Immunoadsorption of Golgi Fractions on Antibody - Coated Beads

Light Golgi fractions (GFI+2) prepared from rat liver homogenates by a modification of the Ehrenreich et al. procedure (J. Cell Biol. 59:45) had significant NADPH-cytochrome P4s0 reductase (NADPH-cyt c reductase) activity if assayed immediately after their isolation. An antibody raised in rabbits against purified microsomal NADPH-cy t c reductase inhibited to the same extent the reductase activity of 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 ~m) polyacrylamide (PA) beads; (b) rabbit anti NADPH-cy t c reductase was immunoadsorbed to the antibody-coated beads; and (c) GFI§ 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% of the NADPH-cy t c reductase (and comparable fractions of NADH-cy t c reductase and glucose-6-phosphatase) but only 20% of the galactosyltransferase activity of the input GFI+2. Differential vesicle counts showed that ~ 7 2 % of the immunoadsorbed vesicles were morphologically recognizable Golgi elements (vesicles with very low density lipoprotein [VLDL] clusters or Golgi cisternae); vesicles with single V L D L and smooth surfaced microsome-like vesicles were too few ( 2 5 %) to account for the activity. It is concluded that NADPH-cytochrome P450 reductase is a Golgi membrane enzyme of probably uneven distribution among the elements of the Golgi