Blue lipophorin from Podisus maculiventris

Abstract

Key words: biliverdin, chromoprotein, lipoprotein INTRODUCTIONThe hemolymph of several insect species displays a distinct blue coloration. Detailed chemical studies have shown that this color is due to a tetrapyrrol pigment, biliverdin IX [l], bound noncavalently to proteins. The first biliprotein characterized in insects was insecticyanin from Munducu sexta [Z], which has since been studied extensively [3-51. A tetrameric protein, it is composed of four identical subunits (M, 20,000), each of which bind one molecule of biliverdin IXy. A similar protein called bilin-binding protein has been isolated from Pieris brussicae [6]. In contrast, Chino et al. [7] found a blue cyanoprotein in the locust, Locustu rnigrutoriu that possessed different properties. This cyanoprotein is also a tetramer; however, it is composed of much larger subunits (83,000) and thus has a native molecular weight of about 350,000. More recently, a structurally similar protein has been found in the bean bug, Riptorsus cluvutus [8].Yet another class of biliverdin binding proteins exist in certain lepidopteran species. We have identified a stage-specific blue chromoprotein in the corn earworm, Heliothis zeu [9]. Belonging to the class of the very high density lipoproteins, this chromoprotein is structurally distinct from all known insect proteins. With a native molecular weight of 560,000 it appears to have four identical subunits surrounding a core containing about 10% lipid. As in all other blue hemolymph proteins, its chromophore biliverdin is noncovalently bound to the chromoprotein. A related protein has subsequently been described in the cabbage looper, Trichoplusiu ni [lo].There are clearly several distinct proteins responsible for biliverdin transport in insect hernolymph. Accordingly, to extend our studies of the structure of blue proteins in insects, we focused on an hemipteran species, the spined soldier bug, Podisus ~f f c~~i~e n~r~s (Say), that also possesses blue hemolymph. We report here the isolation and characterization of the chromoprotein of this species. MATERIALS AND METHODS MaterialsAll solvents were of analytical grade and obtained from Fisher (Ottawa, ON). Silica gel G thin layer plates were from EM Science (Gibbstown, NJ). All other chemicals were obtained from Sigma (St. Louis, MO) unless otherwise indicated. InsectsNymphs of Podisus maculiventris were obtained from Dr. Jeff R. Aldrich of the USDA Insect Physiology Laboratory in Beltsville, Maryland. They were reared on pupae of Tenebrio molitor at 23"C, with a 1623 light-dark cycle. Hemolymph CollectionHemolymph was collected by flushing out the hemolymph from adul t insects through an incision in the neck with PBS' (10 mM sodium phosphate, 154 mM NaC1, pH 7.4) containing 1 mM diisopropylfluorophosphoridate and a few crystals of phenylthiourea. The combined hemolymph of 100 adults (approximately 400 p1 hemolymph) was subjected to density gradient ultracentrifugation. Ultracentrif ugationDensity gradient centrifugation was carried out as described previously [ll]. Potass...