Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.1.5 (neuropathy target esterase)
 1,070 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Resolution for 125I-labeled specimens under electron microscope (EM) autoradiographic conditions was assessed experimentally. With this isotope the size of the silver halide crystal was the most important resolution-limiting factor. Heavy metal staining such as is routinely used in preparing animal tissues for EM autoradiography produced an improvement in resolution of approximately 15-20%. For a 500-1,000-A biological tissue section fixed with OsO4 and stained with uranyl acetate, we obtained resolution (half distance, HD) values of approximately 800 +/- 120 A using Ilford L4 emulsion and 500 +/- 70 A using a Kodak NTE-type emulsion. General aspects of resolution-limiting factors and comparison with 3H and 14C values are discussed.
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PMID:Resolution in electron microscope autoradiography. III. Iodine-125, the effect of heavy metal staining, and a reassessment of critical parameters. 6 63

Fine grain development for electron microscopic radioautography was investigated with two types of radioactive specimens: sections of tritiated methacrylate, which provide a homogeneously labeled source for quantitative evaluation of the radioautographic reaction, and sections of 125I-labeled thyroid. Radioautographs were prepared with Ilford L4, Sakura NR-H2, Agfa-Gevaert NUC 307 or Kodak NTE emulsions. The radioautographs were developed with one of several "solution physical" development procedures (Agfa-Gevaert, phenidone-ascorbic acid, p-phenylenediamine developers) or with arrested "direct" developments (D-19b, Elon-ascorbic acid developers). By arresting each development at an early stage of the reaction and at progressively longer time intervals, it was possible to examine the sequence of shapes in the growth of developed silver deposits for each emulsion-development combination. Thus, conditions which resulted in the development of small, round, compact silver deposits were defined for each emulsion. These developments were used in conjuction with gold latensification, a treatment which increases the sensitivity of the emulsions and thus compensates for the lowered sensitivity of fine grain development procedures. The location of the silver deposits in relation to the silver bromide crystals from which they derive was investigated. The emulsion gelatin surrounding the crystals was stained whereas the spaces, which remained after the crystals were dissolved in the photographic fixer, appeared transparent. This analysis permitted the selection of development procedures in which the single or multiple round silver deposits originating from a single crystal will remain within or on the boundary of this crystal. By this method, quantitation of radioautographic reactions composed of small, round silver deposits was studied by using the uniformly labeled 3H-methacrylate sections as a standard source of radiation. The conditions under which grain counting is feasible are discussed.
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PMID:A comparison of various procedures for fine grain development in electron microscopic radioautography. 118 16

The neurotoxic effects of single subcutaneous injections of 1000 mg triphenyl phosphite (TPP)/kg body weight were investigated in White Leghorn hens. At 7 days postexposure, birds began to show signs of mild to moderate ataxia that progressed to severe ataxia and paralysis at 21 days. Inhibition of whole brain neuropathy target esterase was 85% at 48 hr and 73% by 21 days postexposure. After postexposure periods of 7, 14, and 21 days, hens were killed and their brains and spinal cords were examined for degenerating axons and terminals using the Fink-Heimer silver impregnation method. A small amount of degeneration was noted at 7 days. By 21 days, dense degeneration was noted in the spinal gray matter and funiculi. Degeneration was also present in the granular cell layer of cerebellar folia I-VI and in nuclei and fiber tracts of the medulla. Moderate to dense degeneration was also seen in several forebrain and midbrain areas including the paleostriatum, ansa lenticularis, the dorso-intermediate thalamic nucleus, lateral spiriform, pedunculopontine tegmental, and lateral mesencephalic nuclei and in the deeper layers of the optic tectum. These results indicate that, in addition to affecting the spinal cord and brainstem, exposure to TPP also damages higher order centers responsible for processing and integrating sensorimotor, visual, and auditory information.
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PMID:Neuropathological effects of triphenyl phosphite on the central nervous system of the hen (Gallus domesticus). 160 Dec 12

