Sulfhydryl groups in glycolipid transfer protein: formation of an intramolecular disulfide bond and oligomers by Cu2+-catalyzed oxidation.
Glycolipid transfer protein (GLTP) purified from pig brain facilitates the transfer of various glycolipids between lipid bilayers. Purified GLTP migrates as two bands of different mobility in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions. The slower component and the faster component constituted about 80% and about 15%, respectively, of purified GLTP. Treatment of GLTP with 45 microM CuSO4 resulted in a decrease in the slower component, an increase in the faster component, and the formation of oligomeric components. The faster and oligomeric components were quantitatively converted to the slower component by reduction with 2% 2-mercaptoethanol in the presence of 1% SDS. The formation of oligomeric components was enhanced by increasing the concentration of CuSO4 to 450 microM and 4.5 mM. Oxidation of GLTP catalyzed by CuSO4 resulted in a decrease in the transfer activity and an increase in the apparent binding affinity of GLTP to 1-O-(beta-D-galactopyranosyl)-N-[10-(1-pyrenyl)decanoyl]-D-erythro- sphingosine (PyrGalCer). The oligomeric components and the monomeric components were isolated by chromatography on a Sephadex G-75 column. It was found that GLTP in fractions enriched with the monomeric components had very high transfer activity and is responsible for most of the transfer activity in the oxidized GLTP. Treatment of GLTP with 1.27 mM HgCl2 resulted in a formation of components unresolvable on SDS-PAGE and also resulted in a reduction of the transfer activity to one-third. However, no obvious change in the binding affinity of GLTP to PyrGalCer was observed by HgCl2 treatment. Treatment with 2-mercaptoethanol restored the activity of GLTP inactivated by HgCl2, whereas the activity inactivated by CuSO4 was not restored by treatment with 2-mercaptoethanol. These results suggest that the transfer activity depends on the turnover rate of the GLTP-PyrGalCer complex which is affected by modification of sulfhydryl groups of GLTP. The sulfhydryl group content of GLTP was estimated by the use of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). A value of 2.2 mol sulfhydryl groups per mol of GLTP was found in the presence of 0.5% SDS and one sulfhydryl group in a GLTP molecule was very rapidly oxidized in the native state, from which it is assumed that the slower component contains three sulfhydryl groups per GLTP molecule and the faster component contains one sulfhydryl group and one disulfide bond per GLTP molecule.
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Department of Biochemistry, Sapporo Medical College, Japan.
Sasaki
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D000818:Animals; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002384:Catalysis; D002850:Chromatography, Gel; D003300:Copper / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008623:Mercaptoethanol / Q000494:pharmacology; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013552:Swine
D002352:Carrier Proteins; D004220:Disulfides; D013438:Sulfhydryl Compounds; C030363:lipid transfer protein; D008623:Mercaptoethanol; D003300:Copper