Solubilization and purification

In contrast to the case with the -translocating PPase of R. rubrum chromatophores, the membrane-bound mitochondrial PPase activity is more easily removed from the membrane than the ATPase. Two min sonication of beef heart sub- 

In the first reports [71,74], the two forms of mitochondrial PPase, membrane bound and free, were claimed to be isoenzymes, both having a molecular weight of 75 000. The membrane-bound form, termed pyrophosphatase II in accordance with a suggestion by Iria et al. [75], is a lipoprotein, containing phosphatidylcholine. Moreover, it was shown that the activity of the soluble enzyme, pyrophosphatase I, was greatly enhanced by the addition of mitochondrial lipids [74] and that addition of phosphatidylcholine to PPase I would convert it to PPase II. In this form it could be incorporated into PPase-depleted mitochondrial membranes and catalyze electron transport-linked PPj synthesis [76]. 

In contrast to these results, Volk et al. in a very recent publication [77] using a detergent based solubilization described in [78], found very distinct differences between pyrophosphatases I and II. PPase I has a molecular weight of 60000 and consists of two different subunits, a and PPase II has a molecular weight of 185 000 and is composed of four subunits, a, j8, y and 8. Because of the similarity in mass between subunits a and in the two enzymes, Volk et al. propose that these two subunits are the same in both enzymes and form the catalytic part of PPase II. PPase I from mitochondria thus seems to differ from other soluble pyrophosphatases in that the two subunits are nonidentical. 

It is interesting to note that Volk et al. are able to differentiate between the two enzyme activities by the lack of fluoride inhibition of PPase II, which is in contradiction with the results in [20]. Volk et al. also obtained, though careful differentiation of mitochondrial membranes, a localization of the PPases. They came to the conclusion that PPase I is localized in the mitochondrial matrix. PPase II is situated in the inner mitochondrial membrane, a localization in line with its proposed role as a coupling factor [71]. 

It may appear difficult to reconcile the differences in data from the two Russian groups. But one possible explanation is that Mansurova et al. [71,74] have extracted only part of PPase II, possibly subunits a and )8. The isolation method they actually have used is somewhat undetailed, but if it only consists of a buffer extraction from an acetone powder of mitochondria, without added detergents, one may well envisage that only part of the membrane-bound enzyme is liberated. The same may be true for their sucrose washing of submitochondrial particles with or without sonication. 

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Neisseria meningitidis Types A and Ct, Cultures of N. meningitidis of serotypes A and C 1 Precipitation with hexadecyl-trimethyammonium bromide 2 Solubilization and purification 3 Blending 4 Freeze-drying Estimation of capsular polysaccharide content ... 

Lefkowitz, R. J., Haber, E., and O Hara, D. (1972). Identification of the cardiac beta-adrenergic receptor protein Solubilization and purification by affinity chromatography. Proc. Natl. Acad. Sci. USA 69, 2828-2832. 

Selected entries from Methods in Enzymology [vol, page(s)] Glycosylphosphatidylinositol-specific phospholipase D, 197, 567 solubilization and purification of rat tissue phospholipase D, 197, 575 lysophospholipase D, 197, 583. 

Tsumoto K, Umetsu M, Kumagai I, Ejima D, Philo JS, Arakawa T. Role of arginine in protein refolding, solubilization, and purification. Biotechnol Prog 2004 20(5) 1301-1308. 

The aim of the present Section is to discuss existing information concerning sequences of glycosylation reactions during the assembly of polysaccharide chains. Data concerning identification of intermediates in the process, and on solubilization and purification of the enzymes involved, are also included. 

The enzyme was originally found to be membrane bound and resisted solubilization and purification (26). Lundblad and Moore (27), however, have reported solubilizing it using dilute (5 mM) sodium borate buffer at pH 9 after 16 hr at 37°. Studies on regional and subcellular distribution using density gradient techniques have revealed that the 2, 3 -cyclic phosphate diesterase concentrates in those fractions containing myelin (28, 29), and the conclusion has been reached that the enzyme is localized in the myelin sheath or intimately associated structures. Kurihara and... 

M Crouvoisier, D Mengin-Lecreulx, J van Heijenoort. UDP-N-acetylglucosamine N-acetylmuramoyl-(pentapeptide) pyrophosphoryl undecaprenol N-acetylglucos-amine transferase from Escherichia coli. overproduction, solubilization, and purification. FEBS Lett 449 289-292, 1999. 

Simon EJ, Hiller JM. Solubilization and Purification of opioid binding sites. In Pasternak GW, ed. The Opiate Receptors. Clifton, NJ Humana Press, 1988 165-194. 

Reactions Analytical Methods. Alkaline treatments are used in the food and feeds technology for solubilization and purification of proteins, to destroy toxic contaminants, and to obtain functional properties. These alkaline treatments induce many chemical modifications on the side chains of the amino acid residues, which have been described by many workers. 

Spitsberg, V.L., Gorewit, R.C. 1998. Solubilization and purification of Xanthine oxidoreductase from bovine milk fat globule membrane. Protein Expression Purif. 13, 229-234. 

