Cell surface and Golgi pools of beta-1,4-galactosyltransferase are differentially regulated during embryonal carcinoma cell differentiation.

beta-1,4-Galactosyltransferase (GalTase) has two functionally distinct subcellular distributions. In the Golgi apparatus, GalTase participates in the glycosylation of secretory and membrane-bound glycoproteins, whereas on the cell surface it mediates specific aspects of intercellular adhesion. For this study, a murine GalTase clone was obtained by screening a lambda gt10 cDNA library made from F9 embryonal carcinoma cells with a heterologous bovine GalTase cDNA probe. The murine GalTase cDNA probe was used in conjunction with assays of GalTase activity to investigate the expression and distribution of GalTase during differentiation of F9 stem cells into secretory endodermal epithelium. During the initial phase of F9 cell differentiation, GalTase mRNA levels remained relatively constant; however, as differentiation progressed, as assayed by expression of the differentiation-specific marker laminin B1, GalTase mRNA levels and enzyme activity rose dramatically. Furthermore, subcellular fractionation of these cells showed that the increased GalTase levels were specifically associated with the Golgi apparatus, whereas GalTase specific activity on the plasma membrane remained constant. These results show that levels of cell surface and Golgi GalTase change relative to one another during F9 cell differentiation and suggest that these functionally distinct pools of GalTase are independently and differentially regulated.

Isolation and characterization of UDP-galactose:N-acetylglucosamine 4 beta-galactosyltransferase activity induced in rat parotid glands treated with isoproterenol.

Chronic injection of the beta-adrenergic receptor agonist isoproterenol causes a 6- to 10-fold increase in the specific activity of the Golgi membrane marker enzyme UDP-galactose:D-glucose 4-beta-D-galactosyltransferase (EC 2.4.1.22) in rat parotid glands. Using a combination of size exclusion chromatography and affinity chromatography on alpha-lactalbumin-agarose, the Triton X-100-solubilized enzyme was purified to homogeneity from treated and control animals. The enzyme purified from both sources had a single Mr = 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme isolated from control and isoproterenol-treated animals had identical amino acid compositions and isoelectric points (pI = 5.9). Neutral sugar content as determined by the phenol-sulfuric acid assay was 10%. UDP-galactose:N-acetylglucosamine 4 beta-galactosyltransferase activity purified by alpha-lactalbumin column chromatography was inhibited by the presence of 200 mM N-acetylglucosamine and by the inclusion of alpha-lactalbumin in assays using 10 mM N-acetylglucosamine as acceptor. Increased enzyme activity was immune precipitated from Triton X-100-solubilized membrane prepared from isoproterenol-treated animals versus control animals. This was also true of protein synthesized by in vitro translation of poly(A+) RNA prepared from parotid glands, indicating an increase in mRNA levels for the enzyme. The molecular mass of the immune-precipitated, in vitro translation product was determined to be 45,000 Da.

The asynchronous hormonal induction of lactose synthetase components, alpha-lactalbumin and galactosyltransferase, in relation to lactose secretion by mouse mammary explants.

Hormonal induction of the lactose synthetase components, alpha-lactalbumin and galactosyltransferase, in relation to the induced levels of lactose synthetase activity and lactose secretion by mammary gland explants from mature virgin mice was examined. During 6 days of culture in the presence of insulin, cortisol, prolactin and triiodothyronine mammary explants accumulated progressively increasing amounts of alpha-lactalbumin. By contrast, galactosyltransferase and lactose synthetase activities were maximal on day 3, and then declined; the time course of lactose secretion was similar to that of galactosyltransferase and lactose synthetase activities. Cortisol concentration studies revealed the following dependencies on the steroid: alpha-lactalbumin content was maximal between 0.01 and 0.1 microgram and, at 10 microgram/ml fell below the baseline level observed without cortisol. On the other hand, galactosyltransferase, lactose synthetase and lactose secretion were only slightly increased at 0.01 microgram/ml, were maximal between 0.1 and 1.0 microgram/ml, and at 10 microgram/ml were still considerably above the baseline levels observed without cortisol. These results indicate that lactose formation and secretion by mammary explants from virgin mice correspond more closely to the hormone-induced activity of galactosyltransferase than to that of alpha-lactalbumin, and that asynchrony can arise between the induction of alpha-lactalbumin and the induction of lactose.

Difference between mammary epithelial cells from mature virgin and primiparous mice.

Mammary epithelial cells from mature virgin mice are similar to those from primiparous mice in several respects. However, there is one known difference. The cells from the mature virgin must traverse the cell cycle in order to become competent to make casein and enzymatically active alpha-lactalbumin in vitro; those from the primiparous animal can make these proteins without first traversing the cycle. In this regard, cells from human placental lactogen- and prolactin-treated mature virgins are, after involution, similar to those from primiparous mice. The developemental block in the cells from the mature virgin, imposed by preventing cell cycle traversal, has been partially delineated. It does not appear to reside at the levels of ultrastructural maturation or the formation of casein messenger RNA. Rather, the lesion is postranscriptional and may be at the level of translation, or posttranslational modification, or both.

[Induction of N-acetyllactosamine synthetase activity in the mouse mammary gland by prolactin and placental lactogen hormone]. / Induction d'une activité N. acétyl lactosamine synthétase dans la glande mammaire de souris par la prolactine et l'hormone lactogène placentaire

The authors show that the ovine prolactine promote induction of N. acetyl lactosamine synthetase in tissue culture of mammary glands of pregnant mice. A crude extract of human placenta has also a lactogenic activity as tested by the same method, but in this case the blank values are very high for large concentration of crude extract. The molecular forms of HCS are tested: the slow band has a lactogenic activity, the intermediate band has no activity and the rapid band seems to be inhibitory.