The tanning enzymes of Corcyra cephalonica

In Corcyra cephalonica, the enzymatic synthesis of N-acetyldopamine involves the hydroxylation of tyrosine to dopa, decarboxylation of dopa to dopamine and its subsequent acetylation to N-acetyldopamine. Dopa decarboxylase is mainly present in the epidermal tissues of the rice moth, and phenoloxidase in the haemolymph. These enzymes, along with their substrates, are present in highest concentrations just prior to pupation. An activator for haemolymph phenoloxidase can be detected in the cuticles of the larva and pupa. Some of the characteristics of dopa decarboxylase and diphenoloxidase of Corcyra are similar to that of Drosophila and Calliphora.     

Expression and functions of dopa decarboxylase in the silkworm, Bombyx mori was regulated by molting hormone

Insect molting is an important developmental process of metamorphosis, which is initiated by molting hormone. Molting includes the activation of dermal cells, epidermal cells separation, molting fluid secretion, the formation of new epidermis and old epidermis shed and other series of continuous processes. Polyphenol oxidases, dopa decarboxylase and acetyltransferase are necessary enzymes for this process. Traditionally, the dopa decarboxylase (BmDdc) was considered as an enzyme for epidermal layer’s tanning and melanization. This work suggested that dopa decarboxylase is one set of the key enzymes in molting, which closely related with the regulation of ecdysone at the time of biological molting processes. The data showed that the expression peak of dopa decarboxylase in silkworm is higher during molting stage, and decreases after molting. The significant increase in the ecdysone levels of haemolymph was also observed in the artificially fed silkworm larvae with ecdysone hormone. Consistently, the dopa decarboxylase expression was significantly elevated compared to the control. BmDdc RNAi induced dopa decarboxylase expression obviously declined in the silkworm larvae, and caused the pupae appeared no pupation or incomplete pupation. BmDdc was mainly expressed and stored in the peripheral plasma area near the nucleus in BmN cells. In larval, BmDdc was mainly located in the brain and epidermis, which is consisted with its function in sclerotization and melanization. Overall, the results described that the dopa decarboxylase expression is regulated by the molting hormone, and is a necessary enzyme for the silkworm molting.     

The appearance of dopa decarboxylase activity in imaginal discs of Sarcophaga bullata, undergoing development in vitro

During the postembryonic development of Sarcophaga bullata, two large peaks of dopa decarboxylase activity were observed. These were associated with the sclerotization (hardening) of the puparium and the adult cuticle, respectively. A small peak of activity 5.5–6.5 days after pupariation was possibly associated with the sclerotization of the prothoracic spiracles.A premature increase in enzyme activity was observed in young, third-instar larvae injected with 20 μg of β-ecdysone. However, the advantage of studying the effect of the hormone on enzyme activity in vitro led to an attempt to induce² dopa decarboxylase in cultured wing discs.In the presence of β-ecdysone, wing discs underwent evagination and a substantial increase in dopa decarboxylase activity was observed in these discs. The enzyme activity began to appear after the rupture of the peripodial membrane and reached a maximum about the time disc evagination ceased. We suggest that this enzyme activity was responsible for the slight sclerotization of a fine cuticle secreted by the discs. The cultured imaginal discs underwent changes that are very similar to those which occur in intact animals. Therefore, this system appears promising for further studies on the role in differentiation of the hormonal control of enzyme activity.     

Silencing the genes for dopa decarboxylase or dopachrome conversion enzyme reduces melanization of foreign targets in Anopheles gambiae

The production of melanin is a complex biochemical process in which several enzymes may play a role. Although phenoloxidase and serine proteases are clearly key components, the activity of other enzymes, including dopa decarboxylase and dopachrome conversion enzyme may also be required. We tested the effect of knockdown of gene expression for these two enzymes on melanization of abiotic targets in the mosquito, Anopheles gambiae. Knockdown of dopa decarboxylase and dopachrome conversion enzyme resulted in a significant reduction of melanization of Sephadex beads at 24 h after injection. Knockdown of a third enzyme, phenylalanine hydroxylase, which is involved in endogenous production of tyrosine, had no effect on bead melanization. Quantitative analysis of gene expression demonstrated significant upregulation of phenylalanine hydroxylase, but not the other two genes, following injection.     

