleoyl-CoA transacylation to triacylglycerols. In summary, SCP-2 appeared to exert its greatest effects on microsomal transacylation in vitro by reversing LCFA-CoA inhibition of ACAT and by differentially targeting LCFA-CoA to triacylglycerols. These data suggest that the high affinity interaction of SCP-2s with LCFA-CoA may be physiologically important in microsomal transacylation reactions.
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PMID:Microsomal long chain fatty acyl-CoA transacylation: differential effect of sterol carrier protein-2. 1049 8

The goal of this study was to examine arachidonic acid (AA) metabolism by murine bone marrow-derived mast cells (BMMC) during apoptosis induced by cytokine depletion. BMMC deprived of cytokines for 12-48 h displayed apoptotic characteristics. During apoptosis, levels of AA, but not other unsaturated fatty acids, correlated with the percentage of apoptotic cells. A decrease in both cytosolic phospholipase A(2) expression and activity indicated that cytosolic phospholipase A(2) did not account for AA mobilization during apoptosis. Free AA accumulation is also unlikely to be due to decreases in 5-lipoxygenase and/or cyclooxygenase activities, since BMMC undergoing apoptosis produced similar amounts of leukotriene B(4) and significantly greater amounts of PGD(2) than control cells. Arachidonoyl-CoA synthetase and CoA-dependent transferase activities responsible for incorporating AA into phospholipids were not altered during apoptosis. However, there was an increase in arachidonate in phosphatidylcholine (PC) and neutral lipids concomitant with a 40.7 +/- 8.1% decrease in arachidonate content in phosphatidylethanolamine (PE), suggesting a diminished capacity of mast cells to remodel arachidonate from PC to PE pools. Further evidence of a decrease in AA remodeling was shown by a significant decrease in microsomal CoA-independent transacylase activity. Levels of lyso-PC and lyso-PE were not altered in cells undergoing apoptosis, suggesting that the accumulation of lysophospholipids did not account for the decrease in CoA-independent transacylase activity or the induction of apoptosis. Together, these data suggest that the mole quantities of free AA closely correlated with apoptosis and that the accumulation of AA in BMMC during apoptosis was mediated by a decreased capacity of these cells to remodel AA from PC to PE.
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PMID:A decrease in remodeling accounts for the accumulation of arachidonic acid in murine mast cells undergoing apoptosis. 1102 38

Ameloblastic tissue samples from unerupted bone molars were used to prepare subcellular enamel protein kinase preparations, nuclear + plasma membrane, cytosolic and microsomal, and used in in vitro phosphorylation of purified 20 kDa bovine amelogenin in the presence of 32P-ATP. Both cytosolic and microsomal preparations can phosphorylate purified native amelogenins, the addition of Ca2+ slightly increased the microsomal enzyme activity or at least did not inhibit the activity, whereas the presence of Ca2+ substantially decreased the cytosolic kinase activity towards phosphorylation of amelogenins. A comparative analysis using the enamel microsomal kinase against osteopontin, dephosphorylated casein and bone sialoprotein showed no phosphorylation of the first two proteins, and only minor phosphorylation of the bone sialoprotein. Overall, the present work demonstrates for the first time that the protein kinase responsible for the phosphorylation of amelogenins is a novel kinase, which is not inhibited by Ca2+, unlike the microsomal protein kinase (casein kinase type-II) of bone which phosphorylates secretory proteins osteopontin and bone sialoprotein and is strongly CaZ+ inhibited. The direct phosphoserine analysis on the purified bovine 20 kDa amelogenin indicated the presence of 0.8 moles of phosphoserine/mole protein naturally occurring, consistent with the quantitative analysis of 14C-radiolabeling of phosphoserines by conversion to dehydroalanine and in situ reaction with the thiol agent, 14C-mercaptoethanol, 0.64 moles 14C-incorporated/mole 20 kDa amelogenin. The purified low Mramelogenins 5.3 kDa E4 (TRAP) and 7.2 kDa E3 (LRAP), were also derivatized by 14C-mercaptoethanol, providing 0.46 and 0.88 moles 14C-incorporated/mole respectively. Further studies of the 14C-radiolabeled E4 amelogenin by sequence analysis confirmed one site of label to be at position 16 from the N-terminal and hence provided a direct evidence for the naturally occurring phosphoserine residue at this position.
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PMID:Enamel specific protein kinases and state of phosphorylation of purified amelogenins. 1106 30

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