Tyrosinase catalyzed protein polymerization as an in vitro model for quinone tanning of insect cuticle

The validity of Pryor's widely accepted quinone tanning hypothesis for the sclerotization of insect cuticle was examined using an in vitro model system. Quinones generated in situ by the oxidation of catechols with mushroom tyrosinase and molecular oxygen readily reacted with test proteins such as lysozyme, ribonuclease and cytochrome-c, producing dimers, trimers, and higher oligomers. With the exception of 3,4-dihydroxyphenylalanine, dopamine, and norepinephrine, most other catechols tested participated in protein polymerization. The inability of these three compounds to support oligomerization of test protein was attributed to their high rate of intramolecular cyclization reaction. Radioactive incorporation studies reveal the formation of catechol-protomer adducts, as well as aryl-protein crosslinks in the reaction mixture. The above results strongly support the quinone tanning hypothesis. Based on these findings, the mechanism of cuticular sclerotization is discussed.     

Catechol conjugation with hemolymph proteins and their incorporation into the cuticle of the American cockroach,Periplaneta americana

Newly ecdysed American cockroaches, Periplaneta americana (sixth to last instar) were injected with radioactive dopamine (DA) and hemolymph was collected at 10–60 min post-ecdysis. Size-exclusion chromatography established the presence of at least three proteins that serve as catecholamine carriers. Reinjection of the smaller radiolabeled phenol-bound proteins into newly ecdysed animals results in in vivo aggregation, with the radiolabel bound to large MW proteins (30->200 kDa). In addition, the reinjection of radiolabeled protein of any size resulted in the incorporation of the label into the newly sclerotized cuticle. Hemolymph proteins were synthesized in vivo using [¹⁴C]leucine and subsequently double labeled in vivo with [³H]dopamine. After sclerotization (7 h post-ecdysis) the cuticle was extirpated, hydrolyzed and counted. An identical ratio of ¹⁴C to ³H was found in cuticle extracts as in the double-labeled hemolymph proteins, suggesting that the phenol-bound protein was incorporated in the cuticle unchanged. It appears that the catechol bound to the proteins exists as a β-glucoside.     

Serotonin-immunoreactive visceral nerves and neurohemal system in the cockroach Periplaneta americana (L.)

Summary By use of the indirect immunofluorescent technique applied to whole mounts of tissues and organs of the American cockroach, Periplaneta americana, serotonin-like reactivity has been demonstrated in an extensive meshwork of fibers on the surface of nerves of the subesophageal ganglion and the tritocerebrum. This meshwork appears to serve the neurohemal release of serotonin. In addition, several of these nerves contain two or more centrally located axons that appear to be serotonergic. The corpora cardica show immunoreactivity but do not appear to be a principal release site for serotonin. The nerves and ganglia of the stomadeal nervous system show immunoreactivity for serotonin and provide serotonin-positive innervation to the salivary glands and the visceral muscles of the stomodeum and midgut. The heart, hindgut, Malpighian tubules, fat body, and skeletal muscles all appear to lack serotonin immunoreactivity.     

Taurine-like immunoreactivity in octopaminergic neurones of the cockroach,Periplaneta americana (L.)

Taurine (2-aminoethanesulphonic acid) is reported to interact with the octopaminergic system. The distribution of taurine-like immunoreactivity (-LIR) in relation to octopamine-like immunoreactive dorsal unpaired median (DUM) neurones was investigated with the aim of revealing possible colocalization of these two neuromediators. The specificity of the anti-taurine serum used was demonstrated by dot blot immunoassay and by use of preabsorption controls. There was no crossreactivity with octopamine. The specificity of the octopamine antiserum employed has been described elsewhere. Taurine-LIR could be demonstrated in large dorso-median cells in the suboesophageal and the mesothoracic ganglion as well as in the abdominal ganglia. In addition taurine-LIR is distributed in numerous other regions of the ganglia. A comparison of the immunostaining for taurine and octopamine indicates that several of the taurine-like immunoreactive (-LI) neurones are probably members of the octopamine-immunoreactive DUM cell population. These taurine-LI neurones resemble octopamine-LI DUM cells in soma position and size as well as in the projections of their primary neurites. Colocalization of octopamine-LIR and taurine-LIR within the same neuronal element could be shown by alternate immunostaining of consecutive sections. It is probable that all octopamine-LI DUM neurones also exhibit taurine-LIR, and the possible physiological significance of this coexistence is discussed.     

On the latency and nature of phenoloxidase present in the left colleterial gland of the cockroach Periplaneta americana

The phenoloxidase system responsible for the sclerotization of cockroach ootheca is found to be present as an inactive form in the left colleterial gland of Periplaneta americana. The supernatant fraction obtained by centrifugation of the milky white secretions contained the inactive phenoloxidase which required both sodium dodecyl sulfate (SDS) and the insoluble sediment for exhibiting enzyme activity. Bovine serum albumin could replace the sediment in the activation process. Proteins separated from the supernatant fraction by molecular sieve chromatography on Sephadex G-25 did not require either albumin or the sediment, but required SDS for exhibiting the phenoloxidase activity. Among the detergents tested, SDS (anionic) and cetylpyridinium chloride (cationic) activated the phenoloxidase, but CHAPS (zwitterionic) or nonionic detergents failed to activate the enzyme. The activation caused by SDS occurred well below the critical micellar concentration of SDS indicating that SDS is causing the activation by binding to the protein and altering its conformation. Chloroform-methanol extracts of vestibulum or right gland could replace SDS confirming the presence of endogenous activator(s) of phenoloxidase system. A variety of exogenously added lipids could activate the latent enzyme, among which linoleate, oleate, laurate, linolenate, phosphatidylethanolamine, and phosphatidylglycerol proved to be the effective activators of the latent phenoloxidase. Partially purified phenoloxidase was found to be extremely labile and lost its activity on a) freezing and thawing, b) dialysis, and c) heating for 10 min at 55 degrees C. It exhibited a pH optimum of 7 and was inhibited drastically by phenylthiourea and diethyldithiocarbamate. It readily oxidized a number of o-diphenols such as 3,4-dihydroxybenzylalcohol, 3,4-dihydroxyphenethyl alcohol, catechol, N-acetyldopamine, N-acetylnorepinephrine, dopa, dopamine, etc., but failed to oxidize both 3,4-dihydroxybenzoic acid and 3,4-dihydroxybenzaldehyde. It neither converted the typical laccase substrate syringaldazine to its quinone methide product, nor oxidized the p-diphenols, hydroquinone and methylhydroquinone. Therefore, the enzyme participating in the quinone tanning of cockroach ootheca appears to be a typical o-diphenol oxidase and not a laccase as previously thought.     

