George L. Lookhart

Characterization of haemolymph protyrosinase and a cuticular activator from Manduca sexta (L.)

Abstract A protyrosinase has been isolated from fifth instar larval haemolymph of the tobacco hornworm, Manduca sexta (L.) by ammonium sulphate fractionation, hydroxylapatite chromatography and gel filtration. It exhibited a single band after polyacrylamide gel electrophoresis at pH 8.5 and two bands in the presence of sodium dodecyl sulphate with apparent molecular weights of 7.7 × 10 4 and 7.1 × 10 4 . The proenzyme is a metalloprotein containing 0.18% copper. The activating enzyme was partially purified from pharate pupal cuticle by ammonium sulphate precipitation and hydroxylapatite chromatography. The activation was inhibited by di iso propylphosphorofluoridate and had a pH optimum of 8.8. Chymotrypsin also activated the proenzyme. The cuticular activator is probably a serine protease. Activated protyrosinase exhibited physical, chemical and kinetic properties very similar to those of tyrosinase extracted from pharate pupal cuticle. Haemolymph protyrosinase may serve as a precursor for both haemolymph and cuticular tyrosinases that synthesize catecholamines and quinones used for wound healing, parasite encapsulation as well as for cuticular stabilization and pigmentation.

High-performance capillary electrophoresis of cereal proteins

Cereal grains are widely used of human foods and animal feed throughout the world. Cereals provide dietary protein, which also often has a functional role, as wheat gluten does in bread. Cereal proteins are unique in many ways: they are highly complex and heterogeneous, are often difficult to extract, and aggregate readily, making them difficult to characterize. Because of the economic importance and widespread use of cereal proteins, however, many techniques have been used for their analysis. High-performance capillary electrophoresis (HPCE) is one of the newest techniques to be so used. This review describes the development of charge- and size-based HPCE methods for analysis of cereal grain proteins, and the use of these methods for cultivar identification, classification, and prediction of quality. HPCE is versatile, rapid, easily automated, readily quantified, and provides high-resolution separations. Clearly, HPCE is a valuable addition to other methods of cereal protein analysis and should, in time, be applicable to all protein classes from all cereals.

Associations of Starch Gel Hardness, Granule Size, Waxy Allelic Expression, Thermal Pasting, Milling Quality, and Kernel Texture of 12 Soft Wheat Cultivars

ABSTRACT Starches were isolated from 12 soft wheat (Triticum aestivum L.) cultivars and were characterized for waxy (Wx) allelic expression, thermal pasting characteristics, and starch granule size. Gels were produced from the thermally degraded starches and were evaluated using large deformation rheological measurements. Data were compared with cultivar kernel texture, milling characteristics, starch chemical analyses, and flour pasting characteristics. Larger flour yields were produced from cultivars that had larger starch granules. Flour yield also was correlated with lower amylose content and greater starch content. Harder starch gels were correlated with higher levels of amylose content and softer kernel texture. The cultivar Fillmore, which had a partial waxy mutation at the B locus, produced the highest peak pasting viscosity and the lowest gel hardness. Softer textured wheats had greater lipid-complexed amylose and starch phosphorus contents and had less total starch content. Among these wheats of...

Electrophoresis of cereal storage proteins.

Cereal proteins have been studied by a number of analytical techniques over the years. One of the major methodologies utilized by cereal chemists has been electrophoresis. Starting with moving boundary electrophoresis and progressing to slab gels and high-performance capillary electrophoresis, innovative methods have been developed to provide high resolution separations of difficult to separate proteins. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), acid-PAGE, isoelectric focusing, free zone CE, and even high-resolution two-dimensional HPLC-HPCE methods have been developed to separate cereal proteins. This review focuses on electrophoretic methods for separating and characterizing cereal storage proteins.

Catecholamines and β-alanine in the red flour beetle, Tribolium castaneum: Roles in cuticle sclerotization and melanization☆

Catecholamines and β-alanine titres were measured in a rust-red wild strain of the red flour beetle, Tribolium castaneum Herbst and in a mutant strain which has a black cuticle. During the first three days after adult eclosion, β-alanine and N-β-alanyldopamine (NBAD) are present in the former strain, but they are absent or very low in the latter. Dopamine is approx. 3-fold higher in the black than in the rust-red strain while N-acetyldopamine, the major catecholamine in both strains, is about two times more abundant in the black. Fully sclerotized rust-red elytra contain nearly ten times more NBAD than black elytra, while the latter have about ten times more dopamine. Injection of β-alanine into newly eclosed mutant adults rescues the rust-red phenotype. One-day and two-week-old adults of the black strain exhibit 2 to 3-fold higher levels of catechol oxidase activity than wild-type adults. Cuticle pigmentation is probably determined by the relative concentrations of catecholamines, β-alanine and catechol oxidase which are utilized for production of sclerotin and melanin. High dopamine titres appear to cause the black pigmentation through melanin synthesis while β-alanine and its dopamine conjugate NBAD are associated with the rust-red colouration.

Tyrosine metabolism for cuticle tanning in the tobacco hornworm, Manduca sexta (L.) and other lepidoptera: Identification of β-d-glucopyranosyl-O-l-tyrosine and other metabolites

Abstract When [ 14 C ]tyrosine and [ 14 C ]glucose were fed or injected into feeding fifth-instar larvae of the tobacco hornworm, Manduca sexta (L.), they were incorporated into a conjugate identified in hemolymph and carcass extracts as β- d -glucopyranosyl-O- l -tyrosine. In wandering larvae and pupae, the conjugate was hydrolyzed, and tyrosine was hydroxylated and decarboxylated to dihydroxyphenylalanine and 2-(dihydroxyphenyl)ethylamine. None of these metabolites were formed in fourth-instar larvae or in adults. [ 14 C ]Phenylalanine was hydroxylated to tyrosine in all stages of insect development. β- d -Glucopyranosyl-O- l -tyrosine was also detected in 18 other species of Lepidoptera but not in species from other insect orders. This conjugate appears to be the major tyrosine storage metabolite for production of tanning diphenol substrates in Lepidoptera.

Sequestration of ascorbic acid by the larval labial gland and haemolymph of the tobacco hornworm, Manduca sexta L. (Lepidoptera: Sphingidae)

Abstract Tissues from Manduca sexta were examined for the presence of l -ascorbic acid and l -gulonolactone oxidase. l -Ascorbic acid was found in eggs, larval labial gland, haemolymph, gut, muscle, cuticle, adult nervous tissue and gonads at concentrations ranging from 150 mg per 100 g wet tissue. No ascorbate was detected in larval fat body and Malphigian tubule or adult salivary gland. Concentrations in labial gland and haemolymph increased 80- and 10-fold, respectively, during the fifth larval instar such that the labial gland surpassed all other tissues in ascorbate concentration. Since tissues from insects reared on an l -ascorbate-deficient diet contained no detectable vitamin C and l -gulonolactone oxidase was absent from tissue extracts, the hornworm apparently acquired l -ascorbate solely from the diet.