The first step in the production of eicosanoids and platelet-activating factor is the hydrolysis of arachidonic acid from membrane phospholipid by phospholipase A2. We previously purified from the macrophage cell line RAW 264.7 an intracellular phospholipase A2 that preferentially hydrolyzes sn-2-arachidonic acid. The enzyme exhibits a molecular mass of 100 kDa and an isoelectric point of 5.6. When assayed for other activities, the phospholipase A2 was found to exhibit lysophospholipase activity against palmitoyllysoglycerophosphocholine, and both activities copurified to a single band on silver-stained sodium dodecyl sulfate-polyacrylamide gels. An antibody against the macrophage enzyme was found to quantitatively immunoprecipitate both phospholipase A2 and lysophospholipase activities from a crude cytosolic fraction. When the immunoprecipitated material was analyzed on immunoblots, a single band at 100 kDa was evident, further suggesting that a single protein possessed both enzyme activities. When assayed as a function of palmitoyllysoglycerophosphocholine concentration and plotted as a double-reciprocal plot, two different slopes were apparent, corresponding to concentrations above and below the critical micellar concentration (7 microM) of the substrate. Above the critical micellar concentration, lysophospholipase exhibited an apparent Km of 25 microM and a Vmax of 1.5 mumol/min/mg. Calcium was not required for lysophospholipase activity, in contrast to phospholipase A2 activity. The enzyme, when assayed as either a phospholipase A2 or lysophospholipase, exhibited nonlinear kinetics beyond 1-2 min despite low substrate conversion. Readdition to more substrate after the activity plateaued did not result in further enzyme activity, ruling out substrate depletion. Readdition of enzyme, however, resulted in another burst of enzyme activity. The results are not consistent with product inhibition, but suggest that the enzyme may be subject to inactivation during catalysis.
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PMID:Kinetic properties of a high molecular mass arachidonoyl-hydrolyzing phospholipase A2 that exhibits lysophospholipase activity. 164 32

The intestinal brush-border membranes of rats and guinea pigs possess a high molecular weight, calcium-independent phospholipase B (phospholipase A2 - lysophospholipase activities) with the characteristics of a digestive ectoenzyme. A combination of subcellular fractionation, Triton X-114 phase partitioning, chromatofocusing, and preparative sodium dodecyl sulphate - polyacrylamide gel electrophoresis was used to purify a full-length, although denatured, form of this enzyme from the rat. Renaturation of the gel-purified fraction confirmed that both enzyme activities were associated with this protein. Gel slices containing the purified phospholipase B were used to generate a polyclonal antiserum in rabbits that could be used for immunoblotting. The relative mobility of the phospholipase B during electrophoresis in sodium dodecyl sulphate gels was dramatically affected by the percentage of acrylamide and the presence or absence of reducing agents in the gels. This was true for both the purified protein visualized by silver-staining and following electrophoresis of the total proteins of the membrane, with the phospholipase visualized by immunoblotting. Estimates for the molecular mass of the enzyme varied from 130 to 170 kDa in 7.5% gels and from 120 to 130 kDa in 5-10% gradient gels (with a best estimate of 120 kDa). Upon solubilization from the brush-border membrane by papain digestion, the major immunoreactive band migrated with an apparent mass of 80 kDa in both the 7.5% and 5-10% gradient gels. A major cross-reactive band was detected at 97 kDa following immunoblotting of the papain-solubilized proteins from guinea pig brush-border membranes, in agreement with the size of the purified fragment reported in the literature and at 140 kDa following immunoblotting of the intact proteins. Similar immunoblotting produced reaction with a 135-kDa protein from the rabbit brush-border membrane, as well as 95-kDa protein following papain solubilization. These results suggest that while there are species-specific apparent molecular weights, the intestinal brush-border membrane phospholipase B is conserved among species.
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PMID:Further characterization of a novel phospholipase B (phospholipase A2--lysophospholipase) from intestinal brush-border membranes. 171 22

