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Query: UNIPROT:P06889 (Mol)
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Diisopropyl phosphorofluoridate (DFP), an insecticide, is a potent anticholinesterase that binds essentially irreversibly to acetylcholinesterase, resulting in severe, acute neurologic pathology, and less severe, but longer-lasting, delayed neuropathy. We report here on the short-term effects of bath-applied DFP on extracellularly recorded responses from CA3 and CA1 of rat hippocampus. Exposure to 10 microM DFP evokes low amplitude, spontaneous bursts in CA3 generally within 10 minutes, and the bursting does not reverse with washing. The CA1 neuronal population usually bursts synchronously with CA3, but the population events are of low amplitude and sometimes not detectable, implying a differential sensitivity to DFP. These effects were partially blocked by the muscarinic antagonist atropine, while the cholinergic antagonist gallamine had little effect. Also, the reversible anticholinesterase physostigmine could, within temporal limits, protect slices from DFP's effects, implicating the cholinergic system as the probable mediator in the first stages of DFP-induced epileptogenesis.
Mol Chem Neuropathol
PMID:Effects of diisopropyl phosphorofluoridate (DFP) on CA3 and CA1 responses in rat hippocampus. 209 78

Superfusion of the organophosphorous acetylcholinesterase inhibitor soman (pinacolyl methylphosphonofluoridate; 0.01-25 microM) produced a dose-dependent reduction of extracellularly and intracellularly recorded synaptic responses in the isolated rat superior cervical ganglia at frequencies of orthodromic stimulation that do not normally produce synaptic depression. The magnitude of depression was dependent upon the frequency of stimulation (0.02-1 Hz), was maintained after the removal of soman from the superfusion solution, and recovered by over 65% during periods of inactivity. The depression of synaptic transmission produced by soman was not dependent upon the inhibition of acetylcholinesterase (AChE) activity by this agent. Transmission was increasingly depressed by doses of soman greater than those needed to inactivate all measurable ganglionic AChE activity. Dose-dependent depression of synaptic transmission in soman also occurred after pretreatment with the irreversible AChE inhibitor diisopropylphosphofluoridate (DFP; 100 microM), which inhibited greater than 98% of the AChE activity in the ganglia. Soman produced a decline in the input resistance, resting potential, spike amplitude, and spike threshold and a reduction in the hyperpolarizing afterpotential. Soman-induced depression of synaptic transmission was not due primarily to a blockade of postsynaptic nicotinic receptors. At concentrations of soman which produced significant depression in transmission, ganglionic depolarization produced by bath-applied carbamylcholine (carbachol) was either slightly depressed or facilitated. In the presence of soman, repetitive focal application of acetylcholine or carbachol did not reveal use-dependent desensitization. Muscarinic antagonists, atropine and pirenzepine, protected against the use-dependent depression of synaptic transmission induced by soman. These results suggest that a principal site of action for soman is at the presynaptic terminal and that this site is sensitive to muscarinic receptor blockade.
Cell Mol Neurobiol 1984 Dec
PMID:Noncholinesterase actions of an irreversible acetylcholinesterase inhibitor on synaptic transmission and membrane properties in autonomic ganglia. 615 5

The proenzyme form of C1r was isolated by sequential chromatography from the euglobulin fraction of human serum on DEAE-Sepharose 6B-CL, CM-Sepharose 6B-CL and Sepharose S-300-CL. This C1r had the tendency to spontaneously activate within 60-90 min of incubation at 37 degrees C in presence of EDTA and more slowly in the presence of Ca2+. The spontaneous activation of C1r was found to be a bimolecular process and could be completely inhibited by DFP in the pH range 6-9 and in the presence of Ca2+ without affecting the hemolytic C1r activity. [14C]DFP bound to trace proteins in the 60-90 kD range, but not to C1r proenzyme. The spontaneous activation of C1r was diminished in the presence of EDTA by DFP, but could not be completely suppressed. EDTA acts by removing Ca2+ from C1r, thereby changing the conformation of the protein and causing an increased digestibility of the C1r H-chain. At temperatures above 0-4 degrees C this influence destroyed the ability of C1r proenzyme and enzyme to form macromolecular C1 and thereby abolished its hemolytic activity. We conclude from these results that the spontaneous C1r activation in the pH range 6-9 and in the presence of Ca2+ is due to contaminant proteases. C1r activated also spontaneously at higher pH values between pH 9 and 13.2, but the spontaneous activation ceased abruptly at pH 13.4. An intramolecular process of activation cannot be excluded at these high pH values. It is, however, not clear, whether this activation is a suitable model for the C1r activation in the C1 molecule, because the hemolytic activity of C1r was substantially diminished under the high pH conditions.
Mol Immunol 1982 Mar
PMID:Characterization of the activation of the human C1r complement molecule. 628 81

