Vesicle incubation conditions

Microsomal Incubation Conditions Incubations in animal or human liver microsomes are the most common way to determine activity in the presence of added substrate, UDPGA, Mg, and a buffer. As there is no method available to directly determine enzyme concentration, the incubations are standardized by addition of the same amount of protein (typically 0.25-1.0 mg protein/ImL) after determination of linearity of product formation with respect to protein concentration and time. In general, the enzyme is stable up to 45 min to 1 h. Because of the location of the enzyme, a portion of the microsomal vesicle will be obtained in the normal configuration with the enzyme active site entrapped within the vesicle. Since UDPGA must have access to the active site, and the UDPGA influx transporter is not operative without ATP, it may be necessary to activate or remove latency of the enzyme. In the past this has been achieved by a variety of methods, but most commonly by addition of detergents such as Brij 58, Lubrol, or Triton X... 

Incubation Conditions for Golgi-Membrane Binding and Vesicle Budding Reactions... 

In 1985 Tyminski etal. [55, 56] reported that two-component lipid vesicles of a neutral phospholipid, e.g. DOPC, and a neutral polymerizable PC, bis-DenPC (15), formed stable homogeneous bilayer vesicles prior to photopolymerization. After photopolymerization of a homogeneous 1 1 molar lipid mixture, the lipid vesicles were titrated with bovine rhodopsin-octyl glucoside micelles in a manner that maintained the octyl glucoside concentration below the surfactant critical micelle concentration. Consequently there was insufficient surfactant to keep the membrane protein, rhodopsin, soluble in the aqueous buffer. These conditions favor the insertion of transmembrane proteins into lipid bilayers. After addition and incubation, the bilayer vesicles were purified on a... 

To demonstrate polymerase activity in a model cell, Chakrabarti et al. [79] encapsulated polynucleotide phosphorylase in vesicles composed of dimyris-toylphosphatidylcholine (DMPC). This enzyme can produce RNA from nucleoside diphosphates such as adenosine diphosphate (ADP) and does not require a template, so it has proven useful for initial studies of encapsulated polymerase activity (Fig. 10a). Furthermore, DMPC liposomes are sufficiently permeable so that 5-10 ADP molecules per second enter each vesicle. Under these conditions, measurable amounts of RNA in the form of polyadenylic acid were synthesized and accumulated in the vesicles after several days incubation. The enzyme-catalyzed reaction could be carried out in the presence of a protease external to the membrane, demonstrating that the vesicle membrane protected the encapsulated enzyme from hydrolytic degradation. Similar behavior has been observed with monocarboxylic acid vesicles [80], and it follows that complex phospholipids are not required for an encapsulated polymerase system to function. 

Inside-out vesicles can be perfectly used to study single efflux processes, because preloading of cells is not necessary and because of well-defined study conditions with transporter directly facing compound concentration in the incubation medium in contrast to double-transfected cell lines (3.1.2.4.L). Therefore, these studies allow establishing structure activity relationships (SAR) for one efflux transporter only. 

One approach that has been used quite widely to quantitate neurotransmitter release employs radiolabeled (tritiated) neurotransmitter analogs (e.g.. Reference 67). First, tissue is incubated in a buffer solution that contains tritiated neurotransmitter. During this time, the radiolabeled transmitter is taken up into cells by endogenous plasma-membrane transporters and packaged into vesicles by vesicular transporters. The tissue preparation then is rinsed in buffer to remove extracellular radiolabeled transmitter leaving only that which was taken up into cells. This stored transmitter is then released over time by exocytosis. To quantitate its release, the tissue is continuously perfused with buffer, and time-dependent aliquots are collected. Radioactivity is measured in the aliquots with a scintillation counter and is used as an index of endogenous neurotransmitter release. Rather than estimate absolute neurotransmitter release, this method is typically used to compare the relative release between two or more conditions. 

