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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Organophosphate-inhibited cholinesterases may become progressively refractory to reactivation by nucleophilic compounds due to the dealkylation of an alkoxy group from the covalently bound phosphonate ester. This process is termed "aging". It has been found that "aged" cholinesterases are more resistant to protein unfolding than the non-inhibited ones. The pressure-induced denaturation of the native (non-inhibited) and "aged" tetrameric form of human plasma butyrylcholinesterase was investigated in the presence and absence of a denaturing agent (propylene carbonate). This study was undertaken to determine whether the stability of aged butyrylcholinesterase varies with the structure of the alkyl/aryl (R2) group remaining attached to the phosphorus atom of the organophosphoryl moiety. "Aged" organophosphoryl-cholinesterase conjugates were formed by reacting the enzyme with organophosphates: soman (trimethylpropylmethyl-phosphonofluoridate), sarin (isopropylmethyl-phosphonofluoridate), tabun (ethyl-N-dimethyl-phosphoramidocyanidate), DFP (diisopropyl phosphorofluoridate) and PBPDC (pyrenebutyl-phosphorodichloridate). The dual effects of hydrostatic pressure up to 3.5 kbar and propylene carbonate up to 1.2 M were investigated in 10 mM Tris.HCl (pH 7.0). Non-inhibited and aged enzymes were subjected to pressure/propylene carbonate for 12 hours at 20 degrees C. The perturbing effects of this treatment upon cholinesterase structure were analyzed after pressure release by non-denaturing electrophoresis. Pressure and propylene carbonate induced progressive inactivation of the native enzyme. The loss in activity was correlated with irreversible denaturation of the tetramer and its subsequent aggregation. Similarly, pressure and propylene carbonate induced the formation of irreversibly denatured forms of aged butyrylcholinesterase. These denatured forms are partially unfolded enzyme conformations. The native enzyme was found to be more susceptible to denaturation than aged enzymes, with the exception of the PBPDC-aged enzyme. Methyl phosphono adducts, i.e. soman or sarin-aged conjugates were found to be the most stable aged species. Phenomenological analysis of the pressure/propylene carbonate denaturation maps at half-way of the denaturation process indicated that denaturation is a multistep process. The lowest stability of tabun-aged and DFP-aged conjugates suggested that the size, the orientation and the hydrophobicity of the remaining alkyl/aryl chain (R2) of the organophosphoryl moiety play a role in determining the overall stability of aged enzymes. Molecular modelling of aged adducts shed light on steric constraints exerted by the R2 chain on the salt bridge formed between the negatively charged P-O- of the dealkylated organophosphoryl moiety and protonated His438 N epsilon.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1994 May 06
PMID:Pressure and propylene carbonate denaturation of native and "aged" phosphorylated cholinesterase. 817 37

We describe a strategy for sequential assignment of 31P and deoxyribose 1H NMR resonances in oligode-oxyribonucleotides. The approach is based on 31P-1H J-cross-polarization (hetero TOCSY) experiments, recently demonstrated for the assignment of resonances in RNA [Kellogg, G.W. (1992) J. Magn. Reson., 98, 176; Kellogg, G.W. et al. (1992) J. Am. Chem. Soc., 114, 2727]. Two-dimensional heteroTOCSY and heteroTOCSY-NOESY experiments are used to connect proton spin systems from adjacent nucleotides in the dodecamer d(CGCGAATTCGCG)2 entirely on the basis of through-bond scalar connectivities. All phosphorus resonances of the dodecamer are assigned by this method, and many deoxyribose 1H resonances can be assigned as well. A new three-dimensional heteroTOCSY-NOESY experiment is used for backbone proton 4', 5' and 5" resonance assignments, completing assignments begun on this molecule in 1983 [Hare, D.R. et al. (1983) J. Mol. Biol., 171, 319]. Numerical simulations of the time dependence of coherence transfer aid in the interpretation of heteroTOCSY spectra of oligonucleotides and address the dependence of heteroTOCSY and related spectra on structural features of nucleic acids. The possibility of a generalized backbone-driven 1H and 31P resonance-assignment strategy for oligonucleotides is discussed.
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PMID:Two- and three-dimensional 31P-driven NMR procedures for complete assignment of backbone resonances in oligodeoxyribonucleotides. 821 42

