Philipp Strittmatter

[8] Purification of cytochrome b5

Publisher Summary This chapter provides information on the purification of cytochrome b 5 . The isolation of cytochrome b 5 from liver endoplasmic reticulum in its complete form requires fractionation procedures with detergents at 0° to avoid loss of peptide fragments by endopeptidase activity. The chapter mentions the procedure for the heme peptide and NADH-cytochrome b 5 reductase that can also be used for the complete form of cytochrome b 5 . NADH and the reductase are added to the cytochrome to yield the reduced spectrum. This procedure is the one that gradually evolved from the original procedure described for rabbit liver. To scale the preparation to large amounts of yearling steer liver, CaCI 2 precipitation of microsomes and slightly different column chromatography steps are employed. Cytochrome b 5 serves as an electron acceptor for cytochrome b 5 reductase and an electron donor for stearyl-CoA desaturase where the binding of the protein to phospholipid.

[18] Purification of stearyl-CoA desaturase from liver

Publisher Summary This chapter describes the purification of stearyl-CoA desaturase from liver. The isolation method described in the chapter includes minor modifications of the original procedure that can increase both the reproducibility and the yield of desaturase preparations. The isolation of stearyl-CoA desaturase from liver of rats induced for this enzyme utilizes successive detergent extractions of microsomes to remove other proteins initially. The principle of the rapid spectrophotometric assay method is the measurement of the increase in rate of reoxidation of reduced cytochrome b 5 produced by the addition of stearyl-CoA to a complete desaturase system in the presence of oxygen and limiting NADH. The purified enzyme is relatively labile, particularly in detergents such as Triton X-100 or the deoxycholate concentrations used in the preparation. Even at 0 °, fraction 8 loses 30-50% catalytic activity in 15-20 hrs. This is accomplished by iron loss from the protein.

[21] Incorporation of microsomal electron-transfer components into liposomes: Considerations for diffusion-limited reactions

Publisher Summary This chapter describes the incorporation of microsomal electron-transfer components into liposomes. The procedures described in the chapter employed either relatively lengthy incubations of the proteins with preformed phospholipid vesicles, incubation of vesicles with combinations of two proteins to produce less-stable mixed-protein aggregates, or binding in the presence of detergent followed by removal of the detergent by gel filtration. With such vesicle preparations, both partial and complete electron-transport sequences are prepared to examine protein-protein, protein-lipid, and diffusion-dependent interactions. With vesicle preparations containing a complement of the three enzymes, activity is measured readily by adding a convenient amount of NADH to the vesicle suspension first in the absence and then in the presence of excess stearyl-CoA. The difference in the rate of cytochrome b 5 reoxidation is equal to the rate of formation of an equivalent amount of oleyl-CoA. Reductase vesicles are difficult to prepare because the isolated enzyme undergoes variable and extensive aggregation.

Essential Structural Features and Orientation of Cytochrome b5 in Membranes

It is clear the cytochrome b 5 serves as a mobile electron carrier or shuttle to provide reducing equivalents for a number of oxidation-reduction reactions that utilize cytoplasmically generated reduced pyridine nucleotides as electron sources. Thus, two flavoproteins, NADH cytochrome b 5 reductase (Spatz and Strittmatter, 1973) and NADH cytochrome P-450 reductase (Oshino et al., 1971; Enoch and Strittmatter, 1979a), reduce the membrane-bound heure protein rapidly, the former with a turnover number of 30,000 min−1 at 30°C (Spatz and Strittmatter, 1973) and the latter at 7000 min−1 at 33°C (Enoch and Strittmatter, 1979a). Reduced cytochrome b 5 is the direct electron donor for stearyl-CoA desaturase (Δ9 fatty acyl CoA desaturase) (Holloway and Wakil, 1970; Oshino and Omura, 1973; Holloway and Katz, 1972; Strittmatter et al., 1974; Enoch et al., 1976), and may participate in the reduction of cytochrome P-450 as well (Hildebrandt and Estabrook, 1972; Lau et al., 1974; Saesame et al., 1974; Imai and Sato, 1977). In addition, the microsomal heure protein has been implicated as the reductant in the Δ6 desaturation of fatty acids (Okayasu et al., 1977; Lee et al., 1977), cholesterol biosynthesis (Reddy et al., 1977), plasmalogen biosynthesis (Paultanf et al., 1974), and more recently in the elongation of fatty acids (Keyes et al., 1979). Both the relatively large and nonstoichiometric amount of cytochrome b 5 in endoplasmic reticulum relative to any one of the enzymes with which it interacts, and direct evidence that interaction with NADH cytochrome b 5 reductase in prepared phospholipid and microsomal vesicles is diffusion limited (Strittmatter and Rogers, 1975; Rogers and Strittmatter, 1974) indicate that the translational movement of the heme protein within membrane bilayers is a crucial functional feature of this electron carrier.