Nicholas J. Kuhn

Metabolic significance of milk glucose

The free glucose concentration in the aqueous phase of samples of goat, sheep, cow, rat and rabbit milk was about 0.1-0.3 mM, while that in human milk was about 2mM. During starvation the glucose concentration of goat milk fell considerably (by about 80% in 2 d) in parallel with the decreased rate of lactose production. With rats fed ad lib., glucose concentration in the milk was greater at 12.00 h than at 18.00 h, when lactose synthesis has been shown to decrease. 3-O-Methyl-D-glucose injected into the goat mammary gland via the teat canal specifically entered the blood. These findings support the idea that glucose equilibrates across the apical membrane of mammary secretory cells, so that milk concentrations reflect intracellular glucose concentratioins.

Lactose synthesis and the utilisation of glucose by rat mammary acini

Abstract 1. 1. Glucose uptake and lactose synthesis were measured in acini prepared from lactating-rat mammary gland and suspended in medium 199. 2. 2. Changes in the rate of glucose uptake, due to different concentrations of medium glucose or to the inclusion of phloretin or theophylline. were accompanied by larger changes in the rate of lactose synthesis. 3. 3. No clear evidence for cyclic nucleotide control of lactose synthesis was obtained. 4. 4. Direct measurement of intracellular glucose concentration (0.45 ± 0.08 mM) showed that it must be a rate-limiting factor for lactose synthesis and that glucose transport from the plasma into the mammary cell must be rate-limiting for glucose metabolism in general.

Glucose-6-phosphate hydrolysis by lactating rat mammary gland

Abstract 1. 1. Washed, particle preparations of fully lactating rat mammary gland hydrolysed glucose-6-phosphate at 191 ± 28 nmol/min/g fresh tissue, and a wide range of other phosphates at greater or lesser rates, at pH 6.4 and 37°C. 2. 2. Activity was maximal at pH 5 and pH 10 or above, and minimal at pH 6.4–7.0. 3. 3. Hydrolysis of glucose-6-phosphate at pH 6.4 did not follow Michaelis-Menten kinetics, and the rate at physiological concentrations of glucose-6-phosphate was very low compared with rates of tissue glucose uptake. 4. 4. It is concluded that the observed activity is not due to a specific glucose-6-phosphatase (EC 3.1.3.9), and cannot be utilized as a microsomal marker.

Subcellular compartmentation in the synthesis of the milk sugars lactose and α-2,3-sialyllactose.

SummaryThis paper assembles published and new material to trace out the compartmental aspects of milk sugar synthesis in the mammary gland. The generation of lactose, from glucose and UDP-galactose, in thetrans cisternae of the Golgi stack is well established from biochemical and electron microscopy work. New experiments show that in the rat essentially all this lactose is available for sialyllation to α-2,3-sialyllactose, which accumulates in the same compartment. UDP arising from utilization of UDP-galactose inhibits lactose synthetase, but is removed by nucleoside diphosphatase. Therefore lactose synthetase, sialyltransferase and nucleoside diphosphatase form a coupled enzyme system located on the luminal face of the Golgi membrane. Transport of UMP to the cytosol, followed by its re-conversion via UDP and UDP-glucose to UDP-galactose, defines the operation of a uridine nucleotide cycle that links Golgi and cytosol compartments in the support of milk sugar synthesis. Evidence for carrier-mediated uniport or antiport of UMP and UDP-galactose across the Golgi membrane is discussed, together with the evidence for transport of glucose and inositol by small water-filled pores that forbid back diffusion of lactose, galactinol or sialyllactose. Possible intra-Golgi signals, achieving short-term regulation of milk sugar synthesis, include glucose, submicromolar free Mn(II) ions and cationic activator proteins. Experimental evidence for intracellular and intra-Golgi glucose concentrations in the range 0.1–0.4 mM imply lack of saturation of lactose synthetase. It is shown that lactose synthetase within undamaged Golgi membrane vesicles is about 100 times more sensitive to Mn ions than in damaged or solubilized membranes. Both lactose synthetase and sialyltransferase are greatly stimulated by basic proteins, and similar lactose synthease-activating material is extractable from Golgi preparations. Development of these features may designate the sorts of control exerted upon these Golgi enzymes, and perhaps others. This may uncover the mechanisms by which hormonal and nutritional changes, and manipulation of mammary tissue in vitro, cause profound alterations in milk sugar production.

The Topography of Lactose Synthesis

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 ofthe 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.

Biosynthesis of galactinol by lactose synthetas

1. myo-Inositol was galactosylated by UDP-galactose in the presence of alpha-lactalbumin plus rat mammary Golgi membranes enriched in galactosyltransferase (EC 2.4.1.22). 2. The isolated product migrated on GLC as two peaks of material, apparently identical to galactinol (galactosylinositol) isolated from rat milk. 3. These findings make it likely that in vivo lactose synthetase is responsible for converting inositol into galactinol within the Golgi lumen. 4. This is consistent with the ability of inositol to penetrate the Golgi membrane in vitro, and renders less likely a previously proposed role of beta-galactosidase (EC 3.2.1.23) in mammary galactinol synthesis.