Vesicle acridine orange

Solubilization of an active H,K-ATPase is also a prerequisite for reconstitution of the enzyme into liposomes. With these H,K-ATPase proteoliposomes it is then possible to study the transport characteristics of pure H,K-ATPase, without the interference of residual protein contamination that is usually present in native vesicular H,K-ATPase preparations. Rabon et al. [118] first reported the reconstitution of choleate or n-octylglucoside solubilized H,K-ATPase into phosphatidylcholine-cholesterol liposomes. The enzyme was reconstituted asymmetrically into the proteoliposomes with 70% of the pump molecules having the cytoplasmic side extravesicular. In the presence of intravesicular K, the proteoliposomes exhibited an Mg-ATP-dependent H transport, as monitored by acridine orange fluorescence quenching. Moreover, as seen with native H,K-ATPase vesicles, reconstituted H,K-... 

Membrane vesicles (100 pg protein) from strain KFlOrA (yGln-14—.end) harboring mutant or wild-type plasmids were suspended in buffer containing 1 pM acridine orange, and fluorescence was monitored at 25° C. At the indicated times, 1 mM ATP or 1 pM CCCP was added. Membrane ATPase activities of mutants and wild type are also shown. 

With bilayer lipid membranes it is not possible to achieve a fully asymmetric arrangement of head groups or chains. There is no apparent reason why all the molecules of two independent layers should only concentrate in one layer. Nevertheless, a little asymmetric distribution is found in vesicles made of lipid mixtures. Cerebroside sulfate, an anionic monoglycosyl ceramide was, for example, added exclusively to the outer surface of a performed DPPC vesicle (see Scheme 2.2) which was quantitized by the metachromatic effect of acridine orange. 

Fig. 2. Acidification of small synaptic vesicles by glutamate and chloride in synap-tosomes. The acidification assay was performed as described in section 3.2 of this chapter. Two representative experiments with intact (upper trace) or SLO-permeabilized (lower trace) synaptosomes are shown. The ordinate gives the changes of absorbance obtained (A 492-530). Final concentrations of potassium glutamate (Glut), KCl, and ammonium sulfate (NH/) were 10 mM, 45 mM and 30 mM, respectively. The uptake of glutamate and chloride result in an acidification of the lumen of small synaptic vesicles, which increases the vesicular uptake of acridine orange, resulting in a decrease in the amount of extravesicular dye. This acidification can be only observed when the plasma membrane is permeabilized...

Gastric membrane vesicle preparations enriched with H /K -ATPase have also been used to examine PPIs. The inhibition of hydrogen ion transport by PPIs is measured by use of the initial rate of acridine orange quenching as an index of acidification. However, steady-state acidification, as measured by aminopy-rine accumulation, is inhibited with greater potency and this is consistent with the accumulation of PPIs in the intravesicular acidic space (38). 

An Mg -dependent, proton-translocating ATPase activity has been identified and characterized in surface membrane from L. donovani promastigotes (55). The enzyme has a pH optimum of 6.5, and is inhibited by orthovanadate and dicyclohexylcar-bodiimide. Membrane vesicles, devoid of attached cytoskeletal elements, were shown to contain ATPase activity. These vesicles were incubated in the presence of acridine orange and ATP to demonstrate the proton-pumping activity of the surface membrane ATPase. This experiment revealed an ATP-driven acidification of the vesicles internal space in those vesicles with an inside-out orientation. 

An important information is to find out where the luminescent heli-cates are located within the cytosol of the live cells. Several experiments have been conducted, including colocalization experiments in which the cells were incubated successively with the helicate and with organic stains known to localize in specific compartments of the HeLa cells, such as acridine orange (AO) which stains the cell nuclei. Part of these experiments are shown on Figure 107. In the top part, the helicate is shown, after a short incubation time, to be entrapped into isolated vesicles which diffuse into the cytoplasm and the size of which is usually around... 

Cyanine dyes are avidly accumulated by mitochondria in vivo (7) with a resultant quenching of their fluorescence when assessed in cell suspension (8) however, in the fluorescence microscope the mitochondria appear as brightly fluorescent, filamentous structures (7). Similarly acridine orange and 9-aminoacridine are accumulated by acidic endosomes with a quenching of their blue fluorescence in the fluorescence microscope the vesicles appear as yellow or orange vesicles, this being the characteristic emission of very concentrated solutions of these dyes (1). 

A more qualitative estimate of endosome pH can be obtained by monitoring the distribution of a permeant, fluorescent amine such as 9-aminoacridine or acridine orange whose fluorescence varies with concentration. Such amines acaimulate in acid compartments because the protonated form of the dye is much less membrane permeant than the free base. Indeed, if the latter equilibrates readily across membranes then the pH within the vesicle i.s given by the relationship (1.5) ... 

Hiruma, H. Katakura, T. Takenami, T. Igawa, S. Kanoh, M. Fujimura, T. Kawakami, T. Vesicle disruption, plasma membrane bleb formation, and acute cell death caused by illumination with blue light in acridine orange-loaded malignant melanoma cells. J. Photochem. Photobiol, B Biol. 2007, 86, 1-8. 

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