Reconstitution Studies

The absorption of the aerobically reconstituted erythrocuprein was somewhat lower in the UV region, and Amax in the visible region was shifted to a longer wavelength. Futhermore, the shoulder at 430 nm was not distinct. No spectral changes as compared to the native erythro- 

The purity of the reconstituted erythrocuprein was examined using analytical polyacrylamide electrophoresis (122). All preparations were virtually free from contamination. Again, the 2 Cu-apoerythrocuprein complex, the fully restored erythrocuprein, and the native protein all 

The EPR data supported the electrophoretic, spectroscopic, and enzymic proofs of a successfully reconstituted erythrocuprein. It was surprising that the aerobically reconstituted erythrocuprein and even the 4 Cu2+ apoprotein showed most of the EPR characteristics of the 2 Cu—2 Zn enzyme. Only the A n values of the 4 Cu protein and the aerobically reconstituted erythrocuprein were different. It is suggested that in the last two metalloproteins the Cu2+ ligand distances are somewhat distorted or the ligands are slightly displaced due to conformational changes in the protein portion (Talbe 8). 

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The skeletal muscle Ca channels also can be phosphorylated in vitro by a protein kinase endogenous to the T-tubule membranes [111,115]. This kinase is neither Ca - nor cyclic nucleotide-dependent [115], and is interesting in that it phosphorylates primarily the P subunit while the ai subunit is a poor substrate. However, the amount of this kinase that co-purifies with the T-tubule membranes is variable, and consequently, very few studies have been performed. So far, only low levels of phosphorylation have been obtained (no more than 0.2 mol phosphate/ mol P subunit) and no functional effects of this phosphorylation have been observed in reconstitution studies. 

Schulz, A., Grosse, R., Schultz, G., Gudermann, T., and Schoneberg, T. (2000) structural implication for receptor oligomerization from functional reconstitution studies of mutant V2 vasopressin receptors. J. Biol. Chem. 275, 2381-2389. 

Lambert O, Lev D, Ranck J-L, Leblanc G, Rigaud J-L. A new gel-like phase in dodecyl maltoside-lipid mixtures implications in solubilization and reconstitution studies. Biophys J1998 74 918-930. 

From a chemical point of view, the El/ubiquitin thiol ester should be competent to donate ubiquitin to a substrate amino group. In fact, aminoacyl-errzyme thiol esters are used in exactly this way in non-ribosomal polypeptide synthesis, a process that was discovered around the same time as ubiquitin-protein conjugation [5]. In spite of the attractive simplicity of this model, however, biochemical reconstitution studies showed that besides El two additional fractions were required to conjugate ubiquitin to a model substrate. They were called ubiquitin carrier protein (E2) and ubiquitin-protein ligase (E3), respectively, since the respective factors seemed to act sequentially [6]. Interestingly, the E2 factor apparently formed a thiol ester with ubiquitin. Based on these results, Hershko and co-workers proposed the ubiquitin conjugation cascade (Figure 5.1). 

The contribution of individual core histone N-tails to transcriptional regulation has been recently addressed in a reconstitution study [42]. Varied combinations of recombinant and mutant cores histones have been assembled on circular plasmids, and the resultant nucleosomal arrays were tested for transcriptional... 

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 ... 

Ferreira, V., Ortin, N., Escudero, A., Lopez, R., and Cacho, J. (2002b). Chemical characterization of the aroma of Grenache rose wines. Aroma extract dilution analysis, quantitative determination and sensory reconstitution studies. J. Agri. Food Chem. 50,4048—4054. 

We have begun to characterize the retinoid enhancement factor. Reconstitution studies clearly show that the factor is present in the post-microsomal soluble fraction (Table III) in rat liver (there is a factor(s) present in the 17,000xg pellet which normally inhibits this factor (Table III)). The retinoid enhancing factor is heat sensitive, since its activity in the presence of retinol is lost when supernatant is incubated for 15 minutes at 45°C (Figure 3). Similar results were obtained by incubating the supernatant at 50°C for five minutes. 

The loaf volumes of breads baked from wheat starch coming from different classes of wheat, be it hard red winter, hard red spring, soft red winter or soft white, were similar.436 By contrast, club wheat starch produced a larger loaf volume and durum wheat starch a smaller loaf volume. Other investigators found a range in the loaf volumes of breads baked from wheat starches isolated from different classes of wheat.437 Fractionation and reconstitution studies revealed that rye and barley starches can substitute for wheat starch in producing bread of satisfactory volume. Starches from... 