Induction of dopa (3,4-dihydroxyphenylalanine) decarboxylase in blowfly integument by ecdysone. A demonstration of synthesis of the enzyme de novo.

The activity of the enzyme dopa (3,4-dihydroxyphenylalanine) decarboxylase, present in the epidermis cells of blowfly larvae, increases during the late third instar under the influence of the steroid hormone, ecdysone. By using the double-labelling technique and immune precipitation with univalent antibody to dopa decarboxylase, we demonstrated that the increase in enzyme activity was due to a stimulation of synthesis of enzyme molecules de novo. In this respect, the action of ecdysone is similar to the action of other steroid hormones.     

Induction of tyrosine hydroxylase gene expression by bacteria in the fat body of eri-silkworm, Samia cynthia ricini

A cDNA clone encoding tyrosine hydroxylase (TH) was isolated from larval fat body of immunized Samia cynthia ricini. In naive larvae, the TH gene was expressed only in the brain, but strongly induced in fat body and hemocytes after injecting UV-killed bacteria. The induction of the gene was rather short-lived compared to that of antibacterial protein genes, reaching the maximum levels 6h after bacterial challenge, and then quickly diminished. A strong induction of the gene expression was caused by both Gram-negative and positive bacteria and zymosan, but little if any by soluble peptidoglycan or lipopolysaccharide. A possible role of TH in the fat body of bacteria-challenged larvae would be to supply catecholamines as the substrate for phenoloxidase leading to melanization, working together with dopa decarboxylase.     

In vitro effects on bacterial growth of phenoloxidase reaction products

An active phenoloxidase preparation from the freshwater crayfish Pacifastacus leniusculus exhibited a strong antibacterial effect in vitro on the bacteria Aeromonas hydrophila, Escherichia coli, Streptococcus pneumoniae whereas a weaker but still significant effect against Bacillus cereus, Pseudomonas aeruginosa and Staphylococcus aureus. In most cases reduction of bacterial growth was stronger when dopamine was used as substrate as compared to L-dopa. The effect on bacteria was abolished if no substrate was available for the phenoloxidase or in the presence of the phenoloxidase inhibitor phenylthiourea.     

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.     

Effect of dopa-decarboxylase inhibition on Aedes aegypti eggs: evidence for sclerotization

Newly deposited fertilized and unfertilized Aedes aegypti eggs are soft and white. Within a short time they darken and harden. Injection of a potent dopa decarboxylase inhibitor (dl)-3-(3,4-dihydroxyphenyl)-2-hydrazino-2-methylpropionic acid (α-MDH) into females, subsequent to a blood meal, results in oviposited eggs which are pale in colour. Moreover, such fertilized eggs do not hatch. The severity of both effects seems to be positively correlated and is dependent upon the time of α-MDH injection.Extracts of mature ovaries are capable of converting dopa to dopamine in the absence of a pretreatment with α-MDH. Mature ovaries obtained from females who had been previously injected with α-MDH could not accomplish this conversion. The inhibitor does not seem to have any effect on dopa oxidase activity and subsequent melanization. We conclude that dopamine is synthesized by blood-fed females via decarboxylation of dopa by dopa decarboxylase and propose that the normal darkening and hardening of A. aegypti eggs is a result of sclerotization.     

