Microsomes solubilization

A full understanding of the role of pectin in plant development requires elucidation of the mechanisms that regulate p>ectin biosynthesis (6). Our strategy for studying the biosynthesis of HGA was to 1) establish a PGA-GalAT assay that would allow detection of synthesized HGA, 2) characterize the enzyme in microsomal membranes, 3) characterize the product synthesized by the enzyme in microsomal membranes, and 4) solubilize the enzyme and characterize the solubilized enzyme and its product. 

Fath A, Boiler T. Solubilization, partial purification and characterization of a binding site for a gly copeptide elicitor from microsomal membranes of tomato cells. Plant Physiol 1996 112 1659-1668. 

Recently Bend et al. have succeeded to solubilize and partially purify little skate liver microsomal cytochrome P-l+50 and have thus been able to record the absolute spectra of fish cytochrome P-l+50 (25.). The spectra are very similar to those of rat liver microsomal cytochrome P-l+50 and purified rat liver cytochrome P-1+50 and P-1+1+8 (Table II). 

Omura, T. and Sato, R. The carbon monoxide-binding pigment of liver microsomes. II. Solubilization, purification, and properties. J. Biol. Chem. (1961 ) 239, 2379-2385. 

Hepatic microsomal and solubilized mixed-function oxidase systems from the little skate, Baja erinacea, a marine elasmobranch. In Ullrich, V., Hildebrandt, A., Roots, I., Eastabrook, R.W. (Eds.) Microsomes and Drug Oxidations (1976). Pergamon Press, Oxford, pp 16O-I69. 

In general, our studies with cytochrome P-450-dependent metabolism have emphasized the similarity of the hepatic MFO system in marine fish to that found in mammals. Thus, in the little skate (Raja erinaoea), a marine elasmobranch, enzyme activity is localized in the microsomal fraction, requires NADPH and molecular oxygen for maximum activity, and can be inhibited with CO (1, 2). Moreover, when hepatic microsomes from the little skate were solubilized and separated into cytochrome P-450, NADPH-cytochrome P-450 reductase, and lipid fractions, all three fractions were required for maximal MFO activity in the reconstituted system (3). We have also found, as have others, that the administration of polycyclic hydrocarbons (3-methylcholanthrene, 1,2,3,4-dibenzanthracene [DBA]), 2,3,7,8-tetrachlorodibenzo-p-dioxin... 

Solubilization and Partial Purification of Cytochrome P-450 from Hepatic Microsomes of Male, DBA-Pretreated Little Skates. Washed hepatic microsomes (3) from the livers of 10 skates were suspended in 0.25 M sucrose and frozen under nitrogen (-5 to -10°) at the Maine laboratory. They were then packed in dry ice and transported to NIEHS, Research Triangle Park, NC, within 14 days of preparation and were stored at -62°C until use. Microsomes... 

Figure 3. Elution profile of partially purified Cytochrome P-448 and epoxide hydratase activity of solubilized hepatic microsomes from DBA-treated male skates from a DEAE-cellulose column with Buffer II. Epoxide hydratase activity was determined with BP-4,5-oxide as the substrate (see Materials and Methods).

Very similar results to those described in Fig. 3-6 were obtained when sodium cholate solubilized hepatic microsomes from DBA-treated female little skates were subjected to chromatography on DEAE-cellulose as described above (data not shown). Also not shown are the results obtained with hepatic microsomes from untreated male and female little skates. With untreated animals, 80-90% of the cytochrome P-450 eluted from the DEAE-cellulose column only at higher ionic strength (i.e., with the KC1 gradient). However, in all preparations studied, an appreciable amount of cytochrome P-450 (10-20%), having its absorption maximum in the carbon monoxide-ligated and reduced state at 450 nm, was eluted from the column with buffer II, as was observed with cytochrome P-448 of hepatic microsomes from DBA-treated skates. The further purification of the various forms of cytochrome P-450 from control and DBA-pretreated little skate livers is currently in progress in our laboratory. 

In contrast, the study of proteins present in minute amounts and/or of restricted localization may require selective protein extraction. Several procedures have been described including use of nuclei (Sormenberg et al., 1989) modified by Hope (Hope et al., 1994), sarcolemmal-enriched membrane fractions (Tuana et al., 1987), apoHpoprotein (Peitsch et aL, 1989), or microsome fractions (Kumar et al., 1985) for protein extractions. Numerous others exist and as a general rule, they rely first on the isolation of the cellular compartment of interest followed by the solubilization of the proteins contained in that cellular fraction. 

With albumin-solubilized bilirubin, pH optima of microsomal bilirubin UDP-glucuronyltransferase were 7.4-8.0 for rat (H2, HIO, SIO) and 7.4 for guinea pig (M13) and rabbit (T8). Above pH 8 the enzyme activity decreased abruptly (HIO). In absence of carrier protein, optima were at pH 8 and 8.2 with preparations from liver of guinea pig (P3) and rat (W12), respectively. The activity-pH curve with optimum at pH 8.2 (W12) showed pronounced skewing, with a steady and rather rapid increase of enzyme activity from pH 7.4 to 8.2. One may wonder whether such measurements were influenced by the rapid increase of solubility of the acceptor substrate occurring over the same pH range (B25). 

Isselbacher, K. J., Chrabas, M. F., and Quinn, R. C., The solubilization and partial purification of a glucuronyl transferase from rabbit liver microsomes. J. Biol. Chem. 237, 3033-3036 (1962). 

Preparation of Glucan Synthase Fractions. Microsomal and plasma membranes were isolated by differential and density-gradient centrifugation. CHAPS-solubilized glucan synthase (CSGS) was prepared by the two-step procedure (4,9). In step 1, contaminating proteins were extracted with... 

Rat brain microsome preparations were conveniently stored at -10C with retention of enzyme activity. Solubilization with Triton X-100 appears to be effective and the solubilized enzyme preparation, after filtration once with Amicon XM-300 diaflo membrane, was introduced into an isoelectric focusing column (LKB) with an ampholine pH range of 3.5-10. 

Selander HG, Jerina DM, Daly JW. 1975. Metabolism of chlorobenzene with hepatic microsomes and solubilized cytochrome P-450 systems. Arch Biochem Biophys 168 309-321. 

This interconversion is catalaysed by 17)3-hydroxysteroid dehydrogenase (17/3-HSD), an enzyme generally found in the ER of numerous tissues such as adrenal, liver, testis, ovary and kidney. Like many of the enzymes described above, there appear to be different forms [52,87]. For example, rat adrenal cytosol and ER contain separate 17/3-HSDs, with NADH as the preferred cofactor. The rat testicular enzyme, however, prefers NADPH. Guinea-pig liver also contains two 17j3-HSDs, one solubilized from cytosol, the other associated with the ER [88], These enzymes exhibit different activities towards C19 steroids, the cytosolic one preferring 5/3-reduced 17-oxosteroids and the microsomal counterpart being involved with 5a-reduced steroids, such as 5a-DHT. In this case, the product of the reaction would be 5a-an-drostane-3,17-dione. 

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