Cytosolic components

Experiments in the mid-1980s, analysing the phosphorylation profiles of neutrophils from autosomal recessive forms of CGD, indicated that a component of around 45-47 kDa failed to become phosphorylated during activation with PMA. In normal neutrophils, this component is phosphorylated in the cytoplasm and then translocates to the plasma membrane. Curiously, in CGD neutrophils of patients that lacked the cytochrome b, the 47-kDa component was phosphorylated normally but failed to become incorporated into the plasma membrane. This strongly implied the following  

At around the same time as these discoveries, several groups were trying to determine the ways in which the NADPH oxidase could be activated in vitro (i.e. activated in broken-cell suspensions). The ultimate aim of such studies was to determine the minimal components necessary for assembly and activation of the oxidase in in vivo experiments. Thus, once these minimal constituents were identified, oxidase activity could then be reconstituted in vitro from the individual component parts. The first breakthrough in these studies was 

Most of the patients with autosomal recessive CGD lack pAl-phox whilst the remainder lack p66-phox (details are given in 8.2). Recombinant proteins derived from these full-length cDNA clones can restore activity when added to extracts of autosomal recessive CGD neutrophils, in the cell-free oxidase activation system. 

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Recently, a potential cytosolic component of the MEP precursor pathway, xylulose kinase, has been cloned and tested for function in an Escherichia coli complementation system. " The kinase activates exogenous xylulose in the cytoplasm. DXP is the precursor for DXS, which resides in the plastid, suggesting the activated substrate must be transported into the plastid. Another xylulose kinase homologue in Arabidopsis that contains a plastid targeting sequence was not active in the E. coli system, suggesting that it may have some other function in the plastid. Perhaps plant and bacterial tissue cultures may be fed xylulose to condition accumulation of isoprenoid metabolites. 

Primary and secondary products, and end-products of lipid peroxidation have all been shown to accumulate in senile cataracts (Babizhayev, 1989b Simonelli et al., 1989). Accumulation of these compounds in the lenticular epithelial membranes is a possible cause of damage preceding cataract formation. In senile cataracts there is also extensive oxidation of protein methionine and cysteine in both the membrane and cytosol components (Garner and Spector, 1980), while in aged normal lenses a lesser extent of oxidation was confined to the membrane. The authors therefore suggested that oxidation of membrane components was a precataract state. 

Abo, A., Pick, E. (1991). Purification and characterisation of a third cytosolic component of the superoxide-generating NADPH oxidase of macrophages. J. Biol. Chem. 266, 23577-85. 

Volpp, B. D., Nauseef, W. M Donelson, J. E Moser, D. R Clark, R. A. (1989). Cloning of the cDNA and functional expression of the 47-kilodalton cytosolic component of human respiratory burst oxidase. Proc. Natl. Acad. Sci. USA 86, 7195-9. Watson, F., Robinson, J. J., Edwards, S. W. (1991). Protein kinase C dependent and independent activation of the NADPH oxidase of human neutrophils. J. Biol. Chem. 266, 7432-9. 

Messenger RNA molecules for both subunits of the cytochrome and the two cytosolic components are detectable in unstimulated bloodstream cells. Experiments involving incubation of neutrophil suspensions with the protein synthesis inhibitor cycloheximide indicate that constitutive expression of one or more components of the oxidase is required for the neutrophil to maintain its ability to generate reactive oxidants. For example, when neutrophils are incubated in vitro with cycloheximide, their ability to generate reactive oxidants declines more rapidly than in control cells, as they age in culture (Fig. 7.12). This decline in oxidase activity when protein biosynthesis is blocked is not due to cell death, because cells treated with cycloheximide for this time still exclude trypan blue. Furthermore, when protein biosynthesis is stimulated in neutrophils by the addition of GM-CSF for 24 h in vitro, the ability to generate reactive oxidants is enhanced considerably above the levels observed in untreated cells. 

Figure 12.4 Effector mechanism direct activation of a protein kinase. The protein kinase is the cytosolic component of the hormone receptor. Activation of the kinase results in phosphorylation of a protein which initiates the effects of the hormone. An example of this is the effector mechanism of insulin, which is a tyrosine kinase.

Saitoh H, Pu R, Cavenagh M, Dasso M (1997) RanBP2 associates with Ubc9p and a modified form of RanGAPl. Proc Natl Acad Sci USA 94 3736-3741 Sakata N, Stoops JD, Dixon JL (1999) Cytosolic components are required for protea-somal degradation of newly synthesized apolipoprotein B in permeabilized HepG2 cells. J Biol Chem 274 17068-17074... 

Fatty acid biosynthesis is made irreversible by the specific input of energy. Name the reactions or give equations for those steps in the pathway that require ATP. It is important that you consider both the mitochondrial and cytosolic components of the pathway. 

Fig. 4.1 Mechanism of action of cyclosporine. Cyclosporine readily diffuses into the cytoplasm of the target cells where it binds to cyclophilins. The cyclosporine-cyclophilin complex stably associates with calcineurin and inhibits calcineurin activity. Calcineurin is a Ca2+-dependent enzyme— serine/threonine phosphatase— which after activation by Ca2+, dephosphorylates a cytosolic component of NFAT (NFATc, cytosolic factor of activated T cells). After dephosphorylation, NFATc migrates from the cytoplasm to the nucleus where it associates with NFATn and induces transcription of several cytokine genes including IL-2. Cyclosporine inhibits calcineurin activity after associating with cyclophilins, resulting in the inhibition of IL-2 production and other cytokines (see Color Insert)...

Today, we realize that drug binding/receptor sites that produce pharmacological effects may be part of any cellular constituent for example, nuclear DNA, mitochondrial enzymes, ribosomal RNA, cytosolic components, and cell membranes and wall, to name the most obvious. Nevertheless, in contemporary pharmacology, some authors and researchers apply a more restricted use of the term receptor, reserving it for protein complexes embedded in, and spanning, cellular membranes. However, exceptions to this classification system clearly exist. For example, steroids are known to interact with cytosolic receptors that transport them into the nucleus (their site of... 

Principle In this procedure erythrocytes are treated with Triton X-100 which is reported to solubilize the membrane lipid leaving the underlying cytoskeletal network intact. The cyto-skeletons are separated from cytosolic components, Triton and solubilized lipid by centrifugation through a sucrose solution. The high salt concentration of the sucrose solution ensures the removal of residual lipid and integral membrane proteins from the cytoskeletal network. 

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