Purification and characterization of glycolipid transfer protein from bovine brain

Rhoderick E Brown, Kari L. Jarvis, Kristi J. Hyland

Research output: Contribution to journalArticlepeer-review

33 Scopus citations

Abstract

Bovine brain contains a lipid transfer protein that is specific for neutral glycosphingolipids and gangliosides but does not stimulate phospholipid or neutral lipid intermembrane transfer (Brown, R.E., Stephenson, F.A., Markello, T., Barenholz, Y. and Thompson, T.E. (1985) Chem. Phys. Lipids 38, 79-93). This report describes a new procedure for purifying glycolipid transfer protein from bovine brain as well as a characterization of the resulting protein. Chief among the newly introduced approaches are dye-ligand and fast protein cation-exchange liquid chromatography. Other modifications include increasing the overall scale of purification, incorporating a pH precipitation step and adding different proteinase inhibitors. The resulting procedure simplifies and accelerates the purification process while yielding a homogeneous protein. The purified protein has a molecular weight near 23 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Chromatofocusing reveals that glycolipid transfer protein activity co-elutes with the 23 kDa protein and has an isoelectric point near pH 9.0. A similar isoelectric point is observed using denaturing isoelectric focusing conditions. The protein's amino acid composition reveals high levels of amino acids with non-polar side chains (48%). Based on the findings reported here and on previously published data, bovine brain glycolipid transfer protein has been compared to other lipid transfer proteins as well as lysosomal sphingolipid activator proteins.

Original languageEnglish (US)
Pages (from-to)77-83
Number of pages7
JournalBiochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism
Volume1044
Issue number1
DOIs
StatePublished - May 1 1990

Bibliographical note

Funding Information:
We would like to extend special thanks to Dr. Howard Brockman and Nancy Mizuno for assistance with the FPLC; to Dr. Craig Zwizinski, for helpful comments regarding electrophoresis, to Selene Swanson for performing the amino acid analyses at the Microchemical Facility of the University of Minnesota and to Carmen Perleberg, for secretarial assistance. This research was supported by the Hormel Foundation, by U.S. Public Health Service Program Project Grant HL08214, by a grant-in-aid from the University of Minnesota Graduate School and by American Cancer Society Grant IN-13-Z-22.

Keywords

  • Glycolipid transfer protein
  • Glycosphingolipid
  • Intervesicular lipid transfer
  • Protein purification

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