Presence of NADPH-cytochrome P-450 reductase in rat liver Golgi membranes. Evidence obtained by immunoadsorption method

Abstract

Light Golgi fractions (GF(1+2)) prepared from rat liver homogenates by a modification of the Ehrenreich et al. procedure (J. Cell Biol. 59:45) had significant NADPH-cytochrome P(450) reductase (NADPH-cyt c reductase) activity if assayed immediately after their isolation. An antibody raised in rabbits against purified microsomal and Golgi fractions. To find out whether this activity is located in bona fide Golgi elements or in contaminating microsomal vesicles, we used the following 3-step immunoadsorption procedure: (a) antirabbit IgG (raised in goats) was conjugated to small (2-5 mum) polycrylamide (PA) beads; (b) rabbit anti NADPH-cyt c reductase was immunoadsorbed to the antibody-coated beads; and (c) GF(1+2) was reacted with the beads carrying the two successive layers of antibodies. The beads were then recovered by centrifugation, and were washed, fixed, embedded in agarose, and processed for transmission electromicroscopy. Antireductase- coated beads absorbed 60 percent of the NADPH-cyt c reductase (and comparable fractions of NADH-cyt c reductase and glucose-6-phosphatase) but only 20 percent of the galactosyltransferase activity of the input GF(1+2). Differential vesicle counts showed that approximately 72 percent of the immunoadsorbed vesicles were morphologically recognizable Golgi elements (vesicles with very low density lipoprotein [VLDL] clusters or Golgi cisternae); vesicles with single VLDL and smooth surfaced microsome-like vesicles were too few (approximately 25 percent) to account for the activity. It is concluded that NADPH-cytochrome P(450) reductase is a Golgi membrane enzyme of probably uneven distribution among the elements of the Golgi complex.