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 0.28 mM had very little effect on cell growth over several days of incubation, and cell morphology was unchanged. Chromatographic and mass spectrometric analyses at 6 and 12 h of incubation showed that D,L-alpha-fluoropalmitic acid was taken up by the cells and incorporated without modification as a fatty acyl moiety into select lipids. Significant levels of the compound were found at 12 h in phosphatidylcholine (1.6%) and sphingomyelin (0.6%) fatty acids, but not in those of other phospholipids or neutral lipids. D,L-alpha-Fluoropalmitic acid represented a significant percentage of the fatty acids of neutral glycosphingolipids (1.4%) and ceramides (0.8%) by 12 h. The fluoro fatty acid was not incorporated into long-chain sphingolipid bases, and mass spectrometry failed to reveal additional carbon-2 fluorine-substituted compounds in cellular lipids. Cellular levels of triacylglycerols and phosphatidylcholine remained essentially unchanged, or were slightly increased, while amounts of ceramide and gangliosides were decreased. Comparison of labeled palmitate incorporation into sphingolipid bases and fatty acids of sphingomyelin suggested inhibition of sphingosine synthesis by the fluoro fatty sphingomyelin suggested inhibition of sphingosine synthesis by the fluoro fatty acid. D,L-alpha-Fluoropalmitic acid inhibited the formation of palmitoyl-CoA by Balb/c 3T3 long-chain acyl-CoA synthetase in vitro. The results support involvement of CoA thiol ester-independent steps in modification of membrane lipids.

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 (2.5 X 10(9) M-1) did not vary significantly during mammary development. The number of prolactin receptors, however, expressed by virgin and early pregnant epithelial cells was significantly increased over those from late pregnancy or lactation. Less differentiated cells also respond to growth in pregnant rabbit serum with an increase in specific [125I]-prolactin binding. The diminished receptor expression by cells obtained after 17 days of pregnancy coincides with the attainment of secretory capacity in the animal, and may reflect the influence of the low serum prolactin or high progesterone levels circulating during the last trimester in the rabbit, or be the cultural expression of secretory differentiation.

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 = 1.41-1.85 x 10(9)M-1. Association of lactogen with RMEC receptor followed bimolecular reaction kinetics with rate of 5.17 (+/- 0.75) x 10(5)M-1 sec-1 at 24 C, and 1.03 (+/- 0.11) x 10(6)M-1 sec-1 at 37 C. Dissociation was first order (K-1 = 5.97 (+/- 0.70) x 10(-5) sec-1) and was unaffected by the presence of lactogen. Specific binding determined with an excess of unlabelled bPRL was 66-77% of the total binding, and was optimal at pH 7.4. The binding reaction reached equilibrium in 2 h at 37 C, in 3 h at 24 C, and after 24 h at 4 C. Studies of binding capacity revealed the presence of 4.6-6.3 x 10(3) sites per cell, competition for which was limited to hormones demonstrating lactogenic activity. Recovered lactogen was not degraded by incubation with or dissociation from RMEC. Approximately 25% of the radioactivity remained associated with the cells even upon prolonged incubation. These studies demonstrated several advantages of RMEC for the investigation of hormone-receptor interaction and receptor regulation.

The effect of cyclic nucleotides on virus infection.

The yield of influenza virus from infected primary chick kidney cells (PCKC) was enhanced by treatment with dcAMP. In addition, rapid serial passages of the virus in treated chick cells at low multiplicities did also exhibit maintenance of high virus titers. The action of dcAMP upon viral replication could be reproduced in the chick cells by treatment with various agents known to increase intracellular cAMP levels by differing mechanisms. Furthermore, an adrenergic component in the activation of the PCKC cAMP system is indicated by the effects of certain catecholamines on influenza virus yield. Dibutyryl cAMP was shown in the same host to inhibit multiplication of Herpesvirus hominis Type 2, and its effect on other cell-virus systems indicates that its action consists of some host-specific element(s).