Effects of phlorrhizin and thiol reagents on the galactosyltransferase activity of Golgi membrane vesicles of lactating rat mammary gland

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

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Effects of phlorrhizin and thiol reagents on the galactosyltransferase activity of Golgi membrane vesicles of lactating rat mammary gland

Cited by 5 publications

References 14 publications

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