Purification, characterization, and conformational analysis of rabbit plasma lipid transfer protein.
A procedure for rapid isolation of lipid transfer protein (LTP) from commercially available rabbit plasma is described. Use of protease inhibitors was important for obtaining intact, stable LTP. After lipoproteins were precipitated from the plasma by dextran sulfate, column chromatographies through Butyl-Toyopearl 650M, CM-Toyopearl 650M, and Butyl-Toyopearl 650M were employed. Overall purification from plasma was (3830 +/- 710)-fold with a yield of 3-5%. The isolated LTP migrated as a single band during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r) = 74K and had an NH2-terminal amino acid sequence and amino acid composition closely matching those predicted by its cDNA. This band was recognized by immunoblotting with an anti-human LTP monoclonal antibody, TP2. Gel permeation chromatography revealed that LTP behaved as a globular protein of M(r) = 83K. Isoelectric focusing of the isolated LTP demonstrated a ladder of bands with pI's of 5.7-5.9. The specific activity of rabbit LTP was similar to that of human LTP. Monoclonal antibody TP2, that blocked human plasma LTP activity almost completely, only partially inhibited purified rabbit LTP, and rabbit plasma LTP activity to a similar extent. By a centrifugation binding assay, rabbit LTP was shown to predominantly associate with lipid microemulsion in its presence. Circular dichroism spectroscopy indicated a high content of beta structure, and Provencher and Glöckner analysis gave estimated fractional values of 0.30, 0.39, 0.12, and 0.19 for alpha-helix, beta-sheet, beta-turn, and remainder content, respectively. Upon lipid binding, the helical content did not change drastically, although there was some disordering of beta structure.
Ko
K W
KW
Department of Medicine, University of Alberta, Edmonton, Canada.
Oikawa
K
K
Ohnishi
T
T
Kay
C M
CM
Yokoyama
S
S
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins / Q000097:blood* / Q000737:chemistry* / Q000302:isolation & purification; D008076:Cholesterol, HDL / Q000378:metabolism; D008078:Cholesterol, LDL / Q000097:blood; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004655:Emulsions; D006801:Humans; D015151:Immunoblotting; D007525:Isoelectric Focusing; D008075:Lipoproteins, HDL / Q000097:blood / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011817:Rabbits
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D008076:Cholesterol, HDL; D008078:Cholesterol, LDL; D004655:Emulsions; D008075:Lipoproteins, HDL; C030363:lipid transfer protein