1.Lipoxygenase-mediated N-demethylation of pesticides in vitro.
Chinese Journal of Industrial Hygiene and Occupational Diseases 2002;20(6):409-412
OBJECTIVEIn order to explore the pathway of dealkylation of pesticides other than cytochrome P450 monocoxygenases, lipoxygenase (LOX)-mediated demethylation of aminocarb and some other pesticides were measured.
METHODFormaldehyde generated in the reaction was estimated by Nash reaction to express the rate of demethylation of pesticides mediated by soy lipoxygenase (SLO).
RESULTSN-demethylation of aminocarb mediated by SLO was found to depend on the incubation time, concentration of the enzyme, concentration of aminocarb and hydrogen peroxide. Under optimal conditions, Vmax value of 18 nmol of formaldehyde.min-1.nmol-1 of lipoxygenase was observed. The reaction exhibited Km values of 3.4 mmol/L for aminocarb and 235 mumol/L for hydrogen peroxide. A strong inhibition of the reaction by nordihydroguaiaretic acid, gossypol, and phenidone clearly implicated the lipoxygenase involvement as the protein catalyst. A significant decline in the formaldehyde accumulation in the presence of either reduced glutathione or dithiothreitol suggested generation of a free radical species as an initial oxidation intermediate during the demethylation of aminocarb by SLO. The inhibition of formaldehyde generation by butylated hydroxyanisole(BHT) and butylated hydroxy toluene(BHA) further supported this contention. In addition to aminocarb, seven other pesticides were also found to undergo N-demethylation, albeit at relatively low rates.
CONCLUSIONCertain pesticides may oxidatively undergo dealkylation via the lipoxygenase pathway in animals and plants.
Butylated Hydroxyanisole ; pharmacology ; Butylated Hydroxytoluene ; pharmacology ; Dealkylation ; Free Radicals ; Lipoxygenase ; physiology ; Pesticides ; metabolism ; Phenylcarbamates ; metabolism ; Soybeans ; enzymology
2.O-GlcNAc modification on IRS-1 and Akt2 by PUGNAc inhibits their phosphorylation and induces insulin resistance in rat primary adipocytes.
Seung Yoon PARK ; Jiwon RYU ; Wan LEE
Experimental & Molecular Medicine 2005;37(3):220-229
It has been known that O-linked beta-N-acetylglucosamine (O-GlcNAc) modification of proteins plays an important role in transcription, translation, nuclear transport and signal transduction. The increased flux of glucose through the hexosamine biosynthetic pathway (HBP) and increased O-GlcNAc modification of protein have been suggested as one of the causes in the development of insulin resistance. However, it is not clear at the molecular level, how O-GlcNAc protein modification results in substantial impairment of insulin signaling. To clarify the association of O-GlcNAc protein modification and insulin resistance in rat primary adipocytes, we treated the adipocytes with O-(2-acetamido-2deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), a potent inhibitor of O-GlcNAcase that catalyzes removal of O-GlcNAc from proteins. Prolonged treatment of PUGNAc (100 micrometer for 12 h) increased O-GlcNAc modification on proteins in adipocytes. PUGNAc also drastically decreased insulin-stimulated 2-deoxyglucose (2DG) uptake and GLUT4 translocation in adipocytes, indicating that PUGNAc developed impaired glucose utilization and insulin resistance in adipocytes. Interestingly, the O-GlcNAc modification of IRS-1 and Akt2 was increased by PUGNAc, accompanied by a partial reduction of insulin-stimulated phosphorylations of IRS-1 and Akt2. The PUGNAc treatment has no effect on the expression level of GLUT4, whereas O-GlcNAc modification of GLUT4 was increased. These results suggest that the increase of O-GlcNAc modification on insulin signal pathway intermediates, such as IRS-1 and Akt2, reduces the insulin-stimulated phosphorylation of IRS-1 and Akt2, subsequently leading to insulin resistance in rat primary adipocytes.
Acetylglucosamine/*analogs & derivatives/metabolism/pharmacology
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Adipocytes/*metabolism
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Animals
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Deoxyglucose/pharmacokinetics
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Glycosylation
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Immunoprecipitation
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*Insulin Resistance
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Male
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Monosaccharide Transport Proteins/metabolism
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Oximes/*pharmacology
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Phenylcarbamates/*pharmacology
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Phosphoproteins/*metabolism
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Phosphorylation
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Protein-Serine-Threonine Kinases/*metabolism
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Proto-Oncogene Proteins/*metabolism
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Rats
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Rats, Sprague-Dawley
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Research Support, Non-U.S. Gov't
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Subcellular Fractions/metabolism
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beta-N-Acetylhexosaminidase/antagonists & inhibitors