George Graf

The calcified puparium of the face fly, Musca autumnalis (Diptera: Muscidae)

Abstract The general composition of the calcified puparium of Musca autumnalis was determined. Ash-weight analyses show that 62% of the puparium is inorganic material. The major components of the puparium are calcium, magnesium, phosphate and carbonate. Calcium and magnesium phosphate are the predominant salts in the puparium, with a lesser contribution by their respective carbonates. Scanning electron microscopy revealed that the exocuticle was the site of calcification in the puparium. Inorganic analyses of the insoluble salt isolated from face fly larvae showed it to be compositionally identical with the puparial salt. Major organic components of the puparium are lipid, chitin and protein. The presence of carbonic anhydrase and alkaline phosphatase activity in post-feeding larvae was confirmed.

Uric acid translocation from the fat body of Manduca sexta during the pupal-adult transformation: Effects of 20-hydroxyecdysone

Abstract The uric acid concentration in the fat body and haemolymph of the lepidopteran, Manduca sexta (L.) (tobacco hornworm) was determined for each day between pupation and adult eclosion. Uric acid levels in the fat body remained at about 0.5 μmol/mg dry