Subcellular fractions

In a study of the metabolism of methyl parathion in intact and subcellular fractions of isolated rat hepatocytes, a high performance liquid chromatography (HPLC) method has been developed that separates and quantitates methyl parathion and six of its hepatic biotransformation products (Anderson et al. 1992). The six biotransformation products identified are methyl paraoxon, desmethyl parathion, desmethyl paraoxon, 4-nitrophenol, />nitrophenyl glucuronide, and /wiitrophenyl sulfate. This method is not an EPA or other standardized method, and thus it has not been included in Table 7-1. 

Anderson PN, Eaton DE, Murphy SD. 1992. Comparative metabolism of methyl parathion in intact and subcellular fractions of isolated rat hepatocytes. Fundam Appl Toxicol 18 221-226. 

Vertebrate liver is a very rich source of enzymes that metabolize lipophilic xenobiotics, and subcellular fractions are prepared to study metabolism. Sometimes, other tissues such as brain, kidney, testis, and ovary are also treated in this way. A typical subcellular fractionation of liver might be as follows ... 

HydroxyNPYR [ ] has been identified as a urinary metabolite of NPYR in the rat (up to 1% of the dose), but was not detected when NPYR was incubated with subcellular fractions from rat liver and lung (13, 20). It has been proposed that further metabolism of leads to dimethy 1 amine, [ ], another urinary metabolite of NPYR, 2-Pyrrolidinone [3 ] has also been detected in the urine of rats treated with NPYR. Its origin has not been conclusively established, but it may form from pyrrol id inone-2-oxime (i6). 

Michel XR, PM Cassand, DG Ribera, J-E Narbonne (1992) Metabolism and mutagenic activation of benzo(a)pyrene by subcellular fractions from mussel (Mytilus galloprovincialis) digestive gland and sea bass (Discenthrarcus labrax) liver. Comp Biochem Physiol 103C 43-51. 

Comporti, M., Saccocci, C. and Dianzani, M.U. (1965). EfiFect of carbon tetrachloride in vitro and in vivo on lipid peroxidation of rat liver homogenates and subcellular fractions. Enzymologja 29, 185-204. 

Proteomics is a child of disparate parents the revolutionary advent of genomic sequencing and the evolutionary extension of mass spectrometry to permit the analysis of peptides. The fusion of these advances initially created a vision of full inventories of proteins in a biological unit, such as a cell, a subcellular fraction, or a physiological fluid. Good progress has been made toward this in some cases [11], but the focus is shifting from encyclopedic surveys toward an emphasis on quantitative... 

It has been suggested [6] that these unusual sterols, especially in those cases where these unusual sterols comprise the entire sterol content of the organisms, likely replace conventional sterols as cell-membrane components. Evidence for this comes from subcellular fractionation and subsequent analysis of two marine sponges [10]. The sterol composition of the membrane isolates was found to be identical to that of the intact sponge. Most common variation of the marine sterol is in the side-chain, situated deep in the lipophylic environment of the phospholipid bilayer. This suggests that unusual fatty acids might accompany the sterols, and indeed this is often the case [8]. 

The composition of myelin is well characterized because it can be isolated in high yield and purity by subcellular fractionation 56 Central nervous system myelin is enriched in certain lipids 56 Peripheral and central nervous system myelin lipids are qualitatively similar 58... 

Histamine synthesis in the brain is controlled by the availability of L-histidine and the activity of histidine decarboxylase. Although histamine is present in plasma, it does not penetrate the blood-brain barrier, such that histamine concentrations in the brain must be maintained by synthesis. With a value of 0.1 mmol/1 for L-histidine under physiological conditions, HDC is not saturated by histidine concentrations in the brain, an observation that explains the effectiveness of large systemic doses of this amino acid in raising the concentrations of histamine in the brain. The essential amino acid L-histidine is transported into the brain by a saturable, energy-dependent mechanism [5]. Subcellular fractionation studies show HDC to be localized in cytoplasmic fractions of isolated nerve terminals, i.e. synaptosomes. 

