CT-active

New synthetic transformations are highly dependent on the dynamics of the contact ion pair, as well as reactivity of the individual radical ions. For example, the electron-transfer paradigm is most efficient with those organic donors yielding highly unstable cation radicals that undergo rapid unimolecular reactions. Thus, the hexamethyl(Dewar)benzene cation radical that is generated either via CT activation of the [D, A] complex with tropylium cation,74... 

Recently, it has been shown that cell-permeable cerantides dramatically inhibited the synthesis of the two major membrane phospholipids, PC and PE (Bladergroen et al, 1999b Allan, 2000). The inhibition of phospholipid synthesis was rapid, within 2 h, and resulted in massive apoptosis after 16-24 h. The mechanism by which short-chain cerantides exert their effect on phospholipid synthesis is possibly cell type dependent. In baby-hamster kidney (BHK) fibroblasts rc synthesis was reduced at the level of CT, the putative rate-determining enzyme in the CDP-choline pathway (Allan, 2000). This conclusion was based solely on radio-label studies in combination with an earlier published observation (Wieder et al, 1995) showing that C2-SM (the SM generated from C2-ceramide by SM synthase, which was actively synthesized in the BHK-cells) inhibited CT activity in vitro. On the other hand, data obtained from studies with rat-2 fibroblasts clearly showed that short-chain cerantides regulate the synthesis of PC and PE mainly at the final step of the CDP-pathways. This conclusion was based on the following observations (a) incorporation of [ H]-choline into PC and... 

This is in agreement with data showing that natural ceramides do not have any effect on CT activity in vitro (Sohal and Cornell, 1990). The mechanism by which cell-permeable ceramides exert their effect on CPT and EFT activity and whether natural or intracellular generated ceramides have a similar effect on phospholpid synthesis is at present unknown. 

Fig. 2. Schematic diagrams of the PNM in toluene, with the shaded areas denoting negative values. In Panel (a) the area within a circle on atom i in the mode a is proportional to Ul a, with all diagrams plotted using the same scaling factor, and the CT-active mode phases determined by the convention ii> > 0. The mode ordering is in accordance with increasing values of ha. The numbers reported (a.u.) are ipa (first row), h h, (second row), and F w, (third row, below the diagram)...

Only the ten modes that are symmetric with respect to the symmetry plane of the toluene molecule can be CT-active. The five antisymmetric modes represent... 

The dominant role of the h -hardest mode, a = 15, immediately follows from its wx value and the On vector coefficients (see Fig. 2). The same conclusion, therefore, must follow from the relevant resolution of the pure CT-vector, given by the 0 transformation, uniformly scaled by the factor 1 /m. This feature of the a = 15 mode is also seen in the (AIM + PNM) resolution of the FF vector, shown in Fig. 3b, where all but last CT-active modes mainly describe the CT-induced polarization (explicitly extracted in Fig. 3c). 

It follows from Fig. 11 that the rigid IDM resolution of fCJ (Fig. 9A-c) is more concentrated than that of Fig. 10. The cluster part of the toluene - cluster CT indices has large components 00 only for the softest CT-active modes a = 1 and 3. The toluene fCT part in the strong interaction region is also relatively condensed, being dominated by the P = 4 and 12 modes, which alone explain the main qualitative features of Fig. 9a-c. 

Fig. 16. The EDM resolution, w M), of the FF diagram of Fig. 9 A-e only the CT-active (w / 0, shown below the mode diagram) modes are reported. The mode numbers are the same as in the corresponding parts of Fig. 8...

One also observes that the most important external CT-active EDM in Fig. 14a (the largest wa value), a = 5, corresponds to a net increase in the number of electrons on both reactants when d/V > 0 (inflow of electrons to M). The second mode of the figure in this wa hierarchy, a = 11, represents the... 

Another obvious conclusion that follows from Fig. 18 is that the EDM (-> MEC) are localized, as are the MEC modes that they resemble. This is particularly so in Part b of the figure, where modes a = 14 and 15 represent the corresponding CH fragments of toluene. A reference to the kva values, which reflect a partitioning of the external CT, d/V, for the most important CT-active... 

Fig. 27. (Continued). The two (110)-layer, 147 atom rutile cluster charges (Panel a), AIM FF indices f (Panel b), the most important external CT-active PNM (Panel c), and their contributions to f, W. r (Panel d). The numbers above each diagram in Panel c are the mode number, principal hardness (ordering criterion), and wy...

On the other hand, when highly stabilized carbenium ion results from unimolecular ring opening, the carbenium active species may be present in the system in detectable concentrations. Indeed, in the polymerization of cyclic acetals —O -Ct + active species exist in equilibrium with tertiary oxonium ion active species (cf., Section lI.B.6.b.). 

