The topography of lactose synthesis

Abstract

1. At short incubation times, and under suitable osmotic conditions, the lactose synthesized by Golgi-derived vesicles of rat mammary gland is 85-90% particulate. Evidence is presented for its occlusion within the lumen of the vesicles. 2. Ovalbumin is used as a bulky active-site inhibitor to show that the active site of lactose synthase lies on the inner face of the Golgi membrane. 3. Phlorrhizin and phloretin inhibit lactose synthesis by such vesicles, indicating the presence of a glucose-transport system. 4. The relationship of this topography to the synthesis of N-acetylneuraminyl-lactose and to the secretion of milk sugars is discussed.

Permeability of lactating-rat mammary gland Golgi membranes to monosaccharides

“…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.…”

“…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 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 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). These agents are well-known inhibitors of glucose transport in vertebrate cell membranes.…”

“…The extracted ['4C]lactose was determined by scintillation counting either directly or, in early experiments, after purification by selective precipitation (Kuhn & White, 1975). The latter method was abandoned after it was found that lactose was essentially the only radioactive material that sedimented with the particles.…”