Partial reconstitution

Retention of a protein or protein activity after 105,000y, 1 hr Chromatography on gel filtration columns with large pore sizes Electron microscopy—however, sample preparation may partially reconstitute membranes Decrease in solution turbidity, which may be detected by a diminution in light scattering or an enhancement in light transmission Diffusion of membrane lipids as assayed by nuclear magnetic resonance and electron spin resonance... 

These ideas have been extended by Green, Harbottle, and Maddock (IS) to cases where oxide ions are lost. They supposed that the important reaction of the recoil species at the end of its track was the loss of one or more oxide ions, or conversely, incomplete readdition of oxide ions to the recoil atom. Then each partially-reconstituted species (for example, MnOr, MnOs+, Mn02+ +, etc.) will suffer either hydration (which will reconstitute the parent) or reduction on dissolution in water. The argument runs that the greater the oxidation potential of the parent anion, the sooner will a species be reached (through successive loss of oxide ions) which will inevitably oxidize water on dissolution, and consequently the lower the retention. This rough relationship is indicated by Fig. 1 in... 

Fl-E "kerlund (1981) Partial reconstitution of photosynthetic water splitting in inside-out thylakoid vesicles. In G Akoyunoglou (ed) Proc Intern Congr on Photosynthesis, Vol. 2 465-468... 

M Miyao and N Murata (1984) Partial reconstitution of the photosynthetic oxygen evoiution system byrebinding of the 33 kDa poiypeptide. FEBS Lett 164 375-378... 

Enzymic Properties of the Native Protein, the Partially Reconstituted Protein and the Fully Reconstituted Erythrocuprein... 

CaCl2 washed particles with rebound 33 kDa protein from spinach and E.coli were investigated by O2 evolution assay. The rebinding of the protein from both sources were concomitted by a partial reconstitution of oxygen evolution at the same rates. 

The possible presence of an electron carrier between NADPH-cytochromc c reductase and cytochrome P-450 remains to be established (question mark in Figure 3). It is noteworthy that Lu and Coon have succeeded in solubilizing and partially reconstituting a microsomal enzyme system that catalyses the oi-oxidation of fatty acids and the N-demethylation of drugs but thus far have failed to detect the presence of a carrier. 

Squalene epoxidase, like most enzymes responsible for the later steps of sterol biosynthesis [43, 51], is membrane-bound which makes its purification in native form challenging. The purification is additionally complicated by the presence of a large number of cytochrome P450 and other enzymes that have similar hydro-phobicity and size as squalene epoxidase and are hence difficult to remove [52]. Most studies have been carried out with rat liver microsome squalene epoxidase either partially purified or as a homogenate of the cell membrane fraction. In vitro reconstitution of squalene epoxidase activity is absolutely dependent on molecular oxygen, NADPH, FAD, and NADPH-cytochrome c reductase [52, 53]. In this respect, squalene epoxidase resembles the cytochrome P450 enzymes described... 

Shimomura, O., and Shimomura, A. (1981). Resistivity to denaturation of the apoprotein of aequorin and reconstitution of the luminescent photoprotein from the partially denatured apoprotein. Biochem. J. 199 825-828. 

Biochemical studies with purified preparations incorporated into liposomes have also been performed [32,33,96-98]. Reconstituted receptors from skeletal muscle bound DHPs, PAAs and diltiazem with high affinity and in a 1 1 1 stoichiometry [97], In general, the reconstituted proteins exhibit the characteristic pharmacological properties expected for these channels. In recent studies, our laboratory has reconstituted partially purified channels into liposomes containing the Ca -sensitive fluorescent dye, fluo-3 [33,96]. These channels exhibit Ca influx that is sensitive to activation by Ca channel activators and inhibitors with affinities similar to those observed in intact cells, and the Ca influx is dependent on the establishment of a gradient in the presence of valinomycin [132]. This assay provides a convenient and rapid approach to obtaining a macroscopic picture of the activity of the channels under different conditions, while the more complex studies in lipid bilayers provide a more complete analysis of the single channel behavior. 

Schmidt The liver is sort of an example, and the practical answer to the question of why it is useful to know is because sometimes we want to reconstitute livers. When you do a two-thirds partial hepatectomy all the cells divide. Suppose you push this to a limit and take out 90% of a liver, the hope is that there is something called a liver stem cell which is at something called GO, which is capable of reconstituting 90% as opposed to 67%. Is this a practical definition of GO ... 

After removal of free Emulgen 913 from partially purified hepatic cytochrome P-448 of DBA-treated male skates an active mixed-function oxidase system was reconstituted by preincubating the cytochrome with purified rabbit hepatic NADPH-cytochrome o... 

Figure 7. Profile of radioactive metabolites obtained upon incubation of UC-BP with a reconstituted system containing partially purified Cytochrome P-448 from DBA-pretreatea male little skates.

Figure 4. In vitro reconstituted 30 nm chromatin fiber. Dynamic structural changes in the chromatin fiber in the absence (top) or presence (bottom) of linker histone HI with different NaCl concentration were observed by AFM. Nucleosomes were reconstituted on the 106 kb plasmid and then fixed in the buffer containing 50 mM (top left) or 100 mM NaCl (top right). Nucleosomes were well-spread in 50 mM NaCl but attached each other and partially aggregated in 100 mM NaCl. After the addition of histone HI, the thicker fibers were formed. The width of the fibers is 20nm in 50mM NaCl (bottom left) or 30 nm in lOOmM NaCl (bottom right)...

Simpson RT, Thoma F, Brubaker JM (1985) Chromatin reconstituted from tandemly repeated cloned DNA fragments and core histones a model system for study of higher order structure. Cell 42 799-808 Sugiyama S, Yoshino T, Kanahara H, Kobori T, Ohtani T (2003) Atomic force microscopic imaging of 30 nm chromatin fiber from partially relaxed plant chromosomes. Scanning 25 132-136 Sugiyama S, Yoshino T, Kanahara H, Shichiri M, Fukushi D, Ohtani T (2004) Effects of acetic acid treatment on plant chromosome structures analyzed by atomic force microscopy. Anal Biochem 324 39 4... 

Bergh AF, Strobel HW. 1992. Reconstitution ofthe brain mixed function oxidase system purification of NADPH-cytochrome P450 reductase and partial purification of cytochrome P450 from whole rat brain. J Neurochem 59 575-581. 

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