Na+ uptake by Golgi vesicles isolated from lactating rat mammary gland

Abstract

The initial separation of Na', K' and C1-ions between the aqueous phase of milk and the cytosol of the mammary secretory cell is believed tci occur within Golgi vesicles [ 11. It is predicted that after the production of lactose and the concomitant movement of water into the vesicle lumen, ions diffuse across the Golgi vesicle membrane until equilibrium is reached. There is, however, a paucity of information relating to the permeability of the Golgi vesicle membrane to small monovalent ions. Therefore we have chosen to examine Na' uptake by Golgi vesicles.Vesicles were prepared from the mammary glands of lactating rats ( 10-15 days post parturn) by a modification of the method of West [2] and were suspended in a medium containing 1 mM-NaC1, 10 mM-KCI, 300 mM-sucrose and 5 mM-Tris-Bes, pH 7.0. The activity of galactosyltransferase associated with the Golgi membranes was enriched 20-fold compared with the initial homogenate; there was only a 10% contamination with plasma membranes as judged by 5'nucleotidase activity. Na+ uptake (using ??Na as tracer) was assayed at 20°C according to the method of Gasko et a/. [3], thus intra-and extra-vesicular isotope was separated by applying aliquots of vesicles and medium to columns filled with Dowex 50 W X 8 (Tris form).Na+ uptake (at 1 mM) was found to be time dependent, reaching a point of equilibrium by 15 min. The addition of 0.1 "/o (w/v) Triton X-100 to the incubation medium abolished Na+ influx, suggesting that uptake represents real transport rather than binding of isotope to the membrane. The intravesicular volume calculated from the equilibrium uptake values was 2.64f0.20 pl/mg of protein (mean k s.E.M., n = 6). Na+ uptake was found to be temperature dependent as lowering the incubation medium temperature to 4°C reduced the initial uptake rate by 38.0f6.1% (mean f s.E.M., n = 3).Testing the effect of increasing the extravesicular Na+ concentration on Na+ influx established that uptake was saturable, the K , of uptake calculated from Hanes-Woolf plots being 2 1.4 f 2.5 mM (mean f s.E.M., tz = 4) with a V,,,,, of 29.6 f 4.6 nmol/mg of protein per 3 min ( f s.E.M., 17 = 4).To investigate the specificity of the Na' transporter we tested the effect of adding a range of monovalent cations to the incubation medium. Na+, K', Li' and choline (at 40 mM, as their CIsalts) reduced Na+ influx by 63, 49, 61 and 36"/0, respectively, indicating the order of potency of inhibition to be Na' = Lif > K' > choline. In addition, Rb' and Cs' inhibited Na+ accumulation to the same extent as K'.In order to establish if Na' uptake by Golgi vesicles occurs via a pathway sensitive to changes in membrane potential, diffusion potentials across the membrane were imposed using anions of varying passive permeability. Replacing extravesicular C1-with SCNincreased the initial rate of Na+ influx by 12%, whereas gluconate decreased it by 15%. Frusemide, amiloride and 4,4'-di-isothiocyantostilbene-2,2'-disulphonic acid (all l o -' M ) added to the incubation medium had no significant effect on the rate ...

Archaeology and the Construction of Identities in Medieval North India

“…'Variation in artefacts across space and time arises as a result of an enormous range of different processes, from the problems of coping with a specific environment to the distribution of social power, the organization of material production or changing patterns of religion and iconography.' 56 If we look at archaeological material, it is in actuality difficult to delineate neat, 'bounded entities'. Rather, there are 'an enormous variety of cross-cutting patterns'.…”