Resolution and reconstitution

The PPase activity may be removed from beef heart submitochondrial particles by washing with 0.25 M sucrose. The removal is parallel with loss of PP synthesis [71]. 

The PPase protein isolated from an acetone powder of mitochondrial PPase IT, probably consisting of only the a and /3 subunits of the total membrane bound PPase, can reconstitute both PPase and PPj synthesis activities, the latter to above the original rate [68]. The main difference between PPase IT and PPase T appears 

Since the ATP synthesis is highly active in the resolved particles it seems unlikely that the removal of PPase 11 causes much damage to the membrane structure. It also seems unlikely that the whole membranous protein has been removed, and our guess is that the interconvertibility between the two enzymes described [73] is limited to the two smallest subunits of the preparation described in Ref. 71. 

No -pumping activity has yet been described for either preparation and this aspect of the coupling between electron transport and PP synthesis in the mitochondrial system is thus still an unsolved question. 

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Lu, A. Y. H. and Levin, W. The resolution and reconstitution of the liver microsomal hydroxylation system. Biochim. 

In contrast to the above reductases which contain only a single flavin moiety, NADPH-cytochrome P450 reductase contains both FMN and FAD One flavin may be presumed to accept two reducing equivalents from NADPH while the other serves as a one-electron reductant for the heme iron of cytochrome 450- Flavin resolution and reconstitution studies have shown that the FAD moiety is the low-potential flavin that accepts reducing equivalents from NADPH while the FMN moiety is the high potential flavin that serves as the one-electron reductant for cytochrome 1 450 ... 

Xie, X. S., and Stone, D. K. (1988). Partial resolution and reconstitution of the subunits of the clathrin-coated vesicle proton ATPase responsible for Ca2+-activated ATP hydrolysis. /. Biol. Chem. 263, 9859-9867. 

Fia. 24. Effect of temperature on the resolution of complex II. Complex II was suspended in a solution containing 30 mil/ Tricine, 100 mil/ boric acid, 20 mM succinate and S mM dithiothreitol. pH of the buffer was adjusted to 8.1 with NaOH. Complex II was resolved by addition of 1.0 M NaC10< at the temperatures indicated. After addition of NaClOi the concentration of complex II was 8 mg/ml. (a) Resolution at 0°, 10°, and 30°. (b) Increased resolution and reconstitution as the temperature was changed from (O) 0° to ( ) 30° and from 30 to 0°, respectively. Assays were performed at 38°. From Davis and Hatefi (1S9). 

Fig. 9. (A) EPR spectra of the PS-1 core complex [P700 FeS-X] (a), the complex in which FeS-X was removed (b), and the reconstituted complex (c) (B) Flash-induced absorbance changes monitored at 698 nm for samples (a), (b) and (c) in (A) and their decay kinetics. Figure source Parrett, Mehari and Golbeck (1990) Resolution and reconstitution of the cyanobacteria photosystem I complex. Biochim Biophys Acta 1015 348, 349...

Suzuki I, Chan CW, Takeuchi TL (1992) Oxidation of elemental sulfur to sulfite by Thiobacillus thiooxidans cells. Appl Environ Microbiol 58 3767-3769 Suzuki I, Dular U, Kwok S-C (1974) Ammonia or ammonium ion as substrate for oxidation by Nitrosomonas europaea cells and extracts. J Bacteriol 120 556-558 Suzuki I, Kwok S-C (1970) Cell-free ammonia oxidation by Nitrosomonas europaea effects of polyamines, Mg2+ and albumin. Biochem Biophys Res Commun 39 950-955 Suzuki I, Kwok S-C (1981) A potential resolution and reconstitution of the ammonia oxidizing system of Nitrosomonas europaea. Role of cytochrome c-554. Can J Biochem 59 484-488... 

Breskvar, K., B. Cresnar, and T. Hudnik-Plevnik (1987). Resolution and reconstitution of cytochrome P450 containing steroid hydroxylation... 

The resolution and reconstitution experiments described above indicate that the E. coli pyruvate dehydrogenation complex is a highly organized multienzyme unit. The number of molecules of carboxylase and of flavoprotein per molecule of complex have been calculated. Those numbers are about 12 for the carboxylase and about 6 for the flavoprotein. Each mole-... 

The results of the resolution and reconstitution experiments carried out with the E. coli pyruvate dehydrogenation complex indicate that there are specific binding sites on the lipoic reductase-transacetylase component for the carboxylase and the flavoprotein. In other words, the latter two enzymes appear to be specifically oriented with respect to the lipoic acid bound to the lipoic reductase-transacetylase component. It is evident from the re-... 

RESOLUTION AND RECONSTITUTION OF THE PHOTOSYSTEM I REACTION CENTER COMPLEX IN GREEN PLANTS AND CYANOBACTERIA... 

Yamashita, S., Racker, E. Resolution and reconstitution of the mitochondrial electron transport system. II. Reconstitution of succinoxidase from individual components. J. biol. Chem. 244, 1220-1227 (1969)... 

The fatty acid desaturase system was one of the first microsomal enzymes for which a lipid requirement was established. Using Fleischer s procedure of aqueous acetone extraction (Lester Fleischer, 1961) we were able to reduce the desaturase activity of hen liver microsomes to very low levels and restore activity completely with a mixture of lipids isolated from the microsomes (Holloway Peluffo, 1964 Jones at al., 1969). Shortly after this observation cytochrome bs was implicated in the desaturation process (Oshino et al., 1966). Progress in the isolation and purification of the stearyl CoA desaturase has been slow and it is only in the last two years that the complete resolution and reconstitution of the stearyl CoA desaturase has been achieved by Strittmatter and co-workers (Strittmatter et al., 1974). 

Rabon EC, Smillie K, Seru V, et al. Rubidium occlusion within tryptic peptides of the H,K-ATPase. J Biol Chem 1993 268 8012-8018. Racker E. Resolution and reconstitution of biological pathways from 1919 to 1984. Fed Proc 1983 42 2899-2909. de Reaumur RAF. Sur la digestion des oiseaux. Mem Acad Roy d Sc Paris 1752 266. 

Table I. Resolution and Reconstitution of Photosynthetic Phosphorylation in Chloroplast Membranes of Maize"...

To understand the mechanism of photosynthetic energy transformation, complete knowledge of the components involved is essential. To study the photosynthetic ATPsynthase, resolution and reconstitution experiments have been carried out now for 20 years (Avron,1963). Numerous details and analogies to the ATPsyntha-ses of oxidative phosphorylation in bacteria and mitochondria have been elucidated (Munoz,1982). 

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