Microsomal vesicles

Microsomes Vesicles obtained from homogenized tissues by ultracentrifugation. They are derived mainly from the endoplasmic reticulum in the case of the liver (hepatic microsomes). 

The amount of total enzymatic activity that becomes manifest only after disruption of membranous barriers between enzyme and substrate or upon removal of some otherwise inhibitory factor. Membrane disruption is often achieved by treatment with detergent to solubihze the enzyme. One example is the so-called microsomal glucose-6-phosphatase, an enzymatic activity that is located in the lumen of the endoplasmic reticulum but becomes trapped as a latent activity in microsome vesicles upon mechanical disruption of cells. 

The formation of disulfide bonds in proteins synthesized in vitro can be followed by measuring enzymatic activity or by an increased mobility compared to the reduced protein during SDS-PAGF. This increased mobility arises from the fact that, as disulfide-bonded proteins are intra-molecularly cross-linked, they form a more compact structure and occupy a smaller hydrodynamic volume compared to the reduced protein (Gold-enberg and Creighton, 1984). An illustration of this increase in mobility is shown in Fig. 2. Here the mRNA for preprolactin was translated in a cell-free system optimized for the formation of disulfide bonds, and then analyzed by SDS-PAGF. The translocated protein forms disulHde bonds under these conditions whereas the protein synthesized under the same conditions but in the absence of microsomal membranes does not form disulfide bonds. Thus the nascent protein must be translocated into microsomal vesicles for disulfide bond formation to occur. 

Fig. 2. Cell-free translation of preprolactin niRNA under conditions favoring the formation of disulfide bonds. Translation products were separated by SDS-PAGE after being either reduced and carboxamidomethylated (lanes 1 and 2) or carhoxamidomethylated only (lanes 3 and 4). Lanes 1 and 3, products of translation in the absence of microsomal vesicles lanes 2 and 4, products of translation in the presence of microsomal vesicles.

A sophisticated approach using active site-directed pho-toafflnity substrate analogs has also been applied to study the inward transport in isolated microsomal vesicles. These experiments provided convincing and elegant evidence for the translocation of UDP-glucuronate (48) and of FAD (12) by photo incorporation of the probes into the luminally oriented enzymes in intact microsomes. The drawback of the method is that it is not suitable to determine of the rate or capacity of transport. 

Banhegyi G, Marcolongo P, Eulceri R, Hinds C, Burchell A, Benedetti A. Demonstration of a metabolically active glucose-6-phosphate pool in the lumen of liver microsomal vesicles. J. Biol. 

Csala M, Mile V, Benedetti A, Mandl J, Banhegyi G. Ascorbate oxidation is a prerequisite for its transport into rat liver microsomal vesicles. Biochem. J. 2000 349 413-415. 

Radominska A, Berg C, Treat S, Little JM, Lester R, Gollan JL, Drake RR. Characterization of UDP-glucuronic acid transport in rat liver microsomal vesicles with photoaffinity analogs. Biochim. Biophys. Acta 1994 1195 63-70... 

Battaglia E, Gollan J. A unique multifunctional transporter translocates estradiol-17P-glucuronide in rat liver microsomal vesicles. J. Biol. Chem. 2001 276 23492-23498. 

We focused on the gastric H,K-ATPase as a test protein because i) purified gastric microsomal vesicles are highly enriched in the enzyme (> 85-90% purity), ii) the vesicles are oriented with a common asymmetry i.e. cytoplasmic side out (12), iii) the vesicles are sealed allowing selective cytoplasmic digestion, and iv) there is a pool of existing topological data from other methods (13, 14, 15 16). 

Figure 2. The gastric H,K-ATPase in gastric microsomal vesicles. The H,K-ATPase is a heterodimer composed of an a-subunit and a glycoprotein -subunit, which are asymmetrically oriented.

Preparation of H,K-ATPase enriched microsomal vesicles. H,K-ATPase-containing gastric microsomal vesicles were isolated from rabbit stomach as previously described (12). Crude microsomes were harvested from homogenized mucosa of unstimulated rabbit stomach (H2 receptor-blocked) as the membrane pellet sedimenting between 10 min at 13,000 x g and 1 hr at 100,000 x g. The pellet was resuspended in 10% sucrose, brought to 40% sucrose (9 ml), and overlaid with successive layers of 30% sucrose (11 ml), 10% sucrose (16 ml) [300 mM sucrose, 5 mM tris(hydroxymethyl)aminomethane (Tris), and 0.2 mM EDTA, pH 7.4] in a 37 ml tube. After centrifugation at 80,000 x g for 4 hr, the purified gastric microsomal vesicles were collected from the interface between 10% and 30 % sucrose and stored at 4° C until use. 

Trypsinization of H,K-ATPase-enriched gastric microsomal vesicles. Tubulovesicles (-100 pg of protein) were treated with trypsin (5 pg) in Tris.HCl (20 mM, pH 7.5) at 37°C for 30 min. The vesicles were next centrifuged at 100,000 X g on a TLIOO table top centrifuge for 1 hr at 4°C. The supernatant was carefully separated from the pellet. The supernatant was next boiled for 5 min and stored at -20°C until further analysis. 

Polokoff MA, Bell RM. Limited palmitoyl-CoA penetration into microsomal vesicles as evidenced by a highly latent ethanol acyltransferase activity. J Biol Chem 1976 253 7173-7178. 

The majority of these reactions are catalysed by one enzyme system, the cytochromes P-450 monooxygenase system, which is located particularly in the SER of the cell. The enzyme system is isolated the so-called microsomal fraction which is formed from the endoplasmic reticulum when the cell is homogenized and fractionated by differential ultracentrifugation. Microsomal vesicles are thus fragments of the endoplasmic reticulum... 

As with their animal counterparts, there is evidence that plant G-proteins can modulate the activities of enzymes and ion channels. Dillenschneider et al. [38] showed that GTP.,S promoted the turnover of inositol phospholipids in Acer pseudoplatanus suspension culture cells, whilst Allan et al. [2] demonstrated that GTP was effective in promoting the release of Ca" from Cucurbita microsomal vesicles. In contrast, Melin et al. [96], who... 

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... 

Fig. 5. Schematic model for the ion movements aeross gastric microsomal vesicles. The J values are the ionic fluxes with the superseiipts designating pump flux (P) or leak pathway (L). The model consists of an ATP-driven exchange pump, (K +H )-ATPase, and the passive leak pathways for the...

Having CATa and CATb on either side of the microsomal membrane appears to serve no obvious purpose unless, by analogy with the well-characterized mitochondrial system,these are linked to some form of transport system to move fatty acylcamitines across the membrane. It is impractical to study directly the transport of radiolabelled long-chain fatty acylcamitines into or out of sealed microsomal vesicles because these metabolites bind non-specifically to many cellular proteins. We, therefore, devised a way to do this indirectly, based on the use of the lumenal enzyme ethanol acyltransferase (EAT) as a reporter . In the endoplasmic reticulum EAT ° catalyzes the reaction ... 

Niot, I., Pacot, F., Bouchard, P., Gresti, J., Bernard, A., Bezard, J. Clouet, P. (1994) Biochem. J., 3M, 577-584. Involvement of microsomal vesicles in part of the sensitivity of carnitine palmitoyltransferase I to malonyl-CoA inhibition in mitochondrial fractions of rat liver. 

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