Liver microsomes, electrophoresis

Figure 5.54 Structures of Praziquantel and its metabolites, cis- and fraw5-4-hydroxy-praziquantel. Reprinted from 7. Chromatogr., B, 708, Lerch, C. and Blaschke, G., Investigation of the stereoselective metabolism of Praziquantel after incubation with rat liver microsomes by capillary electrophoresis and liquid chromatography-mass spectrometry , 267-275, Copyright (1998), with permission from Elsevier Science.

Busby WF, Ackermann JM, Crespi CL (1999) Effect of methanol, ethanol, dimethyl sulfoxide, and acetonitrile on in vitro activities of cDNA-expressed human cytochromes P-450. Drug Metab Dispos 27 246-249 Clohs L, Wong J (2002) Validation of a capillary electrophoresis assay for assessing the metabolic stability of verapamil in human liver microsomes. J Cap Elec Microchip Tech 7 113 Coughtrie MWH, Fisher MB (2003) The role of sulfotransferase and UDP-glucuronosyltransferases. In Lee JS, Obach RS, Fisher MB (eds) Drug Metabolizing Enzymes. Marcel Dekker, New York pp 541-575... 

Galeva, N. and Altermann, M. (2002) Comparison of one-dimensional and two-dimensional gel electrophoresis as a separation tool for proteomic analysis of rat liver microsomes cytochromes P450 and other membrane proteins. Proteomics 2, 713-722. 

One of the most interesting problems concerning the cytochrome P-450 system has been its multiplicity, for it is difficult to conceive of a single enzyme system having the ability to oxidise in a specific way such a wide variety of substrates. Current data suggest that there are at least three forms of the enzymes present in rat liver microsomes. Optical spectra reveal two forms of the CO-bound reduced cytochrome P-450, one with a maximum at 448 nm (such as is induced by 3-methylcholanthrene) and one at 450 nm (as is induced by phenobarbital), and SDS (sodium dodecyl sulphate) polyacrylamide gel electrophoresis shows three peaks corresponding to molecular weights of 44000 (phenobarbital), 50000, and 53000 (3-methylcholanthrene) [117]. Differences are also seen in the ESR spectra of P-450 in rat liver microsomes induced by different chemicals and there are further differences when... 

Figure 6. Representation of SDS electrophoresis of liver microsomes prepared from normal and ascorbic acid deficient guinea pigs (32),...

Cardoso, C. D., labor, V. A. R, andBonato, P. S., Capillary electrophoretic chiral separation of hydroxychloroquine and its metabolites in the microsomal fraction of liver homogenates. Electrophoresis, 27, 1248-1254, 2006. 

Clohs, L. and Wong, J., Validation of a capillary electrophoresis assay for assessing the metabolic stability of verapamil in human liver microsomes, J. Capillary Electrophor, 1,113-117, 2002. 

Fig. 9.2 Relative concentrations of P450 in human liver microsomes. a P450s in 60 liver samples were estimated using immunochemical methods (electrophoresis/immunoblot-ting) [52]. Because of cross-reactivity, the individual P450s in subfamilies are not distinguished. The unknown fraction is the difference between the sum of the immunochemi-cally determined forms and the total amount, calculated from Fe -CO versus Fe " difference spectroscopy [53]. b-d Estimates were made using liquid chromatography-mass spectrometry (LC-MS) proteomic analysis with heavy-atom peptides, b Results of an analysis of 50 pooled human liver samples (XenoTech, HLM610 preparation) [54]. c Results reported in the same reference as Part 5 [54] as means from analysis often individual human samples, d Analysis of a pooled set of 23 human liver samples by another laboratory [55]...

Protein modification by phospho adenylylation. When we compared incorporation of adenine equivalents from labeled NAD and labeled NADP into the acid-insoluble fraction of rat liver microsomes, NADP proved to be a much better precursor than NAD. SDS gel electrophoresis revealed a... 

The isoenzymes of rat-liver microsomal /S-D-glucuronidase showed different responses to such dissociating agents as urea, guanidinium hydrochloride, and sodium dodecyl sulphate. Loss of catalytic activity in the denaturing media is accompanied by dissociation of the enzymes into subunits. Gel electrophoresis in the presence of sodium dodecyl sulphate revealed that the enzymes are tetramers consisting of different proportions of three types of glycoprotein subunit (mol. wts. 6.29 X 10 , 6.02 X 10 , and 5.87 x 10 ). 

We have also subjected mouse liver enzyme to electrophoresis at pH 7.5, in contrast to the system already described which runs at pH 9.5. As shown by the gel on the left side of Fig. 11, liver glucuronidase activity of DBA mice is resolved into five components after electrophoresis in the pH 7.5 system. The intensely staining anodal component represents lysosomal enzyme, since it is the only component observed in liver extracts of YBR mice, as shown by the gel on the right side of the Fig. 11. The other four components of DBA enzyme are presumably of microsomal origin, being unresolved in the pH 9.5 system where they migrate as a single component. 

Human intestinal microsomes effectively hydrolyzed frans-permethrin however, bioresmethrin, and deltamethrin were not metabolized in the intestine to any appreciable extent. Human hepatic microsomes and cytosol contained both hCE-1 and hCE-2 when examined by native PAGE (polyacrylamide gel electrophoresis) human intestine contained only hCE-2. Table 8 gives the kinetic parameters obtained by Crow et al. (2(X)7) with tranr-permethrin and liver and intestinal carboxylesterases. 

Gel electrophoresis showed that rabbit Oryctolegus cuniculus) and, probably, rat (Ratius norvegiciis) livers contain variants of jS-D-glucuronidase corresponding to those in mouse Mus musculus) tissues.Microsomal and lysosomal fractions from rabbit liver differed only quantitatively in the j3-D-glucuronidases present. 

The lysosomal form of jS-o-glucuronidase in mouse liver has been purified to homogeneity. Electrophoresis on polyacrylamide gel showed that the enzyme is monomeric. However, microsomal forms of jS-o-glucuronidase are spontaneously converted into the l form (mol. wt. 2.8—3.0 x 10 ), which is composed of four identical subunits (mol. wt. 7.5 x 10 ) and contains D-galactose (0.23%), o-glucose (0.44%), o-mannose (4.52%), and 2-amino-... 

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