Insect Biochem. Vol. 17, No. 2, pp. 353--365, 1987 0020-1790/87 $3.00+0.00 Printed in Great Britain. All rights reserved Copyright © 1987 PergamonJournals Ltd CHARACTERIZATION OF THE NATIVE VITELLOGENIN AND VITELLIN OF THE COCKROACH, NAUPHOETA CINEREA, AND COMPARISON WITH OTHER SPECIES H. IMBODEN, l R. KONIG,1 P. OTT, 2 A. LUSTIG, 3 U. KAMPFER 4 and B. LANZREIN 1 aDivision of Animal Physiology, Institute of Zoology, University of Berne, Erlachstrasse 9a, CH-3012 Berne, 2Department of Biochemistry of the School of Medicine, University of Berne, Bfihlstrasse 28, CH-3012 Berne, 3Biocenter, University of Basle, CH-4056 Basle and 41nstitute of Biochemistry, University of Berne, Freiestrasse 3, CH-3012 Berne, Switzerland (Received 7 November 1985; revised and accepted 14 May 1986) Abstract--In native gradient PAGE vitellogenin of Nauphoeta cinerea showed three closely spaced bands with molecular weights (Mr) of 300,000, 314,000 and 329,000 and a broad band with a Mr of approx. 600,000, and vitellin three closely spaced bands with M# of 274,000, 293,000 and 308,000. Using the same technique vitellin of Leucophaea maderae appeared as several bands with Mrs of 270,000-350,000, Hyalophora cecropia vitellin as a single sharp band with a M r of 520,000 and Manduca sexta vitellin as three bands with MrS of 510,000, 520,000 and 545,000. Investigation of the role of the pH and the ionic strength in the analytical ultracentrifugation revealed a Mr of 244,000 for vitellin (sedimentation equilibrium method) and a s20.w-valueof 9.4 S when analyzed in PAGE buffer (pH 8.25), whereas a Mr of 491,000 and a s20.wof 15 S were found in 0.02 M sodium phosphate, 0.4 M NaCI, pH 7.5. At pH 3.6 aggregation of vitellin was observed. In sucrose density centrifugation addition of Triton X-100 altered the sedimentation coefficient from an apparent value of 15 to 8.4 S. These data suggest that the native vitellin is composed of two monomers with a Mr of 244,000 which assemble to a dimer by hydrophobic interactions. Determination of the Stokes' radius by gel permeation chromatography revealed a value of approx. 7.5 nm for both the monomeric and dimeric form of vitellin and the fractional ratio was calculated to be 1.67 for the monomer and 1.45 for the dimer indicating an elongated shape. Immunodiffusion tests showed the presence of the same two components in vitellogenin and vitellin, one component being much more abundant than the other; data from gel permeation chromatography suggest that the minor component is a smaller molecule with a Stokes' radius of 4.8 nm. Comparative analyses of the carbohydrate, lipid and amino acid composition of vitellogenin and vitellin revealed no major differences although minor differences with respect to the phospholipid composition were observed. Key Word Index: Vitellogenin, vitellin, cockroach, PAGE, analytical ultracentrifugation, sucrose density gradient, Stokes radius, Triton X-IO0, gel permeation chromatography, amino acid composition, lipid, carbohydrate, Leucophaea maderae, Nauphoeta cinerea, Manduca sexta, Hyalophora cecropia INTRODUCTION Vitellogenins are female-specific proteins which are synthesized extraovarially and become the major egg yolk proteins, the vitellins. These are thought to be the nutritive source of amino acids, lipids, carbo- hydrates and phosphorus for the developing embryo. The physico-chemical properties of vitellogenins and vitellins have been investigated in several species (for reviews see Engelmann, 1979; Hagedorn and Kunkel, 1979) and it is apparent that they are high molecular weight lipoglycoproteins. In many insect species, including cockroaches, vitellogenins are synthesized in the fat body under the control of juvenile hormone, released into the haemolymph and taken up selec- tively by the maturing oocyte against a concentration gradient (see Engelmann, 1979, 1983). It is our aim to elucidate the process of vitellogenin incorporation and vitellin formation. For this pur- pose it is important to know the physicochemical properties of these proteins. In our experimental insect, the ovoviviparous cockroach Nauphoeta cin- erea, we recently presented evidence for the existence of specific vitellogenin binding sites on the mem- branes of oocytes at the stage of vitellogenesis (K6nig and Lanzrein, 1985). Here we describe the character- ization of the vitellogenin and vitellin of this species. In view of the lipoglycoprotein nature and the pH and ionic strength dependent solubility and aggre- gation properties of these proteins (Brookes and Dejmal, 1968; Dejmal and Brookes, 1972) we used several methods and various buffers for determining their molecular weight and sedimentation constant. Here we present data obtained by immunodiffusion, different types of native polyacrylamide gel electro- phoresis (PAGE), analytical ultracentrifugation (sed- imentation velocity and sedimentation equilibrium), sucrose density gradient centrifugation in the pres- ence and absence of Triton X-100 and gel permeation chromatography. We also present data on the anal- 353