Permeability of lactating-rat mammary gland Golgi membranes to monosaccharides

Abstract

The Golgi-membrane vesicles present in particulate preparations of lactating rat mammary gland were biosynthetically loaded with [14C]lactose. This lactose was effectively retained by particles sedimented after exposure to 0.25M-disaccharide, but was partly lost after exposure to 0.25 M-glucose or other solutes of similar size. Loss of lactose was time-, concentration-and temperature-dependent and varied with the solute structure. This behaviour is ascribed to the presence of protein in the Golgi membrane, forming a specific carrier or channel that serves to supply glucose for lactose synthesis.The galactosyltransferase (EC 2.4.1.22) of lactating mammary gland makes lactose from UDPgalactose and fl-glucose in the presence of alactalbumin and bivalent metal ions. The enzyme is located on the inner face of the Golgi membrane and the lactose apparently accumulates within the Golgi lumen. In crude or purified particulate preparations of rat mammary tissue, which contain pinched-off vesicles of Golgi membrane, 80-90% of [14C]-lactose newly synthesized from added radioactive substrates remains trapped within the vesicles and can be sedimented by centrifugation (Hill & Brew, 1975;Kuhn & White, 1975, 1977Jones, 1978). Since the Golgi membrane is so impermeable to lactose, the question arises as to how it can admit glucose.The existence of a specific glucose carrier was previously inferred from the inhibition of lactose synthesis in intact vesicles by phlorizin and phloretin (Kuhn & White, 1975 The theory behind this method is that permeant solutes to which the vesicles are exposed, moving down a concentration gradient, cross the membrane and enter the vesicle. As a result water is drawn in and the consequent hydrostatic pressure disrupts the membrane and releases the [14C]lactose. The loss of lactose from the particles is a measure of the permeability of the membrane to a solute. In a report that prompted the present study, Docherty et al. (1979) have described similar experiments to show that stereospecific sugar uptake by rat liver lysosomes occurs by facilitated diffusion. Materials and methods Preparation of [14C]lactose-containing particlesLactating rat mammary tissue (3-5g fresh wt.) was chopped and homogenized with 9 vol. of 0.25 Mlactose in a glass homogenizer with a loosely fitting Teflon pestle. After six to ten strokes of the pestle the homogenate was centrifuged at 10000g for 10min. The pellet was discarded and the supernatant was further centrifuged at 1000OOg for 30min. This pellet was re-suspended by gentle homogenization in the original tissue volume of 0.25 M-lactose containing 5OmM-glycylglycine buffer, pH 7.0. These operations were carried out at 40C.The Golgi-membrane vesicles of this preparation were loaded with ['4Cllactose by mixing 1.7ml with 0.26ml of solution containing glucose (81mM) and MnCl2 (16mM). After

Identification and characterization of glucose transport proteins in plasma membrane- and Golgi vesicle-enriched fractions prepared from lactating rat mammary gland

“…However, we cannot yet be certain that the vesicles containing glucose transporters in the Golgi membrane-enriched fraction actually correspond to those involved in lactose synthesis. Indeed, other studies have reported that the Golgi membrane discriminates only poorly between different sugars ofMr < 300, suggesting the presence of pores, rather than specific transporters for glucose [6,7]. The …”

“…However, the mechanism by which the glucose then crosses the membranes of Golgi vesicles to reach the site of lactose synthesis is less clear. Some studies have yielded evidence that passive sugar uptake into these vesicles occurs via proteinaceous pores that are nonstereospecific, in contrast with the great discrimination between D-and L-glucose shown by the plasma membrane transport system [6,7].…”

Role of Monosaccharide Transport Proteins in Carbohydrate Assimilation, Distribution, Metabolism, and Homeostasis

“…Golgi vesicle fractions from lactating rat mammary tissue transport D-glucose (614) and contain a GLUT1-like protein. It is possible that GLUT1 en route to cell surface expression also transports D-glucose to the site of lactose synthesis (385).…”

Identification of glucose transporters in plasma membrane and Golgi vesicle fractions prepared from lactating rat mammary gland

“…However, there is controversy about the mechanism by which glucose, required for lactose synthesis, crosses the membranes of Golgi vesicles. Some studies have yielded evidence for the presence here of a non-stereospecific pore, rather than a transporter (White et al, 1980). In the present study we sought to clarify this situation by identifying and determining the subcellular distribution of glucose transporters in the lactating rat mammary epithelial cell.…”

“…The sharpness of the bands seen upon Western blotting and photoaffinity labelling suggests that the mammary proteins may be less heterogeneously glycosylated than the erythrocyte transporter. The presence of transporters in the Golgi vesicle fraction is intriguing, given the results of studies which indicate the presence of a non-stereospecific monosaccharide channel in Golgi membranes (White et al, 1980). These transporters cannot be accounted for by contamination with plasma membranes, because 5'-nucleotidase assays revealed that the Golgi vesicles carried only 10"/0 contamination with the latter, while exhibiting a 20-fold enrichment with galactosyl transferase.…”