Oleic anhydride

Poly(A) synthesis also occurred in the second system, but the product remained within the vesicles. Walde also determined the increase of the vesicle concentration, which corresponds to that expected for an autocatalytic process. In this experiment, the enzyme PNPase is first captured by the vesicle envelope, and in the second step, ADP and oleic anhydride are added the anhydride is hydrolysed to the acid. ADP passes through the vesicle double layer and is polymerized in the interior of the vesicle by PNPase to give poly(A). Hydrolysis of the anhydride causes a constant additional delivery of vesicle-forming material, so that the amount of vesicle present increases during the poly(A) synthesis. These experiments demonstrated a model for a minimal cell. Autocatalytically synthesised giant vesicles could be prepared under similar conditions and observed under a microscope (Wik et al., 1995). 

Figure 7.17 Hydrolysis of oleic anhydride catalyzed by spontaneously formed oleic acid vesicles at 40 °C, (a) during the first 3 h, and (b) during a long observation time. A vesicle suspension (10 ml in 0.2 M bicine buffer (pH 8.5)) was overlaid with 0.25 mmol oleic anhydride and 0.025 mmol oleic acid. The increase of the concentration of oleic acid/oleate is plotted as a function of reaction time. Initial concentration of oleic acid/oleate 0 mM ( ), 5 mM ( ), 10 mM (o), 20 mM ( ). For an initial oleic acid/oleate concentration of 20 mM, the concentration of oleic anhydride (A) present in the vesicles during the reaction is also plotted (b, right axis). (From Walde et al, 1994b.)...

The time course of an actual experiment is shown in Figure 7.17, which shows the hydrolysis of oleic anhydride catalyzed by spontaneously formed oleate vesicles. Note the sigmoid behavior, typical of an autocatalytic process. The lag phase is due to the preliminary formation of vesicles, and in fact the length of the lag phase is shortened when already formed vesicles are pre-added, as shown in the hg-ure. Some mechanistic details of these processes will be discussed in Chapter 10. In this work, an analysis of the number and size distribution of vesicles at the beginning and the end of the reaction was also performed by electron microscopy. 

Figure 10.3 Enzymatic synthesis of poly(adenylic acid) in self-reproducing oleate liposomes (redrawn from Walde et al., 1994a). (a) The ADP penetrates (sluggishly) the liposome bilayer, (b) in the presence of polynucleotide phosphorylase, ADP is converted in poly(A), which remains entrapped in the liposome, (c) Polycondensation of ADP goes on simultaneously with the self-reproduction of liposomes (A is the membrane precursor, oleic anhydride, which, once added, induces the self-reproduction of liposomes S, surfactant, in this case oleate, which is the hydrolysis product of A on the bilayer E is polynucleotide phosphorylase).

Simultaneous oleic anhydride hydrolysis resulting in a self-reproducing vesicle system. 

Giant vesicles have been the subject of several international meetings and specialized literature (Luisi and Walde, 2000 Fischer et al., 2000). There are several reasons for this interest. One is that, because of their size, they can be observed by normal optical microscopy, without using the much more expensive and indirect electron microscopy. Figure 10.7 shows, as an example, the transformations brought about by the addition of a water-insoluble precursor (oleic anhydride) to oleic acid giant vesicles (Wick et al., 1995). 

Figure 10.7 Direct observation of transformations in giant vesicles. This results from the addition of oleic anhydride to giant oleate vesicles. (Adapted from Wick...

Oleic acid/oleate vesicles containing the enzyme Q(3 replicase, the RNA template and the ribonucleotides. The water-insoluble oleic anhydride was added externally. 

Growing membrane systems have been used to obtain artificial infrabiological systems. Walde et al. [47] have carried out the synthesis of polyadenylic acid in self-reproducing vesicles [48], in which the enzyme polynucleotide phosphorylase carried out the synthesis of poly-A, and membrane vesicle multiplication was due to the hydrolysis of externally provided oleic anhydride to oleic acid. The snag is that the enzyme component is not auto-catalytic. Enzymatic RNA replication in vesicles [49] suffers from the same problem. It is also not known whether redistribution of the entrapped enzymes into newly formed vesicles occurs or not. An affirmative answer would be evidence for vesicle reproduction by fission. 

The 2-(methyldiphenylsilyl)ethyl (DPSME) group was used to protect a phosphate in a synthesis of Lysobisphosphatidic Acid [Scheme 7.40].82 The phosphate protection was introduced by a phosphite triester approach to give the symmetrical phosphate 40 2. The two isopropylidene groups were removed by acid hydrolysis without harm to the phosphate and the resultant diols selectively protected on their less hindered termini with fert-butyldiphenylsilyl groups. Acylation of the remaining secondary hydroxyls with oleic anhydride afforded the diester 403, whereupon the silyl protecting groups were removed with... 

Figure 3. Freeze-fracture electron microscopy analysis of the vesicle size distribution in the case of the spontaneous vesiculation of oleic acid/oleate. (A) Vesicles formed from the hydrolysis of 25 mM oleic anhydride (overall concentration) at 30 °C, yielding 50 mM oleic acid/oleate. (B) Vesicles extruded throughout 50 nm diameter filters. (C) Vesicles formed upon hydrolyzing 20 mM oleic anhydride (same conditions as in A) in the presence of pre-added extruded vesicles B—all in 0.2 M bicine buffer pH 8.5. For details see ref. 8.

Figure 8. (A) Autocatalytic hydrolysis of oleic anhydride in the presence of 1.9 mM Carbobenzoxy-Phe-His-Leu-OH and Ci e-ONp in 0.02 mM borate buffer pH 8.5. Here the progress of the concentration of formed oleate/oleic acid is reported as a function of time. (B) The simultaneous initial velocity of the hydrolysis of Cl 6-0 Np is measured.

PNPase has been entrapped inside extruded fatty acid vesicles, which are fed by externally added ADP and oleic anhydride. ADP is polymerized inside the vesicles, producing poly(A), whereas - simultaneously - oleate vesicles self-reproduce at the expenses of oleic anhydride. 

QP replicase has been entrapped within extruded oleate vesicles, together with a template RNA and NTPs. Vesicles are fed by externally added oleic anhydride, so that self-reproduction occurs. Simultaneously, RNA is replicated in the vesicle core. This is an example of core-and-shell reproduction. 

The same group also demonstrated that an enzymatic RNA replication can occur within self-replicating liposomes, however, with no dependency between the content and the container during the replication process. As for the previous examples, liposomes were loaded with all the necessary reagents and their self-reproduction occurred using oleic anhydride at 40 °C. This example represents an autopoietic process as the reproduction of the boundary is due to the reaction that takes place within the boundary and is catalytically induced by the boundary itself. This work is presented as the first example in which the reproduction of the membrane and the replication of the internalized RNA molecules proceed simultaneously and thus, the first bridge established between the two more accepted views on the theory of minimal life the RNA world and the cellular autopoietic view. It should be mentioned that in a former... 

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