Purification and characterization of lipopolysaccharide-binding protein from hemolymph of American cockroach Periplaneta americana

A protein having affinity to lipopolysaccharide of Escherichia coli K12 was purified to homogeneity from the hemolymph of Periplaneta americana. This protein, with an average molecular mass of 450 kDa. was a homooligomer of a 28-kDa subunit protein. Comparative studies using lipopolysaccharide molecules of E. coli and Salmonella minnesota suggested that this protein recognizes and binds to a specific carbohydrate structure of E. coli lipopolysaccharide. Ca2+ was required for this protein to bind to lipopolysaccharide, but other divalent cations could not replace Ca2+.     

Sucrose-binding lectin in regenerating cockroach (Periplaneta americana) legs: Its purification from adult hemolymph

Previously, we purified Periplaneta lectin from the hemolymph of adult Periplaneta americana (American cockroach) (Kubo and Natori Eur. J. Biochem. 168, 75–82, 1987). Immunoblotting analysis using antibody against Periplaneta lectin showed that the cockroach hemolymph contains another lectin that cross reacted immunologically with Periplaneta lectin. We have purified this new lectin (regenectin) to homogeneity. Affinity purified antibody against regenectin cross reacted with Periplaneta lectin. Thus, Periplaneta lectin and this new lectin were found to be different lectins sharing common antigenicity. After leg amputation, this new lectin was found to appear transiently at a specific stage of regeneration as revealed by immunoblotting, suggesting that it plays a role in the regeneration process.     

Comparative analysis of the intrinsic leg musculature of the American cockroach,Periplaneta americana (L.)

The American cockroach has a total of 368 muscles inserting on the post-coxal segments of its legs. By using a narrow morphological definition for delimiting individual muscles, it is shown (i) that the protrochanteral musculatures (23 muscles/leg) differ from the essentially identical meso- and metatrochanteral musculatures (24 and 26 muscles/leg) in number and disposition of extensors and in having a completely different flexor composition, and (ii) that the musculatures of the more distal segments of the legs are completely serially homologous, there being 2 muscles for moving each femur, 23 for each tibia, 7 for each first tarsomere, and 5 for each of the paired pretarsal claws. In all six legs, the trochanteral and tibial musculatures each contain single slender muscles that may be acting proprioceptively to measure the angular displacements between, respectively, the coxas and trochanters, and the femurs and tibias. Neurological and phylogenetic considerations are used to demonstrate why a narrow morphological definition should be employed, and why the widely used functional definition of Snodgrass ('35) is not only fallacious on evolutionary grounds, but also leads to making erroneous conclusions regarding the manner in which insect musculature is controlled by the insect central nervous system. Finally, it is hypothesized that the physiological limitations imposed by having an open circulatory system and the problems inherent in the neural control of large muscles may have been major evolutionary factors in forcing insects to use many slender muscles to control their body movements.     

Purification and some properties of a lectin from the hemolymph of Periplaneta americana (American cockroach)

A lectin showing specificity for human A-type red blood cells was purified to homogeneity from the hemolymph of the American cockroach Periplaneta americana by affinity chromatography on bovine submaxillary gland mucin. This lectin was a huge molecule with molecular mass of about 1500 kDa, with a single subunit of 30-kDa protein, and required Ca2+ for expression of lectin activity. Electron microscopic examination showed that these molecules were rods with helical structure with an average length of 50.5 nm and width of 10 nm. The molecule was suggested to contain tandemly aligned basic units of 10 nm length.     

Immunocytochemical identification of proctolinlike immunoreactivity in the terminal ganglion and hindgut of the cockroach Periplaneta americana (L.)

Summary In the American cockroach, the distribution and connections of neuronal elements of the terminal ganglion-proctodeal nerve-hindgut system were investigated by means of immunohistochemical methods and axonal CoCl₂ iontophoresis. Proctolinlike immunoreactivity was localized within neurons of the terminal ganglion projecting into the proctodeal nerve on the one hand, and in nerve cells without a direct connection to this system on the other. Immunohistochemically, in whole mount preparations fibres of the proctodeal nerve and terminal structures in the hindgut musculature exhibit strong proctolinlike immunoreactivity. At the light- and electron-microscopic levels the pathways of about 30 somata of the proctodeal neural system were characterized by cobalt chloride iontophoresis. The relationships of cobalt filled and immunoreactive neuronal structures are discussed.For the preparation of tritiated proctolin we thank Dr. S. Reißmann, WB Biochemie, Sektion Biologie, FSU JenaThe authors wish to thank G. Schörlitz, Film- und Bildstelle, FSU Jena, for photographs of whole mount preparations and Ms. A. Zinßer and Mrs. B. Cosack for excellent technical assistance.