Glycolipids, radiolabeling

Measurements of the quantities of glycolipids inserted into the membrane have also been reported by a technique based on the use of C-labeled lipid anchors. In this method, the carbohydrate (a-o-Man) was covalently coupled to the anchor at the surface of a pre-formed vesicle. Indeed, the liposome structure was shown to remain intact in the treatment. Nevertheless, the measurement of the incorporated mannose was performed after separation of bound and unbound material by centrifugation. The yields of coupling were shown to increase with the increase of the initial mannose/ C-anchor ratio, but non covalent insertions were displayed at high initial mannose concentrations. Therefore, the aforementioned method was not as accurate as could have been expected for the use of radioactive materials [142]. Radiolabeled phospholipids were also used for such determinations thus the amounts of glycosphingolipids incorporated into liposomes were quantified by the use of H-phospholipids whereas the amounts of glycolipids were determined by a sphingosine assay [143]. 

Radiolabeling of Brain Glycolipids. Thy-1 glycolipid was labeled biosynthetically using a previously described method (7). Five to seven day mice of either AKR/J (H-2, Thy-1.1) or ICR Swiss (Thy-1.2) strain mice were used for each preparation. Each mouse pup was injected intracranially with 8 pi of sterile saline solution containing 5 pci [1- 4C]-N-acetylmannosamine (ManNAc) (54.5 mCi/mmol, New England Nuclear, Boston MA). This solution was injected into both sides of the head at a point 1-2 mm anter-... 

A two dimensional TI/ system was developed to attempt the purification of Thy-1 glycolipid using only one plate. The radiolabeled glycolipid material (brain or lymphoma) was spotted in a small area in the corner of the plate. The plate was developed twice in one dimension in solvent 1 (C M W, 50 40 9, 0.02% CaCl2) and once in solvent 2 (C M W, NH4OH, 60 35 6 14) in the second dimension. Each plate was air dried for one hour then dried in vacuo for 45 minutes (between runs) to ensure dryness. 

Radiolabeling and the Analysis of Thy-1 Active Glycolipids. The amount of Thy-1 active glycolipids in brain, thymocytes or lymphoma cells was found to be extremely small and could only be detected by immunological methods and not by chemical means. 

For enzymic studies, the glycolipid can be radiolabeled in the terminal galactose moiety by the galactose oxidase/[ H]borohydride method of Radin (1972a), such as described by Suzuki, Y., and Suzuki, K. (1972) or Dean and Sweeley (1977). 

A number of mono- and oligosaccharides bound to plant lipids have been described. In particular, steryl glucosides (SG) and acylated steryl glucosides (ASG) were found in all plant tissues [1-3]. Kleinig Kopp [4] showed, by radiolabelling of carrot cultures, that SG and ASG represent 0.8 and 1.0 percent, respectively, of carrot membrane lipids. However, relatively little is known on pentose-containing plant glycolipids. A B-sitosterol xyloside (SX) was chemically characterized in Bauhinia candicans [5] and the in vitro formation of pentosyl derivatives has been reported in pea [6]. 

---