Reconstitution of activity

Goloubinoff, P., Christeller, J.T., Gatenby, A.A., Lorimer, G.H. (1989). Reconstitution of active dimeric ribulosebiphosphate carboxylase from an unfolded state depends on two chaperonin proteins and magnesium ATP. Nature 342, 844-889. 

The reconstitution of active E. coli 50 S subunits, in contrast to that of 50 S particles from B. stearothermophilus (Nomura and Erdmann, 1970), requires a two-step incubation procedure (Nierhaus and Dohme, 1974 Dohme and Nierhaus, 1976). The assembly process occurs in four steps from 23 S RNA to 50 S particles, leading to formation of 33 S, 41 S, and 48 S intermediates. The step from 33 S to 41 S consists of a compact folding of the 33 S intermediate, without addition to any protein component. This drastic conformational change has been demonstrated by biochemical and electron-microscopic studies (Sieber and Nierhaus, 1978 Sieber et al, 1980 Nierhaus, 1982). Kinetic analyses performed at... 

Reconstitution of Active Enzyme from Subunit Polypeptide Chains ... 

As discussed above, the purification and reconstitution of active PKSs from a variety of heterologous expression systems (including E. coli) are now feasible. Given the substantial tolerance of PKSs toward altered substrates and intermediates, it should therefore be possible to exploit this catalytic potential in a far more powerful way in cell-free systems than in intracellular systems. The primary limitations are with regard to the scale of synthesis. Attempts to stabilize and reuse the enzymes, in conjunction with the development of cheaper sources of natural and unnatural substrates and recycling systems for NADPH, should go a long way toward ameliorating this limitation. 

Stenmark H, Afanasiev BN, Ariansen SA, Olsnes S (1992) Reconstitution of active diphtheria toxin and its fusion proteins from separate A- and B-fragments. Bio-cbemJ281 619-625. 

The In Vitro Reconstitution Of Active Rubisco. To explore the molecular mechanism by which Rubisco assembly is promoted by the chaperonins, an in vitro system was developed. We chose the dimeric, simplifi form of the Rhodospirilum nibrum Rubisco as our substrate, to be denatured with either 8M urea, 6M guanidium-HCl or acid treatment The E, coli chaperonins, now referred to as cpn60 ( EL) and cpnlO (groES) were purified to homogeneity (22),... 

Figure 2 Chaperonin- and Mg-ATP-dependent in vitro reconstitution of active dimeric Rubisco from denatured Rubisco. a) Rubisco activity, expressed as a percentage of activity observed with an equal quantity of native Rubisco. b) Western blot after non-denaturing PAGE of the reaction mixtures used in a and probed with antibody raised against Rubisco. c) as in b, but probed with antibody raised against cpn60. d) Summary of the reaction conditions. Reprinted by permission from NATURE vol. 342 pp. 884-889. Copyright (C) 1989 Macmillan Magazines Ltd.

Steveninck, J. V. Ledeboer, A. M. (1974). Phase transitions in the yeast cell membrane—the influence of temperature on the reconstitution of active dry yeast. Biochim. Biophys. Acta 352,64-70. 

Subunits - E. coli RNA polymerase is a multi-subunit protein. The five distinct polypeptide subunits of E. coli RNA polymerase are summarized in Table 26.1, Two copies of the ot subunit are present, along with one each of /, [Pg.110]

L.W. Parks, and D.E. Kelly (1995). Purification and reconstitution of activity of Saccharomyces cere-visiae P450 61, a sterol 22-desaturase. FEES Lett. 377, 217-220. 

T. Mild, L.S. Yoshida, and K. Kakinuma, Reconstitution of superoxide-forming NADPH oxidase activity with cytochrome b55g purified from porcine neutrophils. Requirement of a membrane-bound flavin enzyme for reconstitution of activity,/. Biol Chem. 267 18695 (1992). 

Kelly SL, Lamb DC, Corran AJ, Baldwin BC, Parks LW, Kelly DE (1995) Purification and reconstitution of activity of Saccharomyces cerevisiae P450 61, a sterol delta 22-desaturase. FEBS Lett 377 217-220... 

Although no structural information is available yet, it has been well established that both CHS and STS comprise a homodimer of subunits consisting of 388-400 amino acids [76, 127, 133]. To distinguish between independent or co-operative action of the active sites in the CHS and STS dimer, Schroder and co-workers prepared heterodimers in which one of the subunits is inactive (133). This was accomplished by co-expression of the native and mutant subunits under identical promoter-translation start configmation, since in vitro reconstitution of active CHS or STS from subunits has so far been impossible. The heterodimers were indeed fully functional and displayed close to 50% activity of the parent enzyme, as would be expected for a dimer possessing only one active site. These data clearly estabhshed that each subunit performs all the three condensation reactions, with two products being synthesized per dimer and reaction cycle. 

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... 

