Abstract
A glycolipid-specific lipid transfer protein has been purified to apparent homogeneity from pig brain post-mitochondrial supernatant. The purified protein was obtained after about 6,000-fold purification at a yield of 19%. Evidence for the homogeneity of the purified protein includes the following: (i) a single band in acidic gel electrophoresis, in sodium dodecyl sulfate-gel electrophoresis, (ii) a single band in analytical gel isoelectric focusing, (iii) exact correspondence between the glycolipid transfer activity and stained protein absorbance in the acidic gel electrophoresis, and (iv) coincidence between the transfer activity and protein absorption at 280 nm in gel filtration through Ultrogel AcA 54. The protein has an isoelectric point of about 8.3 and a molecular weight of 22,000, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A molecular weight of 15,000 was calculated from AcA 54 gel filtration. The amino acid composition has been determined. The protein binds [3H]galactosylceramide but not [3H]phosphatidylcholine. Under the conditions used, 1 mol of the transfer protein bound about 0.13 mol of [3H]galactosylceramide. The glycolipid transfer protein-[3H]galactosylceramide complex was isolated by a Sephadex G-75 chromatography. An incubation of the complex with liposomes resulted in the transfer of [3H]galactosylceramide from the complex to the acceptor liposomes. The result indicates that the complex functions as an intermediate in the glycolipid transfer reaction. The protein facilitates the transfer of [3H]galactosylceramide from donor liposomes to acceptor liposomes lacking in glycolipid as well as to acceptor liposomes containing galactosylceramide.
Highlights
A sensitiveand specific determination of the glycolipid transfer activity is possible by a modified procedure of the phospholipid transfer assaywhich we previouslydescribed [19]
The transfer of phospholipids between intracellular membranes is presumably mediatebdy the activityof phospholipidtransferproteins.Phospholipidtransferproteins different in their phospholipid specificities have been purified from bovine liver [4, 5], heart [6],and brain [7], rat liver [8,9,10] and hepatoma, yeast [12], and a bacterium
Determination of ~H]GalactosylceramideBinding to the Glycolipid Transfer Protein-Liposomes for the binding experiment were prepared by sonication from a lipid mixture containing phosphatidylcholine, dicetyl phosphate, cholesterol, and [6-3H]galactosylceramide(10' dpm/nmol) in molar ratio of 86:5:45:5
Summary
A glycolipid-specific lipid transfer protein has been interesting to find proteins which facilitate the transfer of purified toapparent homogeneity from pig brain post- glycosphingolipids betweenmembranes because such proteins mitochondrial supernatant. A sensitiveand specific determination of the glycolipid transfer activity is possible by a modified procedure of the phospholipid transfer assaywhich we previouslydescribed [19]. By thusee of this assay, we studied theglycolipid transfer activitypresentinthe postmicrosomal supernatant of rat brainand liver[20, 21]. Incubation of the complex with liposomes resulted in In this paper,we wish to present animproved purification the transfer of [3H]galactosylceramidefrom the com- procedure of pig brain glycolipid transfer protein. Amino acid composition of the protein was detergtwsrolaeymnlclesoafslseiptrtoiodoaftcarcca[ecn3pesHtpfo]etrgorarlrlliieappacooctsostoiosomymnlece.sescTrloaahmncetkiadiipnenrgiofnrtoigenmigngalldfayoacccntooiollsiirpytailldtciepesaor-s-theamamniniidenedt..eTrImthweedapsiraoftoeteuininnd-gttahhleaagtclyttochsoeylilpcpeirdroattmreaiinndsefbecirnodmre[spa3lcHetxi]ognfau.lanccttoiosynlscearsamide
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