A phospholipase A2 activity directed against phosphatidylcholine was previously described in brush-border membrane from guinea pig intestine (Diagne, A., Mitjavila, S., Fauvel, J., Chap, H., and Douste-Blazy, L. (1987) Lipids 22, 33-40). In the present study, this enzyme was solubilized either with Triton X-100 or upon papain treatment, suggesting a structural similarity with other intestinal hydrolases such as leucine aminopeptidase, sucrase, or trehalase. The papain-solubilized form, which is thought to lack the short hydrophobic tail responsible for membrane anchoring, was purified 1800-fold to about 90% purity by ion exchange chromatography on DEAE-Sephacel, gel filtration on Ultrogel AcA44, and hydrophobic chromatography on phenyl-Sepharose. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a main band with an apparent molecular mass of 97 kDa was detected under reducing and nonreducing conditions. In the latter case, phospholipase A2 activity could be recovered from the gel and was shown to coincide with the 97-kDa protein detected by silver staining. The enzyme activity was unaffected by EGTA and slightly inhibited by CaCl2. The purified enzyme displayed a similar activity against phosphatidylcholine and phosphatidylethanolamine, whereas 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine hydrolysis was reduced by 50% compared to diacylglycerophospholipids. Using phosphatidylcholine labeled with either [3H]palmitic acid or [14C]linoleic acid in the 1- or 2-positions, respectively, the purified enzyme catalyzed the removal of [3H]palmitic acid, although at a lower rate compared to [14C]linoleic acid. This resulted in the formation of sn-glycero-3-phosphocholine, but only 1-[3H]palmitoyl-sn-glycero-3-phosphocholine was detected as an intermediary product. In agreement with this, 1-acyl-2-lyso-sn-[14C]glycero-3-phosphocholine was deacylated at almost the same rate as the sn-2-position of phosphatidylcholine. Since upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the two hydrolytic activities were detected at the same position as 97-kDa protein, the enzyme is thus considered as a phospholipase A2 with lysophospholipase activity (phospholipase B), which might be involved in phospholipid digestion.
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PMID:Purification of a new, calcium-independent, high molecular weight phospholipase A2/lysophospholipase (phospholipase B) from guinea pig intestinal brush-border membrane. 272 44

Membrane-bound phospholipase B was purified to a homogeneous state from Torulaspora delbrueckii cell homogenate. Cell homogenate was extracted with Triton X-100, and the enzyme was precipitated with acetone. The acetone powder was washed repeatedly with Tris-HCl buffer (pH 8.0) until no phospholipae B activity was detected in the soluble fraction. The enzyme was extracted with Triton X-100 from the final residue and purified about 1,390-fold by sequential chromatofocusing, Sepharose 6B, and DEAE-Sephadex A-50 column chromatography. The final preparation showed a single broad protein band on SDS-polyacrylamide gel electrophoresis when stained with silver stain reagent and PAS-reagent. The molecular weight of phospholipase B was about 390,000 and 140,000-190,000 as estimated by gel filtration on Sepharose 6B and SDS-polyacrylamide gel electrophoresis, respectively, suggesting that phospholipase B is an oligomeric protein. The isoelectric point was at pH 4.5. Phospholipase B has two pH optima, one acidic (pH 2.5-3.0) and the other alkaline (pH 7.2-8.0). At acidic pH the phospholipase B activity was greatly increased in the presence of divalent metal ions, although metal ions are not a factor for enzyme activity. On the other hand, at alkaline pH the enzyme required Ca2+ or Mn2+ for activity. The pH- and thermal-stabilities at both pHs were similar. The phospholipase B hydrolyzed all diacylphospholipids tested at acidic pH, but hydrolyzed only phosphatidylcholine at alkaline pH. The hydrolysis rates of lysophospholipids were much higher (about 10-fold) than those of diacylphospholipids at both pHs.
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PMID:Purification and some properties of membrane-bound phospholipase B from Torulaspora delbrueckii. 318 65

