Isolation and characterization of plasma membrane from lactating bovine mammary gland

Plasma membranes were isolated from lactating bovine mammary gland. Two crude membrane fractions; medium/d 1.033 (light membrane) and 1.033/1.053 interfaces (heavy membrane), were obtained by Ficoll density gradient centrifugation of osmotically washed microsomal fraction. Two crude membranes were further purified separately by sucrose density gradient centrifugation. Both light and heavy membranes banded at a sucrose density of 1.14. The purified membranes appeared as heterogeneous smooth membrane vesicles on electron microscopy. The contaminating suborganelles were not detected. The yield of the purified membranes relative to the homogenate was 1.2%. The degree of purity of the membranes was shown by a great increase in the specific activity of 5′-nucleotidase over the homogenate of 20-fold for light membrane and of 16-fold for heavy membrane. The relative activities of Mg²⁺-ATPase, (Na⁺ + K⁺)-ATPase, γ-glutamyl transpeptidase, phosphodiesterase I, akaline phosphatase and xanthine oxidase were also high (12–18-times) and nearly 20% of these enzymes was recovered. The activity of marker enzyme for mitochondria, endoplasmic reticulum and Golgi apparatus was very low, while that of acid phosphatase for lysosome was relatively high (5-times). DNA and RNA contents were very low. The major polypeptides rich in other suborganelles were not detected profoundly in the membrane fraction and the polypeptide compositions in both light and heavy membranes were similar upon SDS-polyacrylamide gel electrophoresis.     

Proteomic analysis of microsomes from lactating bovine mammary gland

Mammary gland has multiple metabolic potential including for large-scale synthesis of milk proteins, carbohydrate, and lipids including nutrient triacylglycerols. We have carried out a proteomic analysis of mammary tissue to discover proteins that affect lipid metabolism. Unfractionated microsomes from lactating bovine mammary tissue were analyzed using one-dimensional SDS-PAGE with RPLC-ESI-MS/MS. This approach gave 703 proteins including 160 predicted transmembrane proteins. Proteins were classified according to their subcellular localizations and biological functions. Over 50 proteins were associated with cellular uptake, metabolism, and secretion of lipids, including some enzymes that have been previously associated with breast cancer and potential therapeutic targets. This database develops a proteomic view of the metabolic potential of mammary gland that can be expected to contribute to a greater understanding of gene expression and tissue remodeling associated with lactation, and to further dissection of normal and pathological processes in mammary tissue.     

Characterization of a secretory vesicle-rich fraction from lactating bovine mammary gland.

Fractions enriched in secretory vesicles were obtained from lactating bovine mammary tissue by a straightforward procedure involving gentle homogenization and centrifugation in isotonic milk salt solution containing Ficoll. Secretory vesicle-rich fractions could also be obtained from lactating rat mammary gland by this procedure. With rats, yields of vesicles were substantially increased by administration of colchicine or thioglucose to animals several hours before sacrifice. Isolated fractions were enriched in lactose and consisted predominantly of 0.2–1.2 μm diameter vesicles, many of which contained casein micelles. Enzymatic, compositional and morphological examination revealed vesicle preparations to be largely free of contamination by rough endoplasmic reticulum, mitochondria, nuclei, peroxisomes and lysosomes. Specific activity of several marker enzymes of the secretory vesicle fraction were similar to, or intermediate between, Golgi apparatus and milk lipid globule membranes. Amounts of cholesterol and gangliosides in vesicle fractions approached levels found in plasma membranes. In distribution of major phospholipids, secretory vesicles were intermediate between Golgi apparatus and milk lipid globule membranes. The pattern of polypeptides of secretory vesicle membrane was qualitatively similar to that of Golgi apparatus membranes. While there were similarities between these polypeptide patterns and that of lipid globule membranes, the latter contained relatively more of certain polypeptides, particularly the internal coat-associated polypeptides of the globule membrane. These observations are discussed in relation to the endomembrane hypothesis and the origin of the membrane of milk lipid globules.     

Differences in the ribosomes prepared from lactating and non-lactating bovine mammary gland

1. Ribosomes prepared from bovine lactating mammary gland are able to synthesize protein, whereas similar preparations from non-lactating glands are not. Washing the ribosome suspensions through a medium containing 0.5m-ammonium chloride enhanced their ability to incorporate phenylalanine into polyphenylalanine. 2. Ribosomes isolated from non-lactating bovine mammary gland, in contrast with those from rat liver and lactating mammary gland, contained significant amounts of extraneous nucleases. These enzymes could be removed by washing with a medium A buffer containing 0.5m-ammonium chloride. 3. Only those ribosomes from functionally active tissues were able to bind polyuridylic acid and phenylalanyl-tRNA.     