The activities of hormone-sensitive cholesterol esterase and hormone-sensitive triacylglycerol lipase from rat adrenal glands were enhanced about 2-fold by means of ether stress and showed parallel elution profiles on a Sepharose CL-6B column. Both enzymatic activities were inhibited to a similar extent by DFP after separation from hormone-insensitive lipase on heparin-Sepharose. Fractions from the gel filtration column containing the two hormone-sensitive enzymes showed incorporation of tritium-labelled DFP into only one polypeptide of Mr 84 000. From these results we conclude that both hormone-sensitive activities reside on one polypeptide of Mr 84 000, thus providing further support to the concept that the different hormone-sensitive acylester hydrolase activities in steroid-secreting tissues as well as in adipose tissue are performed by the same bifunctional enzyme. In addition to the hormone-sensitive enzyme, rat adrenals contained high amounts of neutral triacylglycerol lipase activity which was not affected by stress. The latter enzyme was resistant to high salt concentrations, was less susceptible to inhibition by DFP, but could be inhibited completely by the addition of antibodies raised against rat liver lipase, thus most probably representing the adrenal liver lipase-like triacylglycerol lipase.
Mol Cell Endocrinol 1984 May
PMID:Regulation of steroidogenesis in rat adrenal gland: identification of the bifunctional, hormone-sensitive cholesterol esterase--triacylglycerol lipase enzyme protein and its discrimination from hormone-insensitive lipases. 673 27

To explore the molecular basis of the biochemical differences among acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and their alternative splicing and allelic variants, we investigated the acylation phase of cholinesterase catalysis, using phosphorylation as an analogous reaction. Rate constants for organophosphate (DFP) inactivation, as well as for oxime (PAM)-promoted reactivation, were calculated for antibody-immobilized human cholinesterases produced in Xenopus oocytes from natural and site-directed variants of the corresponding DNA constructs. BuChE displayed inactivation and reactivation rates 200- and 25-fold higher than either product of 3'-variable AChE DNAs, consistent with a putative in vivo function for BuChE as a detoxifier that protects AChE from inactivation. Chimeric substitution of active site gorge-lining residues in BuChE with the more anionic and aromatic residues of AChE, reduced inactivation 60-fold but reactivation only 4-fold, and the rate-limiting step of its catalysis appeared to be deacylation. In contrast, a positive charge at the acyl-binding site of BuChE decreased inactivation 8-fold and reactivation 30-fold. Finally, substitution of Asp70 by glycine, as in the natural 'atypical' BuChE variant, did not change the inactivation rate yet reduced reactivation 4-fold. Thus, a combination of electrostatic active site charges with aromatic residue differences at the gorge lining can explain the biochemical distinction between AChE and BuChE. Also, gorge-lining residues, including Asp70, appear to affect the deacylation step of catalysis by BuChE. Individuals carrying the 'atypical' BuChE allele may hence be unresponsive to oxime reactivation therapy following organophosphate poisoning.
Brain Res Mol Brain Res 1995 Jul
PMID:Successive organophosphate inhibition and oxime reactivation reveals distinct responses of recombinant human cholinesterase variants. 747 18

The increasing effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was characterized. The proteolytic activity was markedly elevated by the addition of regucalcin (0.1-0.5 microM) in the absence of Ca2+. This increase was not significantly altered by the presence of diisopropylfluorophosphate (DPF; 2.5 mM)--although DFP caused a significant decrease in the proteolytic activity. Regucalcin (0.25 microM) additively enhanced the dithiothreitol (DTT; 1.0 mM)--increased proteolytic activity, while the regucalcin or DTT effect was completely abolished by NEM (5 mM), indicating that regucalcin may act on the SH group in proteases. Also, regucalcin (0.25 microM) enhanced the effect of Ca2+ (10 microM) increasing liver proteolytic activity, suggesting that regucalcin does not influence on the active sites for Ca2+ in proteases. Moreover, the proteolytic activity of regucalcin (0.25 microM) was significantly decreased by the presence of calpastatin (24 micrograms/ml), an inhibitor of Ca(2+)-activated neutral protease (calpain). Now, regucalcin (0.25 microM) increased about 7-fold the activity of m-calpain isolated from rabbit skeletal muscle. These observations demonstrate that regucalcin directly activates cysteinyl-proteases. Regucalcin may have a role as a potent proteolytic activator in the cytoplasm of liver cells.
Mol Cell Biochem 1995 Jul 05
PMID:Characterization of regucalcin effect on proteolytic activity in rat liver cytosol: relation to cysteinyl-proteases. 747 35