G and a41 Gj have also been purified from rat [77] and porcine [78] brain and shown to be distinct from each other. Both bovine and rat brain G or Gj were shown in reconstitution studies to interact with muscarinic receptors [79-81], by increasing the binding affinity for muscarinic agonists [80,81]. Platelet a2-adrenergic receptors [82] stimulate the GTPase activity of the bovine G and Gj proteins in... 

While the stoichiometries of the Mn SOD enzymes appear to vary, the properties of the Mn-binding site do not. This is borne out in the electronic spectra of these proteins, which display a great degree of similarity despite the diversity of sources from which they have been isolated (Table II). This type of spectrum is distinctive for manganese in the trivalent oxidation state (3). The native enzymes are EPR silent, as might be anticipated if they contained Mn solely as the trivalent ion (S = 2) (1, 6,12,18-20, 24). However, when the enzymes are denatured, the characteristic six-line pattern of Mn(II) (I = 5/2) appears. Magnetic susceptibility studies with the E. coli SOD were consistent with the presence of a monomeric Mn(III) complex with a zero-field splitting of 1 to 2 cm-1 (4). The enzymes are additionally metal specific (however, see Refs. 36 and 37) metal reconstitution studies with E. coli and B. stearothermophilus revealed a strict requirement for Mn for superoxide dismutase activity (2, 22, 23). 

This chapter describes the use of the HPLC method to assay the activity of several enzymes simultaneously. The examples include several different enzymes that can use the same substrate and form the same product, a single enzyme that can use different substrates to form different products and two different activities using the same substrate to form different products. In another example the use of the HPLC method to study metabolic pathways is described through a series of reconstitution studies, and finally the HPLC method is applied to the anabolism of adenosine. 

The enzyme 5 -nucleotidase dephosphorylates IMP to inosine and P. Thus, since this reaction represents a possible fate for the IMP formed by the transferase (Fig. 10.7), reconstitution studies were undertaken with the nucleotidase. These studies were carried out using the HPLC assay method developed for the HGPRTase activity. A reaction mixture was prepared that contained hypoxanthine and PRibPP as substrates. The reaction was started by the addition of purified HGPRTase enzyme. Samples were removed and were analyzed by HPLC. The chromatographic profiles obtained at 0,10, 20, and... 

Structure (II) reacts as a Bronsted acid with the strong base, NH3, causing the disappearance of both the 3660 and 3570 cm i IR bands, but not a weak silanol-type band at 3745 cm i 70). In fact, the 3660 and 3570 cm i bands may be viewed as silanol-type bands whose frequency has been lowered from 3745 cm i by a Lewis acid-bs/se interaction between the electron pairs of the OH group with adjacent 3-coordinate A1 atom 70-72). Thus, it is this interaction and the resulting ability of the AIO4 tetrahedra to reform that contributes to the acidity of this group. Uytterhoeven d al. 70) have demonstrated from NH3 adsorption experiments (Fig. 16) that the transformations (I)f (II) above are essentially reversible. Venuto et al. 75) confirmed this conclusion by gross scale reconstitution studies on a 90% decationated Y sample ... 

Binding of substrates suggests that MFPs might be directly involved in transporting substrates across the periplasm and the outer membrane. The in vitro reconstitution studies support this idea. AcrA stimulates the transport activities of the reconstituted RND transporters AcrB and AcrD [95, 116]. Another MFP MacA is absolutely required for the ATPase activity of MacB, the ABC-type macrolide efflux transporter [122]. 

When the duplicated centrosomes have become aligned, formation of the spindle proceeds, driven by simultaneous events at centrosomes and chromosomes. As just discussed, the centrosome facilitates spindle formation by nucleating the assembly of the spindle microtubules. In addition, the (—) ends of microtubules are gathered and stabilized at the pole by dynein-dynactin working with the nuclear/mitotic apparatus protein. The role of dynein in spindle pole formation has been demonstrated by reconstitution studies in which bipolar spindles form in Xenopus egg extracts in the presence of centrosomes, microtubules, and sperm nuclei. The addition of antibodies against cytosolic dynein to this in vitro system releases and splays the spindle microtubules but leaves the cen-trosomal astral microtubules in position (Figure 20-35). 

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