The structure of insect cuticle has been reviewed briefly and an account given of the processes by which hardening (tanning) occurs after the moult. The sclerotin wall of the cockroach ootheca is formed by the intermingling of the accretions of the left and right colleterial glands. This system, because of its comparative convenience, is studied with a view to shedding light on the sclerotin formation in the cuticle. With reference to their role in sclerotization, the properties of insect blood and cuticle oxidases are reviewed and comparisons made with the animal and plant enzyme grouse they most resemble - tyrosinases and laccases. The function of copper in these oxidases is discussed. A method was developed for determining the protein concentrations, in the presence of phenolic compounds, of the diluted secretion from the left colleterial glands of Periplaneta. This involved digestion (6N-HCl, 6 hours) of the protein followed by estimation with ninhydrin of the total amino acids in the hydrolysate. Investigation of the properties of the copper-containing (0.1-0.2%) phenoloxidase of the left colleterial gland showed that the enzyme is specific towards diphenols (especially ortho-) and is therefore not a tyrosinase. In the majority of its properties the oxidase resembles a laccase, although it is more specific than the plant enzymes. Phenolic substrates bearing an amino group in the side chain are not active - a property which clearly distinguishes the cockroach phenoloxidase from the blood tyrosinase of insects. Towards certain substrates (catechol, hydroquinone, homoprotocatechuic acid) activity is unexpectedly stable to boiling while towards others (protocatechuic acid and its aldehyde) it is destroyed. Possibly non-enzymic, as well as enzymic, activity exists. The effect of some substances (detergents, dopa, catechol, tyrosine, ascorbic acid) on the activity of the phenoloxidase towards protocatechuic acid (3 μmoles) is measured. The stimulation caused by ascorbic acid (0.15 andmu;moles) is related to the possible requirement of cuprous ions for enzyme activity. The phenoloxidase activity which resides in a articulate fraction of the gland secretion, is (unlike other copper oxidases) not inhibited by metal chelating agents, such as phenylthiourea, diethyldithiocarbamate, ethylenediaminetetracetic acid and 4-chlororesorcinol, in the presence of sodium phosphate buffer (0.1M -, pH 6.8). Cyanide and borate are effective inhibitors, however. Varied attempts to purify the participate enzyme and thus to fractionate it from the prosclerotin or structural protein met with little success. The specific activities (Q<sub>O<sub>2</sub></sub>'s) of the supernatants obtained after high speed centrifugation (100,000-182,000 andtimes; g) are approximately five to ten times higher than those (3.93±1.15(10)) of untreated enzyme preparations. An examination of this "purification" led to the conclusion that the enzyme activity appeared to be rather dependent on the state of aggregation of the protein particles. Further evidence of this effect was inferred from the stimulating action of anionic (deoxycholate) and neutral ("Tween") detergents (added prior to buffering) upon phenoloxidase activity. Prior to the institution, as a regular procedure, of dialysis (0.01M-NaCl) of the colleterial gland secretion (to remove endogenous phenolic compounds), a long induction period (ca. 20-60 mins.) before commencement of oxygen uptake by enzyme and substrate was frequently observed. This long period was minimized by the addition of right gland homogenate or ascorbic acid. In section II, the phenolic compounds found in insect cuticle and in cockroach colleterial glands were examined with reference to their biosynthesis and to their role in sclerotization. Protocatechuic acid, its 4-o-β-D-glucoside and p-tyrosine were identified (chromatographically and spectrophotometrically) in homogenates of left colleterial gland. After aerobic incubation (37°C) of these homogenates substances strongly resembling catechol and tyramine were separated by paper chromatography and identified by their u.v. spectral properties after elution. Protocatechuic acid and p-tyrosine, when added (with and without right gland preparation) to homogenates of left glands, increased the production of catechol and tyramine respectively. From densitometric measurements of ferric chloride/ferricyanide sprayed paper chromatograms the aerobic conversion by left gland homogenate of p-tyrosine (1 andmu;mole added) into tyramine (0.62 andmu;moles) was demonstrated, The amine was also separated by absorption on Amberlite-IRC (H<sup>+</sup>). The decarboxylase systems responsible for catechol and tyramine production, whether induced or not, were heat labile. There is some manometric evidence that the former enzyme is located in right gland cells while the latter is present in the larger gland tissue. These findings are made uncertain by the retardation of the reactions in presence of inorganic buffers (and an anaerobic atmosphere) and by extraneous gas evolution particularly during incubation of mixed homogenates of both glands. In the light of some evidence, the possibility that tyramine might be enzymically oxidized by homogenates of left gland is weighed. The significance of such a reaction, together with the decarboxylation of p-tyrosine and protocatechuic acid, is discussed with regard to the biosynthesis of tanning quinones from the p-tyrosine of the insect blood.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:644603 |
| Date | January 1961 |
| Creators | Whitehead, D. L. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:311ee640-4cd1-4e97-8d17-0f2361cddb75 |
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