The crystal structure of beta-lactamase from Staphylococcus aureus inactivated by p-nitrophenyl[[N-(benzyloxycarbonyl)amino]methyl]phosphonate, a methylphosphonate monoester monoanion inhibitor, has been determined and refined at 2.3 A resolution. The structure reveals a tetrahedral phosphorus covalently bonded to the O gamma atom of the active site serine, Ser70. One of the oxygen atoms bonded to phosphorus is located in the oxyanion hole formed by the two main-chain nitrogen atoms of Ser70 and Gln237, and the second bonded oxygen is solvated. The (benzyloxycarbonyl)aminomethyl group is oriented towards the active site gully such that the peptide group forms compensating electrostatic interactions with polar groups on the enzyme. The benzyl group forms a hydrophobic interaction with Ile239 and an aromatic-aromatic edge-to-face interaction with Tyr105, which has undergone a conformational transition relative to the native structure. The mode of binding supports the proposal that on reaction with the enzyme, the phosphonate generates a structure analogous to the tetrahedral transition state/intermediate associated with the acylation step of a normal substrate. The disposition of the phosphonyl group in this complex is the same as that of the corresponding phosphoryl group in the complex resulting from the inhibition of trypsin by diisopropylphosphofluoridate. The structure is consistent with a mechanism of inactivation that follows an associative pathway, proceeding via a transition state/intermediate in which phosphorus is penta-co-ordinated, forming a trigonal bipyramidal geometry with the phosphonyl donor (p-nitrophenol) and acceptor (Ser70 O gamma atom) in apical positions. A model of this transition state can be accommodated in the active site of beta-lactamase without any steric hindrance. A model of the tetrahedral transition state associated with the acylation step by benzyl penicillin has been derived. Because of the conformational rigidity of the fused rings of penicillin molecules, the orientation of the substrate is fixed once the tetrahedral carbonyl carbon and its ligands are superimposed on the phosphonate group. The outcome is that the carboxylate substituent on the thiazolidine ring forms a salt bridge with Lys234, and the preferred puckering of the ring is that observed in the crystal structure of ampicillin, the so-called "open" conformer.
J Mol Biol 1993 Nov 05
PMID:Structure of a phosphonate-inhibited beta-lactamase. An analog of the tetrahedral transition state/intermediate of beta-lactam hydrolysis. 823 Jan 96

Catecholamines can overcome myocardial stunning. However, a previous report on energy metabolism in stunned myocardium during catecholamine infusion was based on the conventional biochemical methods which might affect contractile function. Twenty farm pigs were anesthetized and underwent 15 min coronary artery occlusion and 2 h reperfusion. Ten pigs were given 10 micrograms/kg/min dobutamine from immediately after and throughout the reperfusion (dobutamine group). The other ten pigs were given saline (control group). Phosphorus-31 magnetic resonance spectroscopy and sonomicrometry were done alternately. Dobutamine improved percent segment shortening after reperfusion (control/dobutamine = 3.8%-5.7%/11.7%-13.4%; P < 0.01). At 15 min ischemia, adenosine triphosphate (ATP) decreased (control/dobutamine = 72 +/- 8%/73 +/- 10%, n.s.), and remained depressed after reperfusion in both groups. After reperfusion, phosphocreatine (PCr) returned to and maintained the preischemic value in the dobutamine group, while in the control group, PCr overshoot (112 +/- 5%) was observed. Except for the presence and absence of PCr overshoot, there was no significant difference of ATP and PCr between the two groups, although rate pressure product was significantly higher in the dobutamine group than in the control group. Regional myocardial blood flow after reperfusion was significantly higher in the dobutamine group. Dobutamine may improve "stunning" through effective improvement of energy utilization and production, indicated by the disappearance of PCr overshoot and maintained ATP level.
J Mol Cell Cardiol 1993 Jul
PMID:Dobutamine prevents both myocardial stunning and phosphocreatine overshoot without affecting ATP level. 823 Feb 47

