Structural studies of Manduca sexta lipid transfer particle with apolipoprotein-specific antibodies.
Studies have been conducted to characterize structural and functional properties of Manduca sexta lipid transfer particle (LTP). LTP is a high molecular weight complex of three apolipoproteins and lipid that facilitates the transfer of lipids between lipoproteins and tissues and among lipoproteins in insect hemolymph. Rabbit polyclonal antibodies were raised against each of the three LTP apolipoproteins isolated by preparative electrophoresis. Immunoblot experiments demonstrated that they are apolipoprotein-specific and that LTP apolipoproteins are immunologically distinct polypeptides. Antibody-capture enzyme-linked immunosorbent assay characterization of apolipoprotein-specific IgG demonstrated that each of the three antibodies recognizes native LTP and provided information on the apparent affinity of the antibodies for LTP. Apolipoprotein-specific IgG were then compared in lipid transfer assays to examine the effect of antibody binding on LTP-mediated lipid transfer. Although each of the antibodies inhibited transfer activity, anti-apoLTP-II was capable of nearly abolishing activity at low IgG concentrations (< 26.7 micrograms IgG/micrograms LTP). In contrast, anti-apoLTP-I and anti-apoLTP-III IgG inhibited LTP activity only at much higher concentrations (> 133.3 micrograms IgG/micrograms LTP). These results indicate that apoLTP-II is a catalytically important apolipoprotein. In immunoprecipitation experiments, using 125I-labeled LTP, anti-holoLTP, anti-apoLTP-I, and anti-apoLTP-II were each able to immunoprecipitate all three LTP apolipoproteins while anti-apoLTP-III was not.(ABSTRACT TRUNCATED AT 250 WORDS)
Blacklock
B J
BJ
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Ryan
R O
RO
D000818:Animals; D000906:Antibodies / Q000378:metabolism / Q000494:pharmacology; D001053:Apolipoproteins / Q000737:chemistry / Q000276:immunology*; D002352:Carrier Proteins / Q000737:chemistry* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D007163:Immunosorbent Techniques; D018402:Manduca / Q000737:chemistry*
D000906:Antibodies; D001053:Apolipoproteins; D002352:Carrier Proteins; D007074:Immunoglobulin G; C030363:lipid transfer protein