Cytosol

Since liver is the most important organ for metabolism investigations the procedures described here focus on liver cytosol exemplarily. Liver cytosol fraction contains soluble Phase I and Phase II enzymes which play an important role in drug metabolism (Brandon 2003). These are alcohol and aldehyde dehydrogenases, epoxide hydrolases, sulfotransferases, glutathione S transferase, N-acetyl transferases, and methyl transferases. Therefore, in cytosolic preparations these biotransformation steps can be studied. Cytosolic fractions are commercially available (BDGentest, Invitro Technologies, Xenotech and others) or easy to prepare, alternatively. 

In a typical procedure to study sulfation reactions (Tabrett 2003) the reaction mixtures consists of a 6.25 mM potassium phosphate buffer (pH 7.4), containing 60 pM adenosine 3 -phosphate 5 -phosphosulfate (PAPS) as a cofactor, 75 pg liver cytosolic protein and 0.125-100 pM substrate (4-nitrophenol in the 

Conditions for cytosolic incubations depend on the aim of the assay e.g. to cover specific enzymatic activity present in the cytosol. For this purpose it is essential to fortify the incubation medium with the specific cofactor for the reaction-if needed (Ekins 1999). (J -Nicotinamide adenine dinucleotide (NAD) is needed for alcohol and aldehyde dehydrogenases, flavin adenine dinucleotide (FAD) for polyamine oxidase, P-nicotinamide adenine dinucleotide phosphate (NADPH) for Dihydropyrimidine dehydrogenase. Phase II reactions depend on PAPS (sulfotransferases, 

Xu 2001), glutathione (glutathione S-transferases, Raney et al. 1992 Slone 1995), acetyl-coenzyme A (N-acetyltransferases) or S-adenosylmethionine (methyltransferases). NADPH as a cofactor has to be added if cytosolic reductases are the aim of interest (Inaba 1989). 

Obach (2004) set up a method for inhibition studies in cytosol using phthalazine as a substrate of aldehyde oxidase. In this system 0.05 mg protein/ml was used in 25 mm potassium phosphate buffer ph7.4 containing 0.1 mm ethylendiaminetetraacetic acid. Incubation is terminated after 2.5 min. 

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WS-7528 [132147-69-4][VI] a nonsteroidal estrogen, is an isoflavone which has been isolated from Streptomjces sp. No. 7528 and is an estrogen agonist. It inhibits [3ff]-estradiol binding to its receptor in rat uterine cytosol at an inhibitor for 50% of the rats tested (IC q) concentration of 5.7 nM. It also induces the growth of estrogen-dependent human breast cancer cell line MCE-7 (7). 

The specific role of vitamin A in tissue differentiation has been an active area of research. The current thinking, developed in 1979, involves initial dehvery of retinol by holo-B >V (retinol-binding protein) to the cell cytosol (66). Retinol is then ultimately oxidized to retinoic acid and binds to a specific cellular retinoid-binding protein and is transported to the nucleus. Retinoic acid is then transferred to a nuclear retinoic acid receptor (RAR), which enhances the expression of a specific region of the genome. Transcription occurs and new proteins appear during the retinoic acid-induced differentiation of cells (56). 

Another mechanism in initiating the contraction is agonist-induced contraction. It results from the hydrolysis of membrane phosphatidylinositol and the formation of inositol triphosphate (IP3)- IP3 in turn triggers the release of intracellular calcium from the sarcoplasmic reticulum and the influx of more extracellular calcium. The third mechanism in triggering the smooth muscle contraction is the increase of calcium influx through the receptor-operated channels. The increased cytosolic calcium enhances the binding to the protein, calmodulin [73298-54-1]. 

Spironolactone antagonizes the effects of aldosterone by binding at the aldosterone receptor in the cytosol of the late distal tubules and renal collecting ducts. Side effects of spironolactone are gynecomastia, decreased Hbido, and impotency. 

Figure 12.11 Schematic diagram of the ion pore of the K+ channel. From the cytosolic side the pore begins as a water-filled channel that opens up into a water-filled cavity near the middle of the membrane. A narrow passage, the selectivity filter, links this cavity to the external solution. Three potassium ions (purple spheres) bind in the pore. The pore helices (red) are oriented such that their carboxyl end (with a negative dipole moment) is oriented towards the center of the cavity to provide a compensating dipole charge to the K ions. (Adapted from D.A. Doyle et al.. Science 280 69-77, 1998.)...

While this electron flow takes place, the cytochrome on the periplasmic side donates an electron to the special pair and thereby neutralizes it. Then the entire process occurs again another photon strikes the special pair, and another electron travels the same route from the special pair on the periplasmic side of the membrane to the quinone, Qb, on the cytosolic side, which now carries two extra electrons. This quinone is then released from the reaction center to participate in later stages of photosynthesis. The special pair is again neutralized by an electron from the cytochrome. 

TABLE 5.11 Agents Causing Sustained Elevation of Cytosolic Ca and/or Impaired Synthesis of ATP... 

Nucleus The nucleus is separated from the cytosol by a double membrane, the nuclear envelope. The DNA is complexed with basic proteins (histones) to form chromatin fibers, the material from which chromosomes are made. A distinct RNA-rich region, the nucleolus, is the site of ribosome assembly. The nucleus is the repository of genetic information encoded in DNA and organized into chromosomes. During mitosis, the chromosomes are replicated and transmitted to the daughter cells. The genetic information of DNA is transcribed into RNA in the nucleus and passes into the cytosol where it is translated into protein by ribosomes. 

Osmotic pressure from high concentrations of dissolved solutes is a serious problem for cells. Bacterial and plant cells have strong, rigid cell walls to contain these pressures. In contrast, animal cells are bathed in extracellular fluids of comparable osmolarity, so no net osmotic gradient exists. Also, to minimize the osmotic pressure created by the contents of their cytosol, cells tend... 

FIGURE 9.14 Glycophorin A spans the membrane of the hnman erythrocyte via a single ff-helical transmembrane segment. The C-terminns of the peptide, whose sequence is shown here, faces the cytosol of the erythrocyte the N-terminal domain is extracellnlar. Points of attachment of carbohydrate groups are indicated. 

Knowles, B. H., Blatt, M. R., Tester, M., et al., 1989. A cytosolic 5-endo-toxin from Bacillus thurigiensis var. israelensis forms cation-selective channels in planar lipid bilayers. FEES Letters 244 259-262. 

FIGURE 10.15 A mechanism for Ca -ATPase from sarcoplasmic reticulum. Note the similarity to the mechanism of Na, K -ATPase (see also Figure 10.11). ( Out here represents the cytosol In represents the lumen of the SR.)... 

III. Tyr protein kinases A. Cytosolic tyrosine kinases src, fgr, abl, etc.) B. Receptor tyrosine kinases (RTKs) Plasma membrane receptors for hormones such as epidermal growth factor (EGF) or platelet-derived growth factor (PDGE) Raf (a protein kinase)... 

The cytosolic dyneins bear many similarities to axonemal dynein. The protein isolated from C. elegans includes a heavy chain with a molecular mass of approximately 400 kD, as well as smaller peptides with molecular mass ranging from 53 kD to 74 kD. The protein possesses a microtubule-activated ATPase... 

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