Plasma membrane NADH-AFR reductase

Interestingly, plasma membrane NADH-AFR reductase is inhibited by detergents at concentrations that promote membrane solubilization and cannot then be recovered from the extract, even after removal of detergent. This inhibition is not likely to be due to delipidation of a single transmembrane protein, since addition of sonicated purified phospholipids to the reaction mixture does not restore the activity. These results support the interpretation that the electron flow from NADH to AFR appears to involve more than one protein carrier (Villalba et al., 1993a). NADH-ferricyanide reductase has been purified from erythrocyte plasma membranes as a single polypeptide of 36 kDa and shown to correspond to NADH-cytochrome reductase (Kitajima et al., 1981). Its activity is not affected by WGA (Villalba a/., 1993a). 

The stabilization of ascorbate at the cell surface has been proposed as the expression of NADH-AFR reductase activity (Pethig et aL, 1985 Alcam et aL, 1991), although some controversy was established on its enzymatic character (Rodriguez-Aguilera and Navas, 1994). Addition of CoQ to K562 intact cells increases their capacity to stabilize ascorbate (Fig. 2). The coincidence of a participation of CoQ in both the stabilization of ascorbate at the cell surface and the plasma membrane NADH-AFR reductase further supports the interpretation that ascorbate stabilization is an enzymatic process, probably mediated by the AFR reductase. 

FIGURE 3. Relationship among antioxidant components at the plasma membrane. NADH-AFR reductase activity is carried out by two steps 1) A 34 kDa NADH dehydrogenase (p34) at the cytoplasmic domain of the membrane, which drives electrons to CoQ and 2) a putative CoQ-AFR reductase that will regenerate ascorbate (ASC). Both reduced CoQ (in the interior of the plasma membrane) and ascorbate (outside the membrane) regenerate reduced vitamin E. 

NADH-AFR reductase activity is widely distributed among subcellular membranes such as the outer mitochondrial membrane, endoplasmic reticulum, Golgi apparatus, clathrin-coated vesicles, and the plasma membrane (Sun et aL, 1983). Since NADH-AFR reductase exhibits different properties according to the source membrane, it is likely that activities from different membranes are catalyzed by different enzyme systems (Navas et aL, 1994). 

Plasma membranes from a variety of sources exhibit an NADH-AFR reductase activity ranging from about 2 to 20 nmol min mg depending on the source membrane and the method used for purification. Human erythrocyte membranes catalyze NADH-dependent reduction of AFR at a rate of 2-5 nmol min mg values were calculated to be 1.65 p.M for NADH and 0.25 jxM for AFR with an equilibrium constant of 10 . in rat liver plasma membranes, values of 2 xM for NADH and 0.3 jjlM for AFR have been reported. The optimum pH for NADH-AFR reductase is about 7.4 (Huron et aL, 1987). 

The involvement of coenzyme Q (CoQ) as an intermediate carrier in some plasma membrane-associated redox activities, such as NADH-ferricyanide and NADH-diferric transferrin reductases and NADH-oxygen oxidoreductase, has been demonstrated (Sun et ai, 1992). CoQ is also required for the maintenance of NADH-AFR reductase, and this distinguishes NADH-AFR reductase from other redox activities related to cis electron transport (i.e., with both donor and acceptor sites located on the same side of the plasma membrane) such as NADH-cytochrome... 

CoQ reduction by an NADH dehydrogenase at the plasma membrane would maintain its antioxidant property (Crane et al., 1991). NADH-AFR reductase also would keep ascorbate in its antioxidant form (Navas et al., 1994). Functions of these... 

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