title: Quaternary and subunit structure of Calliphora arylphorin as deduced from electron microscopy, electrophoresis, and sequence similarities with arthropod hemocyanin.
abstract: Arylphorin was purified from larvae of the blowfly Calliphora vicina and studied in its oligomeric form and after dissociation at pH 9.6 into native subunits. In accordance with earlier literature, it was electrophoretically shown to be a 500 kDa hexamer (1 x 6) consisting of 78 kDa polypeptides (= subunits). Electron micrographs of negatively stained hexamers show a characteristic curvilinear, equilateral triangle of 12 nm in diameter (top view) and a rectangle measuring 10 x 12 nm (side view). Alternatively, particles in the top view orientation exhibit a roughly circular shape 12 nm in diameter. Crossed immunoelectrophoresis revealed the presence of a major subunit type; the nature of a very minor and a third immunologically separated component remains unclear. A novel 2 x 6 arylphorin particle was detected and isolated. It comprises less than 10% of the total arylphorin material and shows a long, narrow interhexamer bridge in the electron microscope. An arylphorin dissociation intermediate identified as a trimer (1/2 x 6) was isolated; its possible quaternary structure is discussed on the basis of electron micrographs. The epitope of monoclonal antibody Ec-7 directed against tarantula (Eurypelma californicum) hemocyanin subunit d and also reactive to Calliphora arylphorin was traced to a highly conserved peptide of 27 amino acids localized in the center of the protein. The primary structure of Calliphora arylphorin as published in our preceding paper (Naumann and Scheller 1991) is compared in detail to the sequences of spider and spiny lobster hemocyanin. This revealed a basic framework of 103 strictly conserved amino acids. Isofunctional exchanges are proposed for another 76 positions. On the basis of these similarities, and the published three-dimensional model of spiny lobster hemocyanin, a detailed model of the quaternary structure of Calliphora arylphorin is presented. A second larval storage protein previously termed protein II was purified from Calliphora hemolymph. It was demonstrated to be a 500 kDa hexamer of 83 kDa subunits. In the electron microscope it shows a cubic view 9 nm in length with a large central hole and a rectangular view (9 x 10 nm) with a large central cavity. A morphologically very similar hemolymph protein was detected in Drosophila melanogaster larvae. From its structural appearance it is uncertain whether protein II belongs to the hemocyanin superfamily or not.
authors.affiliation: Institut für Zoologie, Universität Mainz, Federal Republic of Germany.
authors.forename: J
authors.initials: J
authors.lastname: Markl
authors.forename: T
authors.initials: T
authors.lastname: Burmester
authors.forename: H
authors.initials: H
authors.lastname: Decker
authors.forename: A
authors.initials: A
authors.lastname: Savel-Niemann
authors.forename: J R
authors.initials: JR
authors.lastname: Harris
authors.forename: M
authors.initials: M
authors.lastname: Süling
authors.forename: U
authors.initials: U
authors.lastname: Naumann
authors.forename: K
authors.initials: K
authors.lastname: Scheller
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins; D006433:Hemocyanins / Q000737:chemistry*; D007301:Insect Hormones / Q000737:chemistry* / Q000302:isolation & purification; D019476:Insect Proteins; D007313:Insecta / Q000737:chemistry*; D007814:Larva / Q000737:chemistry; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008121:Nephropidae; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013112:Spiders
chemical_list: D006023:Glycoproteins; D007301:Insect Hormones; D019476:Insect Proteins; C041137:storage proteins, Insecta; D006433:Hemocyanins