Purification and characterization of apolipophorin III from immune hemolymph of Heliothis virescens pupae.
Apolipophorin III (ApoLp-III) from Heliothis virescens pupae was purified by heat-treatment followed by Sephadex G-50 filtration and reverse phase-HPLC. The molecular mass of the purified ApoLp-III was determined as 17965.9+/-5 Da by mass spectrometry. The N-terminal sequence confirmed the protein as ApoLp-III with homology of 56-83% to other insect ApoLp-III molecules. The amino acid spatial arrangement of the predicted alpha-helix 1 of Heliothis ApoLp-III was nearly identical to that of the amphipatic alpha-helix 1 of Manduca sexta ApoLp-III. The absorption spectrum from 240-340 nm of the Heliothis ApoLp-III was the same as the UV spectra of ApoLp-III from Manduca sexta and Galleria mellonella, showing absorption maxima at 280, 268, 264 and 259 nm. These results indicated that the primary structure of ApoLp-III is conserved in lepidopterans. The Heliothis ApoLp-III was not a glycoprotein and showed hemagglutination activity against rabbit red blood cells. This hemagglutination activity was abolished by Tween 80, but not by six different carbohydrates. Hydrophobic interaction of ApoLp-III with red blood cells agreed with structural studies since ApoLp-III binds lipid through hydrophobic interaction after conformational change. Bacterial injection apparently increased the amount of ApoLp-III in immune hemolymph when compared with normal hemolymph, and may indicate that ApoLp-III plays a role in insect immunity.
Chung
Kyung Tae
KT
Department of Microbiology and Molecular Cell Sciences, The University of Memphis, Memphis, TN 38152, USA.
Ourth
Donald D
DD
D000595:Amino Acid Sequence; D000818:Animals; D001053:Apolipoproteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000378:metabolism; D006386:Hemagglutination Tests; D006458:Hemolymph / Q000737:chemistry* / Q000276:immunology*; D037102:Lectins / Q000378:metabolism; D007915:Lepidoptera / Q000737:chemistry* / Q000254:growth & development* / Q000276:immunology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011679:Pupa / Q000737:chemistry* / Q000276:immunology; D011817:Rabbits; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013057:Spectrum Analysis
D001053:Apolipoproteins; D037102:Lectins; C042470:apolipophorin III