A1983 review of the molecular properties of opioid receptors that includes an account of solubilization and purification procedures is available.020 ... 

Carbon monoxide inhibited the 6/3-. la-, and 16a-hydroxylation of testosterone by rat liver microsomes to different extents. A C0/02 ratio of 0.5 inhibited the la-, 6/i-, and 16a-hydroxylation reactions by 14%, 25%, and 36%, respectively, and the ratio of C0/02 needed for 50% inhibition of testosterone hydroxylation in the 16a-, 6/3-, and 7a-positions was 0.93, 1.54, and 2.36, respectively (36,48). Studies on the photochemical action spectrum revealed that CO inhibition of the three hydroxylation reactions was maximally reversed by monochromatic light at 450 nm, but there were differences in the shape of the photochemical reactivation spectra for the 6/3-, la-, and 16a-hydroxylation reactions (36,48). The data from our laboratory summarized above and at the First International Symposium on Microsomes and Drug Oxidation in 1968 pointed to multiple cytochromes P450 with different catalytic activities that were under separate regulatory control (36,45,46), and we indicated that the actual number of cytochromes that participate in the multiple hydroxylation reactions must await the solubilization and purification of the microsomal system (36). The use of different inducers of liver microsomal monooxygenases caused selective increases in the concentration of specific cytochromes P450 in fiver microsomes that greatly facilitated the isolation and purification of these hemoproteins. 

Elstner EF, Staffer C, Heupel A (1975) Determination of superoxide free radical ion and hydrogen peroxide as products of photosynthetic oxygen reduction. Z Naturforsch 30c 53-57 Emmel T, Sand W, Konig WA, Bock E (1986) Evidence for the existence of a sulphur oxygenase in Sulfolobus brierleyi. J Gen Microbiol 132 3415-3420 Ensign SA, Hyman MR, Arp DJ (1993) In vitro activation of ammonia monooxygenase from Nitrosomonas europaea by copper. J Bacteriol 175 1971-1980 Erickson RH, Hooper AB (1972) Preliminary characterization of variant CO-binding heme protein from Nitrosomonas. Biochim Biophys Acta 275 231-244 Erickson RH, Hooper AB, Terry KR (1972) Solubilization and purification of cytochrome a, from Nitrosomonas. Biochim Biophys Acta 283 155-166 Evans MCW, Buchanan BB, Amon DI (1966) A new ferredoxin-dependent carbon reduction cycle in a photosynthetic bacterium. Proc Natl Acad Sci USA 55 928-934 Falk JE (1964) Porpyrins and metalloporphyrins. Elsevier, Amsterdam... 

Expression of recombinant aprotinins has been achieved in E. coli K12 as a fusion with parts of the MS-2 polymerase gene [16]. In this case the fusion protein is deposited as inclusion bodies. Functionally active aprotinin can be obtained after solubilization and purification of the fusion protein, CNBr (cyanobromide) cleavage and renaturation of the aprotinin moiety. Low-level periplasmic expression of native, properly folded aprotinin has been shown in E. coli employing the E. coli alkaline phosphatase signal sequence [17]. With respect to expression level and ease of purification, it transpired that secretory expression in the yeast Saccharomyces cerevisiae is by far the most attractive system for the production of this aprotinin variant [18]. In addition, due to the absence of an N-glycosylation site there are no problems with non-human glycosylation of the protein in yeast. 

Investigations to determine whether the transfers to the various acceptors are catalyzed by one nonspecific enzyme, or by several specific enzymes, have been hampered by the particulate (microsomal) nature of active enzyme-preparations and by lability of the preparations obtained when the microsomes are solubilized by conventional methods. However, a solubilization and purification of D-glucopyranosyluronic transferase has been achieved by treating liver microsomes with the venom of Trime-resurus flavoviridis The soluble enzyme showed activity toward phenols and carboxylic acids, but not toward aniline, indicating the existence of at least two different transferases. 

Erni, B. Trachsel, H. Postma, P.W. Rosenbusch, J.P. Bacterial phosphotransferase system. Solubilization and purification of the glucose-specific enzyme II from membranes of Salmonella typhimurium. J. Biol. Chem., 257, 13726-13730 (1982)... 

We have undertaken a biochemical characterization of the Aj adenosine receptor using pig heart as a tissue source. The porcine heart and circulatory system resembles that of man in many respects and this system may provide sufficient material for studies on the solubilization and purification of the receptor protein. The agonist radioligand... 

First described in the literature in 1814, the blue reaction of iodine with starch is the best-known example of a polymerization process induced by supramolecular stabilization. Structural studies indicate that amylose (the linear fraction of starch) forms a helix with six glucose residues per turn to include guest iodine molecules. Inside this helix, iodine molecules form a polymeric chain with a periodicity of 3.1 A, which is much shorter than the nonbonded distance between iodine atoms (4.3 A) but greater than the single I-I bond distance (2.7 A). The amylose-iodine reaction is widely used in analytical chemistry and was utilized for the solubilization and purification of carbon nanotubes. 

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