This assay, which does not require the addition of lipids or detergent, measures the ability of CT or LT to ADP-ribosylate itself in the absence of another ADP-ribose acceptor (except water). ARF added to this assay will also be ADP-ribosylated, but the ADP-ribosylation of it or CT under these conditions does not appear to decrease CT activity or the ability of ARF to stimulate CT (Tsai ef a/., 1991). Reaction products are separated by SDS-PAGE and analyzed by autoradiography as described for Assay 1 (Section 2.3). This assay is the most sensitive of all those listed here and can be used to verify toxin activity observed in other assays. 

It is possible that the toxin was not properly activated, and a new sample of toxin should be activated, using freshly prepared DTT. It is possible that the concentration of NAD in the assay was too low and was hydrolyzed. Because NAD in solution is susceptible to hydrolysis, it should be stored in small portions at -20°C and the stock supply replaced regularly. Mutant toxins may, in fact, be inactive and it is suggested that Assays 3 and 4 be used to confirm or negate this possibility. CT activity may also be affected by salt and/or protein concentrations in the assay. Excessive salt can stimulate toxin activity buffer controls should, therefore, be run in each assay. High protein concentrations may result in decreased or variable toxin activity in Assay 2, the presence of ovalbumin minimizes nonspecific protein effects. 

It is sometimes necessary to modify the conditions of the assays described here. In one specific example, conditions of the agmatine assay were changed to allow the assay of a second enzyme, phospholipase D (PLD), under identical conditions (Massenburg et al., 1994). With the identification of ARF as an activator of PLD (Brown et al., 1993 Cockcroft et al., 1994), it was desired to determine which mammalian ARFs activated PLD, and to identify ARF domains that were involved in the activation of CT and PLD. It was first determined that each ARF sample was active in Assay 2 in the presence of cardi-olipin each ARF was then assayed tor its ability to stimulate PLD in a separate assay (Massenburg et al., 1994). To compare the activities of ARF proteins in the CT and PLD assays, we used conditions under which both enzymatic activities could be measured. With the buffer conditions and sonified lipid vesicles of the composition required tor the pH-choline]-release assay used to measure PLD activity, all reagents required tor Assay 2 were added to create a dual assay system to determine the formation of ADP-ribosylagmatine determined as described above. To perform the PLD assay, radiolabeled vesicles of the same composition replaced the non-radiolabeled vesicles and choline release was determined. Although these conditions resulted in a lower ARF stimulation of CT activity than observed in Assay 2, they allowed direct comparison of ARF activation of CT and PLD. 

Fig. 3. Regulation of PC biosynthesis via the CDP-choline pathway by modulation of the binding of CTP phosphocholine cytidylyltransferase (CT) to membranes. Three different modes of regulation of CT activity are indicated. The abbreviations are CK, choline kinase CPT, CDP-choline 1,2-diacylglycerol cholinephos-photransferase PEMT, phosphatidylethanolamine Af-methyltransferase PC, phosphatidylcholine PE, phos-phatidylethanolamine DG, diacylglycerol.

CT activity was first described by Kennedy and Weiss in 1955. Over three decades later CT was finally purified to homogeneity (P.A. Weinhold, 1987). The CT gene was cloned from Saccharomyces cerevisiae (S. Yamashita, 1987) by complementation of a yeast mutant defective in CT activity and rat liver CT cDNA was subsequently cloned (R.B. Cornell,... 

CT has classically been considered a cytoplasmic enzyme since its activity is found in the cytosol and on microsomal membranes in cellular homogenates. However, Kent and co-workers demonstrated that CT is present in the nuclear matrix and is associated with the nuclear membrane (C. Kent, 1997). To explore the role of the nuclear localization signal in CTa, experiments were performed in a CHO mutant (MT-58) that was temperature-sensitive for CT activity. In MT-58 cells, CT activity is present at low levels and the cells grow at 33°C (C. Raetz, 1980). At the restrictive temperature of 40°C, CT activity is abolished and the cells die via apoptosis (F. Terce, 1996). CTa in which residues... 

CT is recovered from cells and tissues in both the cytosol and microsomal membranes. However, in the early 1980s evidence from several laboratories suggested a close correlation between CT activity on membranes and the rate of PC biosynthesis. The proposed explanation for this observation was that the active form of CT was membrane-bound whereas CT in the cytosol acted as an inactive reservoir (Fig. 3). In agreement with this proposal, cytosolic fractions contain essentially no phospholipid and CT requires phospholipids for activity. Thus, cells have a facile mechanism for altering the rate of PC biosynthesis by reversibly translocating CT between a soluble, inactive reservoir, and... 

CTa contains a domain that is extensively phosphorylated and the state of phosphorylation can affect CT activity (S.L. Pelech, 1982). CT that is bound to membranes is less phosphorylated than soluble CT. Incubation of hepatocytes with oleic acid demonstrated that CT associates with membranes in an active, phosphorylated form and that is subsequently dephosphorylated (M. Houweling, 1994). Thus, a change in the lipid composition of membranes mediates the initial binding of CT to membranes and subsequently CT is... 

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