Alveolar macrophages protect the lungs against noxious agents. Proteases and peptidases are essential for this defense and many metabolic activities. Human alveolar macrophages were evaluated for the presence of six important peptidases. Deamidase, a serine peptidase identical with the lysosomal protective protein and possibly with cathepsin A, had high specific activity in alveolar macrophages and is also present in cultured mouse J774A.1 and human U937 cells, used for the sake of comparison. In fractionated J774A cells, most of the deamidase activity was in the lysosomal fraction and in the final supernatant. Deamidase in human alveolar macrophages, obtained by bronchoalveolar lavage from 23 patients, cleaved dansyl-Phe-Leu-Arg at a rate of 2.26 mumol/h/mg protein and hydrolyzed the chemotactic peptide N-f-Met-Leu-Phe even faster, at a rate of 53.1 mumol/h/mg protein, the highest activity for this enzyme with any of the cells we tested. Rabbit antiserum, elicited with the recombinant partial sequence of the enzyme, immunoprecipitated 77-88% of the macrophage deamidase. In immunocytochemistry, this antiserum localized deamidase within the human macrophages. The enzyme was inhibited by diisopropylfluorophosphate (DFP; 1 mM) and by ebelactone B (10 microM), noncompetitively. The mRNA of deamidase was detected in mouse macrophages by Northern blot; the two protein chains of deamidase were shown in human macrophages by Western blot. In addition, two other serine peptidases were also highly active in macrophages: dipeptidyl peptidase IV (1.38 mumol/h/mg protein) and prolylcarboxypeptidase (0.72 mumol/h/mg protein). The activity of plasma membrane zinc metallopeptidases, neutral endopeptidase 24.11 and carboxypeptidase M, in contrast, was low or absent (angiotensin I converting enzyme; kininase II).(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1995 Aug
PMID:Plasma membrane-bound and lysosomal peptidases in human alveolar macrophages. 762 87

The chymotrypsin in the midgut of Manduca sexta has been purified, characterized and the cDNA encoding the protein has been cloned. The enzyme exists as a monomer of approx. 24 kDa and shows maximal activity between pH 10.5 and 11.0. Kinetic studies reveal that the Michaelis constant (Km) for the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide varies only slightly between pH 7.5 and 11.5 and the Dixon plot shows a kinetically significant pKa at 9.2. The specificity of the purified enzyme was determined to be the peptide bond on the carboxyl side of tyrosine, phenylalanine, tryptophan, histidine, leucine, threonine and glycine. The protease is inhibited by TPCK, PMSF, chymostatin and DFP. A 1 kilobase chymotrypsin cDNA clone was isolated and sequenced. The cDNA sequence encodes a preproenzyme with a putative 17 amino acid signal sequence, a 41 amino acid activation peptide and a mature enzyme of 235 amino acids. The isolated clone encodes the highly conserved active site residues (His, Asp, Ser) and specificity pocket residues present in bovine chymotrypsinogen B. Northern analysis localizes the mRNA for the chymotrypsin to the anterior and middle third of the midgut.
Insect Biochem Mol Biol 1995 Jul
PMID:Purification, characterization and cDNA sequence of an alkaline chymotrypsin from the midgut of Manduca sexta. 763 64

The principal digestive proteinases of the gypsy moth, Lymantria dispar, larval midgut were identified, and the subcellular distribution of the enzyme activities was determined. Proteinase activities of fifth-instar larvae were largely attributed to two luminal serine proteinases, a trypsin-like enzyme (TLE) and an elastase 2-like enzyme (ELA). TLE was purified to homegeneity by benzamidine-Sepharose affinity chromatography. With respect to size (M(r) = 25 kDa), substrate specificity, and interaction with trypsin inhibitors, the gypsy moth enzyme resembled mammalian pancreatic trypsin and trypsin-like enzymes from other insects. Gypsy moth elastase (ELA) was purified from the benzamidine-Sepharose flow-through by mono-Q FPLC. ELA exhibited a slightly smaller size (M(r) = 24 kDa) than TLE. The insect enzyme was inhibited by DFP and chymostatin but was unaffected by TPCK. ELA exhibited little esterolytic activity with BTEE. Succinyl-Ala-Ala-Pro-Leu p-nitroanilide was one of the best substrates for ELA, which is characteristic of elastase 2. TLE and ELA constituted c. 6% of the total soluble protein in midgut lumen of actively feeding fifth-instar larvae. Chymotrypsin and carboxypeptidase activities were not detected in any midgut fraction examined. The brush border membrane (BBM) leucine aminopeptidase (LAP) was isolated from CHAPS-solubilized BBM by FPLC. SDS-PAGE results indicated that the aminopeptidase has an apparent molecular size of c. 100 kDa. The aminopeptidase was inhibited by bestatin and was unaffected by serine proteinase inhibitors.
Insect Biochem Mol Biol 1995 Jan
PMID:Gypsy moth midgut proteinases: purification and characterization of luminal trypsin, elastase and the brush border membrane leucine aminopeptidase. 771 46

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


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