Microsomal protein fraction

Pedron et al generated ABA-protein (ovalbumin or BSA) conjugates through the C-1 carboxyl group or the C-4 carbonyl group of ABA (11 and 12) [20]. ELISA detection showed that these ABA-protein conjugates bound efficiently to the solubilized microsomal protein fraction of Arabidopsis thaliana, independent of the nature of the carrier protein or the ABA-carrier protein linker. Purification of these binding proteins has not yet been reported. 

Recently, Danielsson et al. reported that the activity of a purified 12a-hydroxylat-ing system from rabbit liver microsomes could be modulated by protein fractions from rabbit hver microsomes and cytosol [103]. The microsomal protein fraction had a stimulatory effect whereas the cytosolic protein was inhibitory. Addition of ATP and MgCl2 or NaF had no effect on the activities of the two protein fractions, indicating that phosphorylation-dephosphorylation was of little or no importance. The microsomal 12a-hydroxylase stimulator also stimulated cholesterol 7a-hydroxyl-ase activity. 

There are also numerous enzymes anchored in membranes of the microsomal cell fraction that participate in the metabolism of steroid hormones. Thus, those of the p450 family, which carry out molecular oxidation, or the sulfatases and sulfotransferases, more or less specific to several hormones (Pasqualini et al. 1995). The affinity of steroid hormones for proteins of the membrane (Kd between 10 and 100 nM) is frequently greater than that which some of these enzymes present for their substrates (Luzardo et al. 2000). Therefore, it is unlikely that a part of the proteins of the membrane that bind steroids is in reality enzymes metabolizing these hormones. 

Each pellet ( dirty microsomal fraction ) was resuspended in 2 mL of ice-cold 1.15 % KCl, collected together and ultracentrifuged again at 100 000 x g for 45 min at 0 °C. The supernatant was discarded and the pellet was rinsed thrice with ice-cold dilution buffer. The rinsed pellet was resuspended in ice-cold 1.15 % KCl to yield a final concentration 20 mg mL of microsomal proteins. The microsomal suspension was immediately aliquoted, frozen and stored at —80 °C. 

Previous studies by Hoagland et al. (13), Zamecnik et al. (24), and in this laboratory (9, 10) demonstrated that the transfer of amino acid from isolated sRNA-amino acid to microsomes required GTP, ATP, an ATP-generating system, and a soluble portion of the cell. Most of the aminoacyl-transferring activity present in the homogenate supernatant was recovered in the pH 5 Supernatant obtained after precipitation of the amino acid-activating enzymes at pH 5. A protein fraction, 500- to... 

Figure 2. Subtractive proteomics. It is impossible to purify NEs to homogeneity because of the many connections to both the nucleoplasm and the cytoplasm. Thus, biochemically purified NEs are expected to be contaminated with chromatin and cytoskeletal proteins and with vesicles from organelles such as mitochodria and ER. In contrast, some of these expected contaminants can be purified free of NE contamination. One such contaminant is ER, which can be isolated as microsomes. Another is mitochondria, which has a well characterized protein complement. Therefore NE and microsomal membrane fractions are separately isolated and analyzed for protein content by MudPIT. All proteins appearing in both fractions are removed from the NE dataset because they could be due to ER vesicles sticking to the isolated nuclear NEs. Similarly, known mitochondrial proteins are removed. Because ER and mitochondria are the only expected membrane contaminants of NEs, all remaining integral membrane proteins in the NE fraction should be NE-specific in theory. After prediction by computer algorithm for membrane-spanning segments, an in silica purified NE transmembrane protein list is obtained. A limitation of this approach is that it discounts any proteins that are found both within the ER and the NE membranes (e.g. solid black triangles).

With the piupose of protein fraction isolation of aminoacyl-tRNA-synthetases (ARS), the tissue was triturated from the frozen state, the produced powder was extracted in weak saline neutral buffer in the presence of protease inhibitors [4]. After depositing microsome and membrane fractions, total ARS fraction was obtained by bringing pH to 5.0, protein deposit was dissolved in 0.05 M tris-HCl-buffer, 0.1 mM 2-mercaptoethanol, and 30% glycerin and stored at -20 °C. 