M thermoautotrophicum cells synthesize ATP in the presence of an artificially imposed pH gradient [18], Proton uptake is not detected following acidification. The addition of valinomycin results in the synthesis of ATP and is accompanied by the extrusion of K but not protons. ATP synthesis is unaffected by DCCD and is stimulated by uncouplers such as 2,4-dinitrophenol and m-chlorophenyl hydrazone. Membrane vesicles from M thermoautotrophicum synthesize ATP when conditions are anaerobic in response to the membrane potential since the addition of suppresses synthesis. ATP synthesis is inhibited by 100 pM CCCP and partially inhibited by DCCD (53% at 100 pM). ATP synthesis also takes place in response to a ApH produced by the oxidation of hydrogen. In this case, ATP synthesis is inhibited by lOpM DCCD and CCCP. Unlike cells, vesicles do not synthesize ATP in response to an artificially imposed ApH or in the presence of valinomycin [54]. M. thermoautotrophicum membranes have an ATPase activity that hydrolyzes ATP, GTP, and UTP at approximately the same rate. The enzyme loses activity at -90°C which is due to aggregation, and activity is restored following sonication. ATPase activity is partially inhibited by DCCD (40% at lOOpM) when membranes are incubated at pH 8 for 10 min [18]. A similar ATPase is found in a different strain of M. thermoautotrophicum [29]. The enzyme is most active at an alkaline pH and it is not significantly inhibited by ADP, 5mM NEM, or 150 pM DCCD. The absence of NEM inhibition suggests that the enzyme may not be a V-type ATPase. 

The leakage of small water-soluble dyes encapsulated in the aqueous interior of liposomes during their preparation is often used as a method to study their membrane integrity during incubation under various conditions (temperature, pH, presence of serum proteins, etc.). In the case of ARSL s, the release of CF or calcein has been used as a measure of the vesicle membrane integrity, during incubation of ARSL in buffer or in presence of serum proteins [80% FCS] at 37°C under mild agitation. Calcein (or CF) is encapsulated in the vesicles in a quenched concentration (100 mM), and, therefore, its release from the membrane can be calculated without separation of free and liposomal dye, as reported before (23). In brief, 20 pL of the incubated ARSL dispersion are drawn out from each incubation tube and diluted with 4 mL of PBS, pH 7.40. The fluorescence intensity of the samples is then measured (EM 490 nm, EX 520 nm, slit-slit 10-10), before and after the addition of Triton X-100 at a final... 

The ATP-vesicles were also tested for their effect on cell survival during varions ischemic conditions. Using HUVECs incubated with ATP-vesicles, we tested the effect of 6-h hypoxia (<0.5% O ) on cell viability as measured by adherence. The experiments were repeated three times for each condition... 

Acid CEH has an optimum pH of 4.5 and is located within lysosomes (Fig. 1). Like many lipolytic enzymes, it is water soluble whereas its substrate is not. For this reason, devising a reliable assay method is difficult, and results should be viewed with caution [26,35]. Recently developed conditions foimd to yield satisfactory linearity with both time of incubation and enzyme concentration are 12.7 jitM cholesteryl oleate, dispersed in 1.27 mM egg PC 50 mM acetate buffer 2.0 mM sodium taurocholate 0.005% digitonin pH 3.9 [36]. In aortic cells these conditions gave an apparent of 1.5 /iM for cholesteryl oleate. It should be noted, however, that the nature, physical form, and molecular organization of the CE and associated molecules can have a critical effect on the activity of any preparation of CEH [37]. Rat liver acid CEH, for example, shows K s of 15.3, 14.3, and 7.3 jaM for cholesteryl oleate when the latter is present in vesicles, micelles, and emulsions, respectively [35]. 

Several investigators have studied the physiological relation between these activities under a variety of in vitro conditions. Zilversmit and colleagues [68] demonstrated that transfer of CE from reconstituted lipoproteins to small, unilamellar vesicles proceeded on an equimolar, reciprocal basis with TG transfer. However, Fielding and coworkers [74] clearly showed that CETP can mediate the net transfer of CE in the complete absence of TG. Generating CE by co-incubating LCAT, A-1 and UC-PC liposomes, they found that CE accumulated up to a finite limit that was... 

---