Genetic elements near the structural gene modulate the level of dopa decarboxylase during Drosophila development

Summary Measurement of dopa decarboxylase (DDC) levels in 109 strains of Drosophila melanogaster isogenic for second chromosomes isolated independently from natural populations was undertaken. One of the most extreme variants, designated Ddc ⁺⁴, was shown to have about 20% more DDC activity at adult eclosion than a standard laboratory strain used for comparison. The DDC overproduction was shown to segregate with the second chromosome and was mapped to a position within 0.15 map units of the DDC structural gene. The variant was shown to be an underproducer of DDC activity at pupariation and the genetic element responsible for this trait mapped in an identical fashion to that causing overproduction. The temporal phenotype described above was observed in the epidermis but DDC activity levels in neural tissue were normal. Examination of CRM levels at pupariation and eclosion revealed that altered DDC protein levels were responsible for the variant DDC activity levels. Electrophoretic analysis under both denaturing and non-denaturing conditions indicated that the DDC molecules in Ddc ⁺⁴ and the laboratory strain were indistinguishable. These results suggest that alterations in a genetic element (or elements) lying in close proximity to the structural gene are responsible for the complex temporal phenotype of DDC activity exhibited in the variant Ddc ⁺⁴.Abbreviations CRM cross-reacting material - DDC dopa decarboxylase - PTU phenylthiourea     

Dopa decarboxylase activity in Aedes aegypti: A preadult profile and its subsequent correlation with ovarian development

Dopa decarboxylase activity was monitored throughout the entire life cycle of Aedes aegypti. Peaks of activity were detected at each larval molt, at the larval-pupal ecdysis, and at eclosion. The dopa decarboxylase activity in adults was high right after eclosion, but it then dropped rapidly and after 5 days very little activity was detectable. This activity, however, was persistent and remained essentially constant, albeit low, for up to 15 days of adult life. Throughout this part of the study no sex differences in enzymatic activity were observed.A dramatic increase in the level of dopa decarboxylase was noted after adult females were allowed to blood feed. Since a blood meal is necessary in order to initiate ovarian development in this species and since the rate of increase of enzymatic activity paralleled oocyte maturation a causal relationship was indicated. Specifically, we suggest that the dopa decarboxylase is incorporated into the eggs to be used later for subsequent sclerotization.Injection of the molting hormone β-ecdysone into non-blood fed females resulted in a marked stimulation of dopa decarboxylase activity. No such stimulation was observed in saline-injected adult females. The adult female enzymatic activity profile obtained with time after hormone injection was qualitatively the same as that seen after a blood meal. The possibility that ecdysone or an ecdysonelike hormone is necessary for normal ovarian development in Aedes aegypti is discussed.     

Insect melanogenesis. III. Metabolon formation in the melanogenic pathway-regulation of phenoloxidase activityy by endogenous dopachrome isomerase (decarboxylating) from Manduca sexta

Tyrosinase initiates melanogenesis in a variety of organisms. The nature of melanin formed is modified subsequently by dopachrome isomerase and other melanogenic proteins. Earlier, we reported the partial purification of dopachrome isomerase (decarboxylating) from the hemolymph of Manduca sexta and demonstrated the generation of a new quinone methide intermediate during melanogenesis (Sugumaran, M., and Semensi, V. (1991) J. Biol. Chem. 266, 6073-6078). In this paper, we report the purification of this enzyme to homogeneity and a novel inhibition mechanism for regulation of phenoloxidase activity. The activity of phenoloxidase isolated from M. sexta was markedly inhibited by purified dopachrome isomerase. In turn, phenoloxidase also reciprocated by inhibiting the isomerase activity. Preformed dopaminechrome did not serve as the substrate for the isomerase; but dopaminechrome that generated in situ by phenoloxidase was readily converted to melanin pigment by the phenoloxidase/isomerase mixture. Furthermore, the isomerase, which has a molecular weight of about 40,000 in native state, exhibited retardation during affinity electrophoresis on sodium dodeyl sulfate (SDS)-polyacrylamide gel electrophoresis gel copolymerized with tyrosinase and migrated with a molecular weight of 50,000, indicating complex formation with phenoloxidase. Electrophoresis of pupal cuticular extract on polyacrylamide gel, followed by activity staining revealed the presence of a protein band carrying both phenoloxidase and isomerase activity. Accordingly, a high-molecular-weight melanogenic complex was isolated from the pharate cuticle of M. sexta. The complex catalyzed the generation of melanochrome from dopa, while the free phenoloxidase produced only dopachrome from the same substrate. When the complex was treated with trace amounts of SDS, which inhibited the activity of dopachrome isomerase present in the complex, then only the conversion of dopa to dopachrome was observed. These studies confirm the formation of a melanogenic complex between phenoloxidase and dopachrome isomerase. By forming a complex and regulating each other's activity, these two enzymes seem to control the levels of endogenous quinones.     