Purification and characterization of casein kinase II from lactating rabbit mammary gland

• 1.1. Highly purified 200 kDa casein kinase II from rabbit lactating mammary gland (MG-CK II) was obtained by means of a new purification procedure consisting of one phosphocellulose and three Mono Q steps.
• 2.2. Its K_m for ATP was 2.22 μM and 0.57 mg/ml and 0.13 mg/ml for partially dephosphorylated casein and phosvitin respectively. Stathmine was also suitable as substrate. 2-aminopurine and 6-dimethylaminopurine inhibited efficiently MG-CK II K_i = 5 and 1 mM respectively).
• 3.3. MG-CK II autophosphorylated on its α-, α '- and β-subunits. The β-subunit auto-phosporylation was enhanced in presence of exogenous substrate. Its modulation was highly dependent on ATP concentration.
• 4.4. The effects of basic compounds which affected dramatically the phosphorylation of dephosphorylated casein in presence of various ATP concentrations were reported.

     

EGF receptors on plasma membranes purified from bovine mammary gland of lactating and pregnant animals

A procedure for purification of plasma membranes from bovine mammary gland has been developed. The binding capacity of EGF to the plasma membranes from mammary tissue of pregnant cows was equal to 335 fmol per mg of protein thus being twofold higher than in membranes from lactating gland. The KD values were not changed. Autoradiographs of the membrane receptor linked to [125J]-EGF revealed three labeled bands corresponding to 160 kDa, 145 kDa and 115 kDa polypeptides. The main band of 145 kDa was labeled stronger in membranes from pregnant animals. The results suggest that the EGF receptor level is enhanced in fast proliferating normal mammary tissue.     

The lactose synthetase particles of lactating bovine mammary gland. Preparation of particles with intact lactose synthetase

1. The particulate form of lactating bovine mammary lactose synthetase activity is shown to be more highly organized than previously reported. 2. A novel method of shattering frozen mammary tissue with effective cell disruption is described. 3. The apparent subcellular distribution of lactose synthetase was shown to reflect the method of homogenization. 4. After mild homogenization particles associated with a high content of intact lactose synthetase activity sedimented in the lysosome size range between 5×10⁴ and 3×10⁵g-min. 5. Lactose synthetase was dissociated and solubilized by VirTis homogenization and ultrasonic treatment. The activities and behaviour of UDP-galactose hydrolase, succinate dehydrogenase, β-glucuronidase and phosphodiesterase I were compared. 6. Inhibition of UDP-galactose hydrolase by UTP and α-lactalbumin was observed.     

Physicochemical properties of isocitrate dehydrogenase from lactating bovine mammary gland: effect of substrates and cofactors

The influence of substrates and cofactors on the oligomeric structure of the cytosolic form of NADP+-specific isocitrate dehydrogenase (IDH) from lactating bovine mammary gland was investigated using analytical ultracentrifugation and kinetic methods. In guanidine-HCl, the monomer molecular weight for reduced and carboxymethylated IDH was found to be 50,000 to 52,000. In nondenaturing solvents IDH behaves as a homogeneous solute with a molecular weight of 97,200. When added separately, manganous isocitrate, isocitrate, manganous citrate (substrate analog), and a mixture of the substrate analog and NADP+ do not significantly alter the sedimentation coefficient or the molecular weight of IDH as judged by direct observation of the enzyme at 0.1 to 3 microM using sedimentation velocity and equilibrium. Active enzyme sedimentation (AES) was used to assess the degree of dissociation of IDH at lower concentrations, and Kd for the dimer-monomer equilibrium was estimated to be 2 nM. In enzymatic studies, the specific activity at several levels of substrate does not vary as the subunit concentration of enzyme is reduced from 10 to 0.3 nM. Estimates for Kd by AES indicate the presence of a significant fraction of monomer at assay concentrations of 1 nM and below, where the weight fraction of monomer is predicted to be 0.6. If the monomer has a lower activity than the dimer, a drop in specific activity is expected below 1 nM. Significant decreases occur only when the IDH is not protected from denaturation. The concentration of cytoplasmic IDH in bovine mammary tissue is estimated to be 5.7 microM, at least 100-fold greater than our estimates of Kd. Since over 90% of the enzyme is present in the dimeric form, ligand-induced changes in aggregation state cannot play a significant role in the regulation of the cytosolic form of IDH in situ in this tissue.     

Metabolism of arginine in lactating rat mammary gland.

Significant activities of the four enzymes needed to convert arginine into proline and glutamate (arginase, ornithine aminotransferase, pyrroline-5-carboxylate reductase and pyrroline-5-carboxylate dehydrogenase) develop co-ordinately in lactating rat mammary glands in proportion to the increased production of milk. No enzymes were detected to carry out the reactions of proline oxidation or reduction of glutamate to pyrroline-5-carboxylate. Minces of the gland converted ornithine into proline and into glutamate plus glutamine. These conversions increased during the cycle of lactation in proportion to the increased milk production and to the content of the necessary enzymes. The minced gland did not convert labelled ornithine into citrulline, confirming the absence from the gland of a functioning urea cycle, and did not convert labelled proline or glutamate into ornithine. A metabolic flow of labelled arginine to proline and glutamate in mammary gland was confirmed in intact animals with experiments during which the specific radioactivity of proline in plasma remained below that of the proline being formed from labelled arginine within the gland. It was concluded that arginase in this tissue had a metabolic role in the biosynthesis of extra proline and glutamate needed for synthesis of milk proteins.     

