Cell-free systems properties

The increase in levels of tissue CAT is compatible with previous results which showed that LLLT induced an increase in CAT activity of irradiated isolated cardio-myocytes compared to controls. It was suggested that laser therapy efficacy in chronic wounds and ulcers can be attributed to the activation of CAT in tissue fluids [62]. He-Ne laser has been shown to cause photoactivation and structural modifications of catalase enzymes that positively correlated with its functional properties in cell free system [63]. 

That some cell-free systems were incomplete became evident when the role of UDP-Glc was appreciated (see Refs. 127-135). Only in the presence of UDP-Glc (in addition to UDP-GlcNAc and GDP-Man, when no endogenous acceptors are present) was a lipid-linked oligosaccharide obtained that had the same chemical properties as the D-... 

The catalytic mechanisms and molecular recognition properties of peptide synthetases have been studied for several decades [169]. Nonribosomal peptides are assembled on a polyenzyme-protein template, first postulated by Lipmann [170]. The polyenzyme model was refined into the thiotemplate mechanism (Fig. 11) in which the amino acid substrates are covalently bound via thioester linkages to active site sulfhydryls of the enzyme and condensed via a processive mechanism involving a 4 -phosphopantetheine carrier [171-173].The presence of a covalently attached pantetheine cofactor was first established in a cell-free system that catalyzed enzymatic synthesis of the decapeptides gramicidin S and tyrocidine. As in the case of fatty acid synthesis, its role in binding and translocating the intermediate peptides was analyzed [174,175]. 

It has been shown that combretastatin A-4 disrupts the microtubules of human umbilical vein endothelial cells (HUVECs) in culture, thus confirming that the tubulin binding properties shown in cell-free systems are retained when the compound enters cells and that tubulin binding is a significant component of the biological activity. Also 3-fluoro- and 3-chloro derivatives retained activity in human umbilical vein endothelial cells. This kind of activity against endothelial cells is extremely important, as endothelial cells play a key role in the angiogenic process. 

From Solarium tuberosum, Slabnik and Frydman isolated a unique phosphorylase that has no requirement for the primer addition for formation of an amylopectin-like polysaccharide in a cell-free system. The properties of this enzyme were found to differ from those of the usual potato phosphorylase this new enzyme is assumed to be a glycoprotein, the glycosidic component of which acts as the primer. The activity of this phosphorylase disappeared at 55 , in contrast to the usual phosphorylase activity (which withstands this temperature). There was also good correlation between formation of polysaccharide and appearance of inorganic phosphate in the absence of primer. The polysaccharide formed de novo" was shown to be an eflScient primer for starch synthesis. ... 

Beyond understanding pathway operation in a cell-like extract environment, crude lysate systems have also been exploited to create other building blocks. For example, the synthesis of triketone precursors, essential for some antibiotics, have been pursued in cell-free systems [73], In E. coli crude lysates, enzymes from Bacillus subtilis were overexpressed to achieve triketide lactone production. While the scientists briefly use this system to characterize enzymes in the pathway, this is a good example of how different properties such as chirality can be achieved through manipulation of enzyme activities. However, this system does not take advantage of any of the benefits of crude lysate and therefore could be performed in a purified system as well. This still remains an important finding in the growing body of CFME literature. 

A possible transfer of mannose from retinyl mannosyl phosphate to polypeptide was first indicated by the studies of Maestri and De Luca (1973), though these did not prove that retinyl mannosyl phosphate was the mannosyl donor. In a more detailed analysis Rosso etaL (1977) used a radioactive label largely free of dolichyl mannosyl phosphate in a cell-free system and obtained a clear-cut incorporation of mannose from retinyl mannosyl phosphate into a substance with the solubility properties of protein and which was labile to proteolysis. Some free mannose, its 1-phosphate and retinyl mannosyl phosphate were also present at the end of the incubation, but no large mannolipid was detected. Dolichy CJmannosyl phosphate did not give any transfer of [ C] mannose to protein and further support for a distinctive retinol pathway came from the effects of EDTA. This totally failed to block transfer from retinyl [ mannosyl phosphate, under conditions where block... 

The procedures reported here have been used to investigate the development of male pronuclei in cell-free systems of sea urchins and, to a lesser extent, surf clams. Pronuclear formation in vitro is a slower process than that in vivo. This property has been used advantageously to examine the steps of pronuclear formation. Each step can be easily manipulated, but the methods described here may require some adjustments for other organisms. Sea urchin and surf clam male pronuclei formed in vitro are virtually complete, with decondensed chromatin, nuclear envelopes, pores, and lamina. 

RNA of these particles possesses other properties characteristic of information RNA or D-RNA. It hybridizes with homologous DNA to the same extent as nuclear D-RNA isolated by the hot phenol fractionation procedure. There is a strong cross-competition for binding sites on DNA between D-RNA isolated from the SOS particles and D-RNA isolated by hot phenol fractionation. This means that the RNAs prepared by the two procedures contain a similar population of molecules (Samarina et al., 1965a, 1967a). RNA prepared for nuclear extracts or from SOS particles effectively stimulates amino acid incorporation in a cell-free system (Jacob and Busch, 1967 Samarina, 1969). It is labeled strongly after short intervals, and its specific activity is of the same order as the specific activity of nonextractable RNA of the cell nucleus. The specific activity of this RNA increases with the increase of the number of extractions. 

The enzymes catalyzing the steps after GAi2-aldehyde are 2-oxoglutarate-dependent dioxygenases, a type of enzymes known particularly well from the work with prolyl 4-hydroxylase in animal systems. In the case of GA biosynthesis, the participation of this class of enzymes was first shown for the entire pathways in cell-free systems from Cucurbita maxima [10] and Pisum sativum [12] and for 2 -hydroxylation in cell-free systems from peas and beans [11]. Their properties and distribution in different organisms were surveyed by MacMillan [15]. 

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