Activated phenoloxidase from Tenebrio molitor larvae enhances the synthesis of melanin by using a vitellogenin-like protein in the presence of dopamine.

One of the biological functions of activated phenoloxidase in arthropods is the synthesis of melanin around invaded foreign materials. However, little is known about how activated phenoloxidase synthesizes melanin at the molecular level. Even though it has been suggested that the quinone derivatives generated by activated phenoloxidase might use endogenous protein components for melanin synthesis in arthropods, there is no report of protein components engaged in melanin synthesis induced by activated phenoloxidase. In this study, to isolate and characterize proteins involved in melanin synthesis, we prepared in vitro prophenoloxidase activating solution (designated G-100 solution), specifically showing phenoloxidase activity in the presence of Ca2+ and beta-1, 3-glucan, from the hemolymph of larvae of the coleopteran Tenebrio molitor by using a Sephadex G-100 column. When G-100 solution was incubated with dopamine to induce melanin synthesis in the presence of Ca2+ and beta-1,3-glucan, four types of protein (160 kDa, prophenoloxidase, phenoloxidase and 45 kDa) disappeared from SDS/PAGE under reducing conditions. Under identical conditions, but including phenylthiourea as a phenoloxidase inhibitor added to the G-100 solution, three of these proteins (160 kDa, phenoloxidase and 45 kDa) did not disappear. To characterize these melanization-engaging proteins, we first purified the 160-kDa melanization-engaging protein to homogeneity and raised a polyclonal antibody against it. Analysis of the cDNA revealed that it consisted of 1439 amino-acid residues and showed partial homology with Caenorhabditis elegans vitellogenin precursor-6 (19.7%). Western blot analysis showed that it disappeared when active phenoloxidase induced melanin synthesis. Furthermore, when the purified 160-kDa melanization-engaging protein was added to a G-100 solution deficient in it, melanin synthesis was enhanced compared with the same solution without the protein. These data support the conclusion that the 160-kDa vitellogenin-like protein is involved in arthropod melanin synthesis.     

Dopa decarboxylase from Drosophila melanogaster

Summary Dopa decarboxylase (EC 4.1.1.26) has been purified to near homogeneity from mature larvae of Drosophila melanogaster. The enzyme has a molecular weight of 113,000 measured by sucrose gradient sedimentation and 102,000 measured by variable porosity acrylamide gel electrophoresis. Electrophoresis under denaturing conditions revealed the enzyme consists of two subunits of molecular weight 54,000. The affinity of the enzyme for L-dopa is 30-fold greater than for L-tyrosine. Activity is strongly inhibited by heavy metal ions and the sulfhydryl reagent N-ethylmaleimide. N-acetyl dopamine acts as a competitive inhibitor of the enzyme.Antibodies were elicited against the purified enzyme and measurements of the amount of cross-reacting material (CRM) in two groups of mutants were made. The first group comprised the recessive lethal mutants l(2)amd. Heterozygous mutant stocks are hypersensitive to -methyl dopa, an inhibitor of dopa decarboxylase. These stocks were found to have nearly normal amounts of CRM and enzyme activity.A second group of recessive lethal mutants, characterized by lower levels of dopa decarboxylase, was also analysed. These mutants, designated l(2) Ddc, as heterozygotes exhibited CRM levels between 25 and 75% of normal. Although they are alleles at a single locus, they were classifiable into three distinct groups whose properties readily could be ascribed to a homodimeric structure of the enzyme. This structure would also account for the pattern of intracistronic complementation exhibited by the mutants. Finally, the severity of the mutant defects, as judged by our measurements of CRM and activity, closely parallels that deduced from their complementation pattern. We conclude that these mutations are lesions in the structural gene for dopa decarboxylase.     

