Solubilization by detergents

Figure 12.2 (a) Schematic drawing of membrane proteins in a typical membrane and their solubilization by detergents. The hydrophilic surfaces of the membrane proteins are indicated by red. (b) A membrane protein crystallized with detergents bound to its hydrophobic protein surface. The hydrophilic surfaces of the proteins and the symbols for detergents are as in (a). (Adapted from H. Michel, Trends Biochem. Sci. 8 56-59, 1983.)... 

Table 3, which summarizes some recent applications of detergents to solubilizing particulate plant proteins, provides an indication of the diversity of proteins that can be solubilized by detergent action. 

Although the majority of the lipids in M. laidlawii membranes appear to be in a liquid-crystalline state, the system possesses the same physical properties that many other membranes possess. The ORD is that of a red-shifted a-helix high resolution NMR does not show obvious absorption by hydrocarbon protons, and infrared spectroscopy shows no ft structure. Like erythrocyte ghosts, treatment with pronase leaves an enzyme-resistant core containing about 20% of the protein of the intact membrane (56). This residual core retains the membrane lipid and appears membranous in the electron microscope (56). Like many others, M. laidlawii membranes are solubilized by detergents and can be reconstituted by removal of detergent. Apparently all of these properties can be consistent with a structure in which the lipids are predominantly in the bilayer conformation. The spectroscopic data are therefore insufficient to reject the concept of a phospholipid bilayer structure or to... 

Peripheral Mitochondrial ATPase, alkaline phosphatase Readily solubilized by detergents... 

Much more is known about the interaction of cells with laminin. A high-affinity (Ad = 1 x 10 9 to 4 x 10-9) receptor for laminin has been found on many cells including myoblasts, tumor cells, and macrophages (Lesot et al., 1983 Rao et al., 1983 Malinoff and Wicha, 1983). The laminin receptor (Mr 67K) is solubilized by detergent and has all the characteristics of an integral membrane protein. A partial amino acid sequence of the receptor has been deduced from the nucleotide sequence of cDNA clones. These show a possible transmembrane sequence and suggest that this receptor has substantial cytoplasmic and extracellular domains (Wewer et al., 1986). 

During cell fractionation, nearly all the cyclopenase activity was found in a fraction containing the cell wall together with the cytoplasmic membrane (83,84). From this fraction, the enzyme could be partially solubilized by detergents (e.g., Triton X-100) to yield a protein-phospholipid complex. By treatment with M-butanol, the solubilized enzyme preparation was split into the lipid fraction and the enzyme protein which retained a considerable part of the total enzyme activity. Compared with that of the membrane-bound enzyme, the substrate affinity of the solubilized protein-lipid complex was decreased, whereas... 

Initially, this process was thought to be concerted in nature, but it has been demonstrated that oxidation and cyclization are different steps by the synthesis and isolation of 2,3-oxidosqualene as an intermediate and the use of inhibitors of the cyclization reaction such as 2,3-iminosqualene [92]. The enzymes for the oxidation and cyclization differ in their susceptibiUty to solubilization by detergents. The partially purified cyclase M, 90000) aggregates and is inactivated upon removal of the detergent. The oxidation is Oj dependent while cyclization is anaerobic with no cofactors required. 

CD spectroscopy has been used to study the conformation of a number of membrane proteins. The use of CD for determining the secondary structure of membrane proteins has been reviewed. Membrane proteins which have been solubilized by detergents generally present no special difficulties for CD analysis, but the conformation is likely to be different from that in the native environment. The study of membrane proteins in situ by CD has been the subject of considerable controversy. Two kinds of artifacts which may afflict such studies have been identified. 

Ghoroghchian PP, Frail PR, Susumu K, Park TH, WU SP, Uyeda HT, Hammer DA, Therien MJ (2005) Broad spectral domain fluorescence wavelength modification of visible and near-infrared emissive polymersomes. J Am Chem Soc 127 15388-15390 Ghoroghchian PP, Jin JJ, Brannan AK, Frail PR, Bates FS, Therien MJ, Hammer DA (2006) Quantitative membrane loading of polymer vesicles. Soft Matter 2 973-980 Pata V, Ahmed F, Discher DD, Dan N (2004) Membrane solubilization by detergent resistance conferred by thickness. Langmuir 20(10) 3888-3893... 

The chemical shifts of various phospholipids in organic solvent and Triton X-100 micelles are shown in Tables 9.13 and 9.14. A detailed discussion on P-NMR of phospholipids in micelles, including applications to membrane solubilization by detergents and phospholipase activity may be found in the recent review of Dennis and Pliickthun (1984). 

Chloroplasts of Saxifraga stolonifera convert cis-caffeic acid into esculetin [M.Sato Phytochemistry 6 (1967) 1363-1373], The enzyme responsible is a phenolase (ortho-diphenol Oj reductase), which has been solubilized by detergent treatment of chloroplasts. It is proposed that the ortho-quinone from cis-caffeic acid converts spontaneously to esculetin, following spontaneous hydroxylation by water in the ortho position (Fig. 2). During synthesis in the plant, the 3,4,6-trihydroxy-cis-coumaric acid is presumably trapped as its 2-glucosyloxy derivative. A different biosynthetic route has been proposed for dalrubone, and some C. may arise from the A ring of anthot a-nins [D.L.Dreyer etal. Tetrahedron 31 (1975) 2OT-293]. 

C.hj from the microsomal fraction of bird and mammal livers is thought to deliver electrons to the fatty acid desaturase system of the endoplasmic reticulum. The heme group is noncovalently bound to the protein via histidine and does not react with O . It also protects the C.b molecule from denaturation and proteolytic attack. C.b solubilized by detergent treatment is an oligomer (A/, 120,000) of several monomers (M, 16,000, 126 amino acids), while C.65 solubilized by protease or lipase treatment has only 82 to 98 amino acids, depending on the species. The primary sequences of these fragments are known. They have a-helix and 25% -structure. 

Biuret method This method is simple and reasonably specific as it depends on the reaction of cop-per(II) with four N atoms in the peptide bonds of proteins. Compounds containing peptide bonds give a characteristic purple colour when treated in alkaline solution with copper sulfate. This is termed the biuret reaction because it is also given by the substance biuret NH2CONHCONH2. For a wide variety of proteins, 1.0 mg of protein in 2 cm of solution results in an absorbance at 540 nm of 0.1. Many haemoproteins, for example, give spurious results due to their intrinsic absorption at 540 nm, but modifications which overcome this difficulty are known (either removal of the haem before protein estimation or destruction of the haem by hydrogen peroxide treatment). The protein content of cell fractions such as nuclei and microsomes can be estimated by this method after solubilization by detergents such as deoxycholate or sodium dodecyl sulfate. 

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