Lipid binding capacity of spider hemocyanin.
The spider hemocyanin capacity to bind different lipid classes was evaluated by measuring some binding kinetic parameters. A very high lipoprotein (VHDL) which contains hemocyanin, was isolated from Polybetes pythagoricus hemolymph plasma and delipidated. Hemocyanin was bound separately to labelled palmitic acid, phosphatidylcholine, cholesterol, and triolein resulting in several artificial lipoprotein structures. It was possible to corroborate in vitro the lipid-hemocyanin interactions which had been previously observed and, consequently, the apolipoprotein role played by the respiratory pigment of spiders. Lipoproteins were analysed by gel filtration chromatography, and three subfractions with different hemocyanin structures were obtained. The four lipid classes were only bound to the hexameric structure (420 Kda), possibly to low polarity sites. Upon radioactivity measurements of the protein-associated lipids, maximal binding ratios (Mr), dissociation constants (Kd), and the maximal binding effectiveness at low lipid concentrations (Eo) were calculated. Lipid/protein ratios were increased proportionally to each available lipid concentration, following a hyperbolic binding model. Values of saturation, affinity, and maximal binding efficiency to hemocyanin were found to be different for each lipid class assayed. The highest lipid/protein ratio (41.5) was obtained with the free fatty acid and the lowest (7.2) with triolein. Phosphatidylcholine and cholesterol showed the highest relative affinities for hemocyanin (Kd = 63 x 10(-5) M and 74 x 10(-5) M, respectively). Phosphatidylcholine at low concentrations, similar to the physiological ones, presented the highest Eo value. Maximal lipid/protein ratios reached in vitro, were greater than those in P. pythagoricus VHDL, pointing out that hemocyanin could play the apolipoprotein role even under physiological conditions with a very high plasma lipid concentration. J. Exp. Zool. 284:368-373, 1999.
Cunningham
M
M
Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP), UNLP - CONICET and Facultad de Ciencias Naturales y Museo, UNLP, 1900-La Plata, Argentina.
Gómez
C
C
Pollero
R
R
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000378:metabolism; D000818:Animals; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006433:Hemocyanins / Q000378:metabolism*; D006458:Hemolymph / Q000378:metabolism*; D050356:Lipid Metabolism; D019308:Palmitic Acid / Q000378:metabolism; D013112:Spiders / Q000378:metabolism*; D014304:Triolein / Q000378:metabolism
D014304:Triolein; D015060:1,2-Dipalmitoylphosphatidylcholine; D019308:Palmitic Acid; D006433:Hemocyanins; D002784:Cholesterol