Purification of phenolic compounds and a phenoloxidase from larval cuticle of the red-humped oakworm,Symmerista cannicosta francl

A phenoloxidase has been extracted, purified, and characterized from cuticle of last-instar larvae of the red-humped oakworm, Symmerista cannicosta. It is a typical tyrosinase (EC 1.10.3.1., o-diphenol:O₂ oxidoreductase), active toward o-diphenols but not p-diphenols, inhibited by thiourea and phenylthiourea, with a pH optimum between 6.0 and 7.2. In these respects it resembles enzyme A of C. vicina, one of the few species from which this presumed wound healing enzyme has been purified and characterized. Hydrolysis of either exuviae or intact cuticle from last instar larvae yielded a number of ketocatechols of which the most abundant, 2-hydroxy, 3′,4′-dihydroxyacetophenone, represented 2.9% of the dry weight of head capsule exuviae, 0.3% of exuviae from the remainder of the body, and 4.6% of the dry weight of head capsule cuticle from previously frozen intact larvae. Differences in the type and amount of ketocatechol recovered from these cuticles are described.     

保幼激素类似物及蜕皮甾类对亚洲玉米螟幼虫酚氧化酶活性的影响

分别用1 μg/头、0.1 μg/头和0.01 μg/头浓度的保幼激素类似物methoprene（蒙五一五）体外处理亚洲玉米螟5龄幼虫,测定幼虫体壁组织、血清和血细胞溶离物中酚氧化酶的活性。结果表明： 1 μg/头 methoprene处理组和0.1 μg/头处理组幼虫体壁组织中酚氧化酶活性与对照组相比有显著提高（P<0.01）,血清和血细胞溶离物中酚氧化酶活性也显著上升（P<0.01）。将含有20-羟基蜕皮酮的人工饲料饲喂亚洲玉米螟5龄幼虫,处理组幼虫体壁组织的酚氧化酶活性下降(P<0.05),血清和血细胞溶离物中的酚氧化酶活性均低于对照组 （P<0.01）。这些结果表明methoprene可以诱导亚洲玉米螟5龄幼虫体内酚氧化酶活性的上升,而20-羟基蜕皮酮则抑制了酚氧化酶的活性。     

Kinetic properties of catecholoxidase activity of tarantula hemocyanin

Phenoloxidases occur in almost all organisms, being essentially involved in various processes such as the immune response, wound healing, pigmentation and sclerotization in arthropods. Many hemocyanins are also capable of phenoloxidase activity after activation. Notably, in chelicerates, a phenoloxidase has not been identified in the hemolymph, and thus hemocyanin is assumed to be the physiological phenoloxidase in these animals. Although phenoloxidase activity has been shown for hemocyanin from several chelicerate species, a characterization of the enzymatic properties is still lacking. In this article, the enzymatic properties of activated hemocyanin from the tarantula Eurypelma californicum are reported, which was activated by SDS at concentrations above the critical micellar concentration. The activated state of Eurypelma hemocyanin is stable for several hours. Dopamine is a preferred substrate of activated hemocyanin. For dopamine, a K(M) value of 1.45 +/- 0.16 mm and strong substrate inhibition at high substrate concentrations were observed. Typical inhibitors of catecholoxidase, such as l-mimosine, kojic acid, tyramine, phenylthiourea and azide, also inhibit the phenoloxidase activity of activated hemocyanin. This indicates that the activated hemocyanin behaves as a normal phenoloxidase.