Fig. 6.3.9 An illustration showing the mechanism of the reconstitution and luminescence of symplectin. The binding of dehydrocoelenterazine with the SH group of a cysteine residue of aposymplectin (left) results in the formation of active symplectin (center). Symplectin is oxygenated at the C2 position, resulting in the formation of coelenteramide bound to aposymplectin (right), accompanied by the emission of light. From Isobe et al., 2002, with permission from Elsevier.

Thus three lines of evidence define the rapidly dissociating receptor as the LR complex. Conditions known to uncouple R from G--first, guanine nucleotide and second, pertussis toxin—produce LR third, reconstitution of G protein restores receptor affinity, sensitivity to guanine nucleotide, and effector activation. In this sense, the ligand and binding behavior of this system is analogous to that of the beta-adrenergic receptor, where the LR and LRG complexes have already been studied with purified proteins and reconstituted membrane preparations (2,i0). 

Solubilization of an active H,K-ATPase is also a prerequisite for reconstitution of the enzyme into liposomes. With these H,K-ATPase proteoliposomes it is then possible to study the transport characteristics of pure H,K-ATPase, without the interference of residual protein contamination that is usually present in native vesicular H,K-ATPase preparations. Rabon et al. [118] first reported the reconstitution of choleate or n-octylglucoside solubilized H,K-ATPase into phosphatidylcholine-cholesterol liposomes. The enzyme was reconstituted asymmetrically into the proteoliposomes with 70% of the pump molecules having the cytoplasmic side extravesicular. In the presence of intravesicular K, the proteoliposomes exhibited an Mg-ATP-dependent H transport, as monitored by acridine orange fluorescence quenching. Moreover, as seen with native H,K-ATPase vesicles, reconstituted H,K-... 

ATPase also catalyzed a passive Rb -Rb exchange, the rate of which was comparable to the rate of active Rb efflux. This suggested that the K-transporting step of H,K-ATPase is not severely limited by a K -occluded enzyme form, as was observed for Na,K-ATPase. Skrabanja et al. [164] also described the reconstitution of choleate solubilized H,K-ATPase into phosphatidylcholine-cholesterol liposomes. With the use of a pH electrode to measure the rate of H transport they observed not only an active transport, which is dependent on intravesicular K, but also a passive H exchange. This passive transport process, which exhibited a maximal rate of 5% of the active transport process, could be inhibited by vanadate and the specific inhibitor omeprazole, giving evidence that it is a function of gastric H,K-ATPase. The same authors demonstrated, by separation of non-incorporated H,K-ATPase from reconstituted H,K-ATPase on a sucrose gradient, that H,K-ATPase transports two protons and two ions per hydrolyzed ATP [112]. 

Using the reconstitution approaches described above, we have demonstrated that phosphorylation of the skeletal muscle Ca channels by PKC results in activation of the channels [108], In the fluo 3-containing liposomes, channels phosphorylated by PKC exhibited a two-fold increase in the rate and extent of Ca " influx [108], Using the lipid bilayer-T-tubule membrane reconstitution system we are currently analyzing the effects of PKC-catalyzed phosphorylation at the single channel level [133], The demonstration that these channels undergo phosphorylation as a result of activation of PKC in intact skeletal muscle cells has not yet been achieved. 

Ratnatilleke A, JW Vrijbloed, JA Robinson (1999) Cloning and sequencing of the coenzyme B,2-binding domain of isobutyryl-CoA mutase from Streptomyces cinnamonensis. Reconstitution of mutase activity and characterization of the recombinant enzyme produced in Escherichia coli. J Biol Chem 274 31679-31685. 

Superoxide is produced by the NADPH oxidoreduc-tase (oxidase), which is a membrane-bound enzyme complex containing a flavoprotein that catalyses the transfer of single electrons from NADPH in the cytosol to extracellular oxygen. NADPH is mainly supplied by the hexose monophosphate shunt. In resting cells, the oxidase complex is inactive and disassembled, but is rapidly reconstituted and activated by chemotactic mechanisms or phagocytosis (Baggiolini and Thelen, 1991). 

Most proteins are not sufficiently stable in aqueous solution to allow formulation as a sterile solution. Instead, the protein is freeze-dried and reconstituted before use. Development of a freeze-dried protein formulation often requires special attention to the details of the freezing process (potential pH shifts and ionic strength increase with freezing) as well as to potential loss of activity with drying. Formulation additives, such as sugars and polyhydroxy compounds, are often useful as cryoprotectants and lyoprotectants. Residual moisture may also be critical to the stability of the dried preparation [33],... 

Protein fragment complementation assays are based on an enzyme reassembly strategy whereby a protein-protein interaction promotes the efficient refolding and complementation of enzyme fragments to restore an active enzyme. The approach was initially developed using the reconstitution of ubiquitin as a sensor for protein-protein interactions (Johnsson and Varshavsky, 1994). Ubiquitin is a 76 amino acid protein that... 

Aspinall-O Dea, M., M. Wentworth, A. Pascal, B. Robert, A. Ruban, and P. Horton. 2002. In vitro reconstitution of the activated zeaxanthin state associated with energy dissipation in plants. Proc. Natl. Acad. Sci. USA 99 16331-16335. 

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