Many rate-limiting enzymes are modulated by reversible phosphorylation-de-phosphorylation. Recently, it was suggested that cholesterol 7a-hydroxylase is subject to such modulation. Sanghvi et al. reported that the activity of cholesterol 7a-hydroxylase in crude microsomes was increased after inclusion of ATP, Mg and a cytosolic protein fraction in the incubation [90]. There was a loss of enzyme activity in the presence of E. coli alkaline phosphatase which was proportional to the amount of phosphatase. Much of this loss was recovered upon addition of ATP, Mg and the cytosolic protein fraction. Similar results were reported in a later publication by Goodwin et al. [91]. In contrast, Kwok et al. reported that ATP as well as ADP had an inhibitory effect on 7a-hydroxylation of cholesterol. AMP and cyclic AMP were found to be stimulatory [92]. The inactivation by ATP was dependent on Mg and a cytosoUc factor [92]. 

Staple et al. showed that the conversion of 3a,7a,12a,24-tetrahydroxy-5/8-cholestanoic acid into cholic acid (cf. Fig. 3) can occur in rat liver microsomes or in cytosolic fractions fortified with NAD" or NADP and that propionic acid is released [42,43]. Pedersen and Gustafsson showed recently that the peroxisomal fraction had a high capacity to convert 3a,7a,12a-trihydroxy-5 8-cholestanoic acid into cholic acid [150]. Later, Kase et al. found that the peroxisomal fraction was more active than the microsomal and the mitochondrial fractions and that 3a,7a,12a-24-tetrahydroxy-5j8-cholestanoic acid was an intermediate in the conversion [151]. Thus, some of the previous contradictory results may be explained by varying degrees of contamination of the microsomal and mitochondrial fractions with peroxisomes. In the work by Kase et al. it was shown that the over-all conversion of 3a,7a,12a-trihydroxy-5 -cholestanoic acid into cholic acid in the peroxisomes was absolutely dependent upon the presence of Mg ", CoA, ATP and NAD. The reaction was stimulated by FAD, by cytosolic protein, by microsomal protein and by bovine serum albumin. It is possible that the stimulatory effect of the microsomes and cytosol was imspecific and due to the increased protein concentration per se. The stimulatory effect of FAD was taken as evidence that 3a,7a,12a-tri-hydroxy-5yS-cholestanoyl-CoA oxidase is a FAD-containing protein. There was a lag phase in the reaction, possibly due to the activation step, and it was suggested that the activation was rate limiting. Also in this case, it was not possible to isolate a A -unsaturated intermediate in the reaction. The participation of a desaturase and a hydratase was proved by the incorporation of from H20 into 3a,7a,12a,24-te-trahydroxy-5/8-cholestanoic acid (Bjorkhem, Kase and Pedersen, unpublished study). 

Monoamine oxidases are integral outer mitochondrial membrane proteins that catalyze the oxidative deamination of primary and secondary amines as well as some tertiary amines. MAO occurs as two enzymes, MAO-A and MAO-B, which differ in substrate selectivity and inhibitor sensitivity (Abell and Kwan, 2001 Edmondson et al., 2004 Shih et al., 1999). A number of MAO inhibitors have been developed for clinical use as antidepressants and as neuroprotective drugs. Clinically used drug substances include, among others, moclobemide, a relatively selective reversible MAO-A inhibitor, and L-deprenyl, an irreversible selective inhibitor of MAO-B. In vitro, clorgyline and L-deprenyl are used as selective irreversible inhibitors of MAO-A and B, respectively. (Note For in vitro studies using irreversible inhibitors, preincubation of the irreversible inhibitor with the enzyme prior to initiation of the substrate reaction is required for optimal inhibition.) Expressed MAO-A and MAO-B are not readily available via commercial resources however, MAO-A and MAO-B have been evaluated and are active in subcellular fractions. While monoamine oxidases are located in the mitochondria, many microsomal preparations are contaminated with monoamine oxidases during the preparation of the microsomal subcellular fraction and thus microsomes are sometimes used to evaluate monoamine oxidase activity in combination with selective inhibitors. 

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