Identification and characterization of microRNAs from the bovine adipose tissue and mammary gland

We report the identification of bovine miRNAs by cloning small RNAs from adipose tissue and the mammary gland. Fifty-nine distinct miRNAs were identified, five of them were not homologous to known mammalian miRNAs, and many of them had 3' and/or 5' end variants. Ribonuclease protection assays indicated that miR-23a and miR-24, whose genes are closely located on the same chromosome, were co-expressed in different tissues. The assays also suggested a role for several miRNAs in the mammary gland and a role for miR-133, a previously known skeletal and cardiac muscle-specific miRNA, in the rumen, an organ unique to the ruminant.     

Lipid synthesis in lactating mammary gland

The mammary gland o f a high-yielding cow may produce as much as 1–2 kg o f fat in a day. Studies reveal unique mechanisms at work to produce typical milk lipid.     

Properties of ornithine decarboxylase-antizyme from mammary gland of lactating rats

Ornithine decarboxylase-antizyme was induced in mammary gland of fasted lactating rats by administration of 1,3-diaminopropan-2-ol. Antizyme from mammary gland showed similar chemical and kinetic behavior to that previously reported by Canellakis and co-workers for antizyme from liver [J. S. Heller, W. F. Fong, and E. S. Canellakis (1976) Proc. Natl. Acad. Sci. USA 72, 1858-1862]; specifically the inhibitor was nondialyzable, heat labile, and ribonuclease insensitive, and the inhibition was time independent, proportional to the concentration of antizyme present, and noncompetitive with respect to the substrate, ornithine. However, ornithine decarboxylase-antizyme from mammary gland eluted from Sephadex G-75 with an apparent molecular mass of 55 kDa, compared with 27 kDa, for antizyme from liver under identical conditions. The elution pattern was unaffected by the presence of high salt concentrations, indicating that the larger size was not due to macromolecular complexes. The presence of antizyme-ornithine decarboxylase complex was detected in mammary gland of untreated lactating rats fasted for 6 or 24 h, thus indicating that antizyme plays a role in the regulation of ornithine decarboxylase in mammary gland under physiological conditions.     

Oestrogen receptor of mammary gland. Inhibition of aggregation and characterization of receptor from lactating gland in the presence of sodium bromide

1. When NaBr, a chaotropic salt, is added, in concentrations ranging from 0.5m to 2m, to low-salt mammary cytosol, (i) age-dependent aggregation of oestrogen receptor is inhibited, (ii) the receptor sediments as a sharp peak at 4.2S on sucrose-gradient centrifugation, with complete disappearance of heavier forms, and (iii) on gel filtration with Sephadex G-200, the receptor is included in the gel matrix. On a calibrated column, the receptor has a Stokes radius of 3.7nm (±6%). 2. Because NaBr inhibits interaction of receptor with other components of cytosol, the values of the sedimentation coefficient, measured by sucrose-gradient sedimentation, and of the Stokes radius, measured by gel filtration, can be accepted with confidence. From these values, it can be computed that the oestrogen-receptor form in NaBr has a mol.wt. of 64000, with a frictional ratio of 1.4. 3. Also, inhibition of aggregation by NaBr allows a 30–90-fold purification of oestrogen receptor. Analysis of this partially purified receptor by sucrose-gradient sedimentation and gel filtration in NaBr gives the same results as for receptor in crude cytosol. On electrofocusing on a pH5–8 gradient, the partially purified oestrogen receptor focuses at pH6.2. On removal of NaBr, receptor aggregates even in this partially purified state. It seems likely that at the protein and ionic concentrations of cytoplasm in vivo, the 64000-mol.wt. receptor form is part of higher states of self- and/or hetero-association with other cytoplasmic components. 4. NaBr up to a concentration of 2m does not inhibit binding of oestrogen by receptor, nor does it decrease the affinity of the interaction (K_D8.9×10⁻¹⁰m). The total number of binding sites in cytosol, however, decreases by approx. 10%, but this decrease may actually be the result of elimination of lower-affinity binding by non-receptor components of cytosol. 5. NaSCN, another chaotropic salt, was also tested but gave less satisfactory results with the mammary cytosol than with uterine cytosol. EDTA was omitted from the buffers because it favours aggregation of mammary oestrogen receptor. KCl (0.4m), sucrose (15%) and ZnSO₄ (3mm) did not prevent aggregation of receptor.