We recently reported that modulation of anion homeostasis by substitution of extracellular chloride by nitrate prevents ischaemia- and reperfusion-induced ventricular fibrillation (VF) in rat and rabbit in vitro by an unknown mechanism independent of haemodynamic changes but related to widening of QT interval (Ridley and Curtis 1991). In the present study we have examined three possible explanations for the mechanism: modification of membrane anion permeability, alteration of cyclic nucleotide homeostasis and alteration of intracellular pH. In isolated Langerdorff-perfused rat heart (n = 12/group), substitution of chloride in modified Krebs perfusion solution by anion surrogates (methylsulphate, bromide, nitrate or iodide) inhibited left regional ischaemia- and reperfusion-induced arrhythmias only when the membrane permeability of the surrogate was greater than that of chloride (e.g., nitrate, bromide, iodide); the least permeant anion, methylsulphate, was proarrhythmic during ischaemia. Rank order of arrhythmia susceptibility correlated with the relative permeability of each anion, with near abolition of both ischaemia- and reperfusion-induced VF (P < 0.05) by the most permeant anions (iodide and nitrate). Arrhythmia suppression occurring in the iodide and nitrate groups was accompanied by significant widening of QT interval at 90% repolarization, with effects substantially more marked during ischaemia than before ischaemia. In separate studies using the same model we determined cardiac cyclic (c) AMP and cGMP content and their molar ratios by radioimmunoassay of biopsies before, during and after ischaemia. There was no meaningful relation between cyclic nucleotide content and rank order of arrhythmia susceptibility ruling out changes in the former as a contributory mechanism to the latter. In further studies we measured intracellular pH in the isolated perfused rat heart by phosphorus NMR spectroscopy. Nitrate caused a slight intracellular acidosis which was exacerbated when hearts were made globally ischaemic, indicating that its antiarrhythmic activity was not a consequence of alkalinisation (e.g., via inhibition of chloride-bicarbonate exchange). To test for inherent adverse effects on cardiac contractile function we analysed Starling curves in isolated rat hearts perfused under conditions equivalent to those used for arrhythmia studies. There was no relationship between perfusion anion composition and systolic (developed pressure at constant intraventricular volume, and pressure-volume slope) or diastolic function (end-diastolic pressure at constant intraventricular volume). In conclusion, alteration of membrane permeability is a mechanism which may be sufficient to explain modulation of arrhythmias by manipulation of extracellular anion content, appears to be devoid of deleterious effects on contractile function, and may represent a focus for future antiarrhythmic drug development.
J Mol Cell Cardiol 1993 Apr
PMID:Anion manipulation, a novel antiarrhythmic approach: mechanism of action. 839 92

CpG sites in DNA are hotspots for mutations leading to human genetic disorders. However, the structural basis for these events were still unclear and necessitated a deeper evaluation. Our experiments with phosphorus-31 nuclear magnetic resonance, ultraviolet-melting and circular dichroism on two related CpG-containing octanucleotide duplexes show that CpG is a malleable step whose conformation and thermal stability are strongly dependent on the nature of its flanking steps. We conclude that the CpG step may exert a deleterious structural influence on the helix very much like the mismatch containing steps. This peculiar property of CpG should constitute a molecular basis for its recognition by various ligands as well as for mutations affecting CpG and hence an explanation for its rarity in vertebrate genomes.
J Mol Biol 1993 Mar 20
PMID:Structural deviations at CpG provide a plausible explanation for the high frequency of mutation at this site. Phosphorus nuclear magnetic resonance and circular dichroism studies. 846 51

We investigated changes in pHi during ischaemia-reperfusion of isolated rat hearts using phosphorus nuclear magnetic resonance spectroscopy (31P NMR). Hearts were separated into three groups according to the perfusion buffer: bicarbonate-buffered Krebs solution, HEPES-buffered Krebs solution, or bicarbonate-buffered Krebs solution plus 10(-6) M 5-(N-ethyl-N-isopropyl) amiloride (EIPA). In HEPES buffer and in bicarbonate buffer plus EIPA, pH at the end of 30 min of ischaemia and pH oscillations observed during early reperfusion were lower than in bicarbonate buffer. Thus, the presence of two pH regulation mechanisms (Na(+)-H+ antiport and Na(+)-HCO3- symport) was confirmed in the isolated rat heart, while in HEPES buffer, pH was regulated by Na(+)-H+ antiport, and in bicarbonate buffer plus EIPA, by Na(+)-HCO3- symport. When cardiac contraction was inhibited by 10 mM 2, 3-butanedione 2-monoxime (BDM), we observed, in all cases, a less pronounced decrease in pHi at the end of ischaemia, and in pHi oscillations at the onset of reperfusion. These effects were similar to those observed with 150 x 10(-8) M verapamil and might thus be related to a decrease in intracellular calcium. However, with BDM, a greater reduction in the pH recovery rate was observed only in HEPES buffer, suggesting a possible phosphatase-like effect affecting the Na(+)-H+ exchange. Whatever the buffer used, the protective effect of BDM was reflected by an increase in the rate pressure product, which was not observed with verapamil.
J Mol Cell Cardiol 1995 Aug
PMID:pH regulation during ischaemia-reperfusion of isolated rat hearts, and metabolic effects of 2,3-butanedione monoxime. 852 32