Lysophospholipase released from rat platelets upon activation with thrombin has been purified to near homogeneity by sequential column chromatography on heparin-Sepharose, CM-Sephadex C-50, and TSK gel G2000SW. The final preparation showed a single band with a molecular mass of 32,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. The purified enzyme was heat-labile and inactivated after 5 min at 60 degrees C. It showed a broad pH optimum (pH 6-10) and required a divalent cation, such as Ca2+, for the optimal activity. Appreciable activity, however, was observed in the presence of EDTA. Lysophospholipase activity was inhibited by diisopropylfluorophosphate and dithiothreitol. This enzyme activity was retained by a concanavalin A-Sepharose column and eluted with methyl-alpha-D-mannoside. Treatment of lysophospholipase with peptide: N-glycosidase F gave degraded products, suggesting that this protein contain N-linked carbohydrate chains. The purified enzyme was specific to 1-acyl-sn-glycero-3-phospho-L-serine; none of lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, and 1-acyl-sn-glycero-3-phospho-D-serine was hydrolyzed appreciably.
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PMID:Purification and characterization of lysophospholipase released from rat platelets. 339 99

Rabbit myocardial cytosolic acyl coenzyme A (acyl-CoA) hydrolase activity was purified to near-homogeneity by ammonium sulfate precipitation and ion-exchange, gel filtration, chromatofocusing, and hydroxylapatite chromatographies. Kinetic analysis of the purified protein demonstrated a maximum velocity of 24 mumol/(mg . min) and an apparent Michaelis constant of 50 microM. Cytosolic acyl-CoA hydrolase and lysophospholipase activities cochromatographed in every fraction of every step. The purified protein was a single band (Mr 23 000) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. These results suggest that cytosolic lysophospholipase and palmitoyl-CoA hydrolase activities are catalyzed by a single polypeptide with dual activities. Palmitoyl-CoA competitively inhibited lysophospholipase activity (Ki = 4 microM). Low concentrations (20 microM) of lysophosphatidylcholine or L-palmitoylcarnitine increased palmitoyl-CoA hydrolase activity at low palmitoyl-CoA concentrations but had little effect at high concentrations of palmitoyl-CoA. In contrast, high concentrations (100 microM) of lysophosphatidylcholine or L-palmitoylcarnitine inhibited palmitoyl-CoA hydrolase activity. The results suggest that interactions between endogenous cardiac amphiphiles and palmitoyl-CoA hydrolase contribute to the regulation of intracellular long-chain acyl-CoA concentrations and therefore potentially modulate fluxes of fatty acid through several biochemical pathways.
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PMID:Purification of rabbit myocardial cytosolic acyl-CoA hydrolase, identity with lysophospholipase, and modulation of enzymic activity by endogenous cardiac amphiphiles. 614 28

Rabbit myocardial lysophospholipase-transacylase was purified 69,000-fold to near homogeneity by ammonium sulfate precipitation, DEAE-Sephacel, hydroxylapatite chromatography, and high precision liquid chromatography. The purified protein was a single band (Mr = 63,000) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. It had a specific activity of 4 mumol/mg/min for fatty acid release and 2 mumol/mg/min for phosphatidylcholine synthesis. Both its hydrolase and transacylase activities were saturated at a lysophosphatidylcholine concentration of 20 microM and transacylation was prominent at submicellar concentrations of substrate (2 microM). Fatty acid release obeyed Michaelian kinetics, but Line-weaver-Burk plots of transacylase activity were parabolic. In contrast, plots of the reciprocal of the initial reaction velocity of phosphatidylcholine formation (1/V) versus 1/[S]2 were linear. Computer simulations of a reaction mechanism in which two molecules of substrate formed a ternary complex with the enzyme resulted in linear Lineweaver-Burk plots for fatty acid release and linear 1/V versus 1/[S]2 plots for phosphatidylcholine synthesis. Low concentrations of long chain acylcarnitine (5-20 microM) markedly inhibited both fatty acid release and phosphatidylcholine synthesis. Inhibition of lysophospholipase-transacylase by L-palmitoylcarnitine was reversible by dilution or dialysis. Since long chain acylcarnitines increase in the cytosolic compartment of ischemic myocardium, these results suggest that inhibition of lysophospholipase-transacylase by long chain acylcarnitines contributes to the accumulation of lysophosphoglycerides in ischemic myocardium with consequent deleterious effects on membrane function.
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PMID:Rabbit myocardial lysophospholipase-transacylase. Purification, characterization, and inhibition by endogenous cardiac amphiphiles. 665 12


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