Regulation of membrane-bound enzymes of glycosphingolipid biosynthesis.

The descriptive phase of glycosphingolipid biochemistry has blossomed over the past decade with the application of exquisitely sensitive analytical methods of mass spectrometry, NMR, and monoclonal antibody technology. Consequently, appreciation of the interrelation among the complex carbohydrate structures that are expressed on the cell surface has focused attention on the regulation of the glycosyltransferases responsible for such diversity. This review explores some aspects of several potential molecular mechanisms of regulation--compartmentation of glycosyltransferases and effect of the immediate lipid environment; modulation of enzyme activity by acceptor substrate, divalent cations, and availability of sugar nucleotides; modifications of the enzyme operon elicited by viral transformation integration sites, or to the enzyme by phosphorylation-dephosphorylation--which are presently at the cutting edge of glycosphingolipid science.

D,L-alpha-Fluoropalmitic acid inhibits sphingosine base formation and accumulates in membrane lipids of cultured mammalian cells.

D,L-alpha-Fluoropalmitic acid was synthesized by tosylation of methyl-D,L-alpha-hydroxypalmitate, and displacement of the tosylated function by tetrabutylammonium fluoride in acetonitrile. Uptake and utilization of the compound by cultured Balb/c 3T3 cells were studied after presentation of the fluoro fatty acid analogue complexed with bovine serum albumin. A concentration of 0.

Prolactin receptor expression in monolayer cultures of rabbit mammary epithelial cells: pre- and postpartum [125I]-prolactin binding activity.

Expression of specific [125I]-prolactin-binding sites under culture conditions has been investigated for isolated mammary epithelial cells from virgin, pregnant, and lactating rabbits. Primary monolayer cultures were obtained by sequential enzymatic dispersion of mammary tissue followed by 48 hr incubation in a medium selective for epithelial cells. Scatchard analyses of binding data obtained from these cultures indicated a single class of receptor sites, the affinity constant of which (2.

Kinetic characterization of [125I] iodo-prolactin in binding to primary monolayer cultures of rabbit mammary epithelium.

Monocellular suspensions of epithelial cells from mammary glands of rabbits at 20-22 days of pregnancy were prepared by sequential dissociation with collagenase-hyaluronidase followed by Pronase. Maintenance in D-valine-substituted minimum essential medium (D-valine-MEM) supplemented with 10% dialyzed calf serum yielded monolayers enriched for rabbit mammary epithelial cells (RMEC). RMEC specifically and reversibly bound bovine PRL with Ka = 1.