Enhanced detection of lipid transfer inhibitor protein activity by an assay involving only low density lipoprotein.
Lipid transfer inhibitor protein (LTIP) activity has been typically quantitated by its ability to suppress lipid transfer protein-mediated lipid movement between low density lipoprotein (LDL) and high density lipoprotein (HDL). In an attempt to establish an LTIP activity assay that is more sensitive, we have exploited the reported preference of the inhibitor protein to interact with LDL. A lipid transfer assay was established that involves LDL as both the donor and the acceptor; LDL in one of these two pools was biotinylated to facilitate its removal with immobilized avidin. Compared to the standard LDL to HDL assay, LTIP inhibited lipid transfer from radiolabeled LDL to biotin-LDL 7-fold more. In the absence of LTIP, lipid transfer activity was the same in both assays. An added benefit of this assay was the near linearity (up to 85%) of the inhibitory response, in contrast to the highly curvilinear response of LTIP in LDL to HDL transfer assays. The high sensitivity of the LDL to biotin-LDL transfer assay in measuring LTIP activity could not be duplicated by other transfer assays including assays containing only HDL (HDL to biotin-HDL), assays between liposomes and LDL, or assays between LDL and HDL where the concentration of lipoproteins was reduced 10-fold. Thus, LTIP activity is most effectively measured in homologous lipid transfer assays involving only LDL (and its biotin derivative). This increased sensitivity to LTIP suggests that the inhibitor binds more avidly to the LDL surface than does lipid transfer protein.
Morton
R E
RE
Department of Cell Biology, Cleveland Clinic Foundation, OH 44195.
Greene
D J
DJ
D001692:Biological Transport; D001710:Biotin; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000097:blood; D002788:Cholesterol Esters / Q000097:blood; D006801:Humans; D008075:Lipoproteins, HDL / Q000097:blood; D008077:Lipoproteins, LDL / Q000097:blood; D008081:Liposomes; D014316:Tritium
D002352:Carrier Proteins; D002788:Cholesterol Esters; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D008081:Liposomes; C030363:lipid transfer protein; D014316:Tritium; D001710:Biotin