2'-Deoxythymidine 5'-triphosphate and 2'-deoxyadenosine 5'-triphosphate analogs containing a methylene group between the alpha phosphorus and 5' oxygen were synthesized. The substrate properties of these compounds toward some mammalian DNA polymerases and retroviral reverse transcriptases were evaluated using a system containing phage M13mp10 DNA, a synthetic oligonucleotide, and the enzyme. The compounds containing a hydroxyl at the 3' position were incorporated into the DNA chain by DNA polymerase alpha and terminal deoxynucleotidyl transferase, but were not recognized by retroviral reverse transcriptases and mammalian DNA polymerases epsilon and beta. The selectivity of the compounds synthesized was capitalized on during simultaneous isolation of DNA polymerases alpha and epsilon from human placenta. A methylene group was also introduced into the acyclovir molecule. It was shown that this modification inactivates furanose-related nucleotide analogs, but has a minor effect on the substrate properties of acyclic nucleotide analogs.
Mol Biol (Mosk)
PMID:[New nucleotide inhibitors of human DNA polymerase alpha]. 855 70

The alteration of Ca(2+)-binding protein regucalcin mRNA expression in the kidney cortex of rats administered cisplatin and cephaloridine, which can induce kidney damage, was investigated. Cisplatin (0.25, 0.5 and 1.0 mg/100 g body weight) or cephaloridine (25, 50 and 100 mg/100 g) was intraperitoneally administered in rats, and 1, 2 and 3 days later they were sacrificed. The alteration in serum findings after the administration of cisplatin (1.0 mg/100 g) or cephaloridine (50 and 100 mg/100 g) demonstrated chemically induced kidney damage; blood urea nitrogen (BUN) concentration increased markedly and serum inorganic phosphorus or calcium concentration decreased significantly. Moreover, the administration of cisplatin (1.0 mg/100 g) or cephaloridine (100 mg/100 g) caused a remarkable increase of calcium content in the kidney cortex of rats, indicating kidney damage. The expression of regucalcin mRNA in the kidney cortex was markedly reduced by the administration of cisplatin or cephaloridine in rats, when the mRNA levels were analyzed by Northern blotting using rat liver regucalcin cDNA (0.9 kb). The mRNA decreases were seen with the used lowest dose of cisplatin or cephaloridine. The present study clearly demonstrates that the mRNA expression of Ca(2+)-binding protein regucalcin in the kidney cortex of rats is decreased by chemically induced kidney damage.
Mol Cell Biochem 1995 Oct 04
PMID:Suppressed expression of calcium-binding protein regucalcin mRNA in the renal cortex of rats with chemically induced kidney damage. 858 14

A condition similar to insulin-dependent diabetes mellitus (IDDM) was induced in male CD-1 mice by injection of streptozotocin (STZ). Five weeks after treatment, the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles were isolated for analysis. Phosphorous metabolites were quantified by 31P-NMR and HPLC, native myosin was characterized electrophoretically, and activities of metabolic enzymes were measured spectrophotometrically. Relative to control animals, STZ-diabetes resulted in a significant 32% decrease in the FM1 isoform of myosin in EDL and a 24% decrease in IM myosin of SOL. Mass-specific activities of phosphofructokinase, citrate synthase, and cytochrome oxidase were significantly lower in SOL from STZ-diabetic mice than in controls by 23, 18, and 36%, respectively. Intracellular ATP was significantly lower in SOL from STZ-diabetic mice than in controls (3.44 +/- 0.20 mumol g-1 wet weight vs. 4.61 +/- 0.20 mumol g-1, respectively), as was creatine phosphate (11.98 +/- 0.80 mumol g-1 wet weight vs. 14.22 +/- 0.44 mumol g-1). In contrast to results from SOL, there were no significant changes in phosphorus metabolites or enzyme activity in EDL. These results show that the effects of IDDM on levels of phosphorus containing metabolites and maximal activities of key regulatory enzymes in muscle are markedly fiber-type specific. It is suggested that the muscle type-specific effects of STZ-diabetes may be a consequence of differential accumulation of intracellular fatty acids.
Mol Cell Biochem 1995 Jul 19
PMID:Responses of mouse fast and slow skeletal muscle to streptozotocin diabetes: myosin isoenzymes and phosphorous metabolites. 859 19


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