Considerable evidence suggests that the major site of calcium sequestration and the source of intracellular calcium for signaling is the ER. In addition to the points already made, subcellular fractionation studies have demonstrated positive correlations of ATP-dependent Ca2+ accumulation and of IP3-mediated Ca2+ release with classical enzymatic markers for ER. Calcium uptake into the IP3-sensitive store is augmented by oxalate this augmentation is a property generally associated with the ER. 

Birkle, D. L. and Bazan, N. G. Effect of bicuculline-induced status epilepticus on prostaglandins and hydroxyeicosatet-raenoic acids in rat brain subcellular fractions /. Neurochem. 48 1768-1778.1987. 

Neumann, P.E. and F. Taketa. 1987. Effects of triethyltin bromide on protein phosphorylation in subcellular fractions from rat and rabbit brain. Mol. Brain Res. 2 83-87. 

Farrow, J. T., and Van Vunakis, H. (1973) Characteristics of D-lysergic acid diethylamide binding to subcellular fractions derived from rat brain. Biochem. Pharmacol., 22 1103-1113. 

Lau, D. T. W., Benet, L. Z., Nitroglycerin metabolism in subcellular fractions of rabbit liver. Dose dependency of glyceryl dinitrate formation and possible involvement of multiple isoenzymes of glutathione S-transferases. Drug Metab. Dispos. 18 (1990), p. 292-297... 

However, the reaction of NP with thiols may be a necessary but not sufficient cause for the release of NO from the ion as there are many thiols in frog heart tissue and NP is a vasodilator only under illumination. Furthermore Sogo et al. [50] could not detect NO generation from NP in human plasma containing cysteine, glutathione, homocysteine and reduced cysteine residues. Therefore, there must be a unique component of mammalian tissues which is involved in the release of NO from NP, and this reaction comes after reaction with thiol. Kowaluk et al. [51] report that NP is readily metabolised to NO in subcellular fractions of bovine coronary arterial smooth muscle and that the dominant site of metabolism is in the membrane fraction. This led to the isolation of a small membrane-bound protein or enzyme that can convert NP into NO. The mechanism shown in Scheme 8.2 combines the thiol reaction and that with an enzyme. 

Cribb AE, Nuss CE, Alberts DW, et al. Covalent binding of sulfamethoxazole reactive metabolites to human and rat liver subcellular fractions assessed by immunochemical detection. Chem Res Toxicol 1996 9(2) 500-507. 

The secretory vesicles have recently been discovered by Borregaard and co-workers (Sengelpv, Nielson Borregaard, 1992). These are very difficult to separate from the plasma membrane on density gradients. They possess latent alkaline phosphatase activity (i.e. subcellular fractions must be incubated with detergents such as Triton to release activity) and albumin, whilst the membranes contain CR1, CR3 and the fMet-Leu-Phe receptor. They are endocytic vesicles but can be rapidly translocated to the plasma membrane. 

Experiments using fluorescein-labelled fMet-Leu-Phe indicate that the majority of re-expressed receptors that appear on the plasma membrane within 4-10 min after stimulation arise from the mobilisation of internal pools. Subcellular fractionation studies indicate that the pools of these receptors are the membranes of specific granules, although it is possible that these are on other membranes (e.g. on gelatinase-containing granules or se-... 

Charles AK, Rosenbaum DP, Ashok L, et al. 1985. Uptake and disposition of mirex in hepatocytes and subcellular fractions in CD1 mouse liver. J Toxicol Environ Health 15 395-403. 

Dinitrobenzene is an intermediate employed in chemical syntheses of a large number of compounds used in the dye, explosives and plastics industry. The compound is known to induce methemoglobinemia and to cause testicular toxicity with the Sertoli cell being the major target. Nitro reduction was observed in erythrocytes, in rat Sertoli-germ cell cocultures and in rat testicular subcellular fractions, and it was shown that 3-nitrosonitrobenzene was formed that was considerably more toxic. Testicular toxicity was enhanced when the intracellular thiol levels were reduced by pretreatment with diethylmaleate. In turn, pretreatment with cysteamine or ascorbate reduced the toxicity of 1,3-dinitrobenzene and 3-nitrosonitrobenzene. 

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