Permeabilization import reaction

Procedure. The addition of recombinant nucleoplasmin core protein and tRNA to the permeabilized cells prior to the initiation of the import reaction... 

Furthermore, the type of enzyme formulation (free enzyme, immobilized enzyme, or whole cells) plays a key role in determining the progress of the overall reaction. For most applications, lyophilized enzyme powders have been used with good results presumably they dissolve into the liquid phase. When poorly soluble products are formed, the enzyme can be recovered by washing with water [52]. For co-factor-dependent reactions permeabilized cells may be used [44]. When using immobilized enzymes, it has been demonstrated that the chemical nature and the pore size of the support are very important parameters to consider [8, 41]. 

The titanium compounds CpTiCl3, Cp2TiCl2, and CH2(C5H4)2TiCl2 have been used as catalysts for the diastereoselective epoxidation of allylic alcohols.1932 [TiCp2(LL)]2+ (LL = 1,10-phenantroline, 2,2 -bipyridine) (Scheme 758) have been studied in order to determine the importance of the ancillary chelated ligands versus the metal center for the membrane-permeabilizing action and their effects on lipid epoxidation reactions.1933... 

A typical transport reaction (12 /u.1) contains 0.5 mM ATP, 0.5 mM GTP, 10 mM creatine phosphate, and 50 figjml creatine phosphokinase, 2 fi of Xeno-pus egg extract for protein import or 4 /il for snRNP import and 0.5-1 fig of fluorescent BSA-NLS conjugate or fluorescently labeled snRNPs to a final concentration of 30 fig/ml and 80 /xg/ml respectively 1 x 10 permeabilized cells. The amount of extract may need to be increased to bring about efficient import if reticulocyte lysates or somatic cell extracts are used. 

As an alternative to quantitative fluorescence microscopy, a flow cytometer can be used to measure the levels of accumulation (Paschal and Gerace, 1995). The permeabilized cells used in the import assay behave essentially as intact cells and are used unflxed. A standard 20-min incubation reaction time is used and the permeabilized cells are washed once in 4 ml of ice cold transport buffer and resuspended in 0.5 ml of the same buffer. The fluorescence of 10 cells is measured for each sample (Sweet and Gerace, 1996). 

There are different techniques to overcome the cell membrane barrier and introduce exogenous impermeable compounds, such as dyes, DNA, proteins, and amino acids into the ceU. Some of the methods include lipofection, fusion of cationic liposome, electroporation, microinjection, optoporation, electroinjection, and biolistics. Electroporation has the advantage of being a noncontact method for transient permeabilization of cells (Olofsson et al., 2003). In contrast to microinjection techniques for single cells and single nuclei (Capecchi, 1980), the electroporation technique can be applied to biological containers of sub-femtoliter volumes, that are less than a few micrometers in diameter. Also, it can be extremely fast and well-timed (Kinosita et al., 1988 Hibino et al., 1991), which is of importance in studying fast-reaction phenomena (Ryttsen et aL, 2000). 

Another bio-inspired approach is to design polymersomes as enclosed reaction compartments for the development of nanoreactors, nanodevices, or artificial organelles, in which active compounds are not only protected from the environment, but also allowed to act in situ. For such function, membrane permeability is of crucial importance, since it allows the exchange of substrates/products with the environment of the pol)maersomes. Various methods have been reported to generate polymersomes with permeable membranes (i) polymers forming intrinsically porous membranes, (ii) polymer membranes that are permeable to ions as e.g. specific oxygen species, (iii) pore formation in pH responsive polymer membranes by chemical treatment, (iv) polymer membrane permeabilization by UV-irradiation, and (v) biopores or membrane proteins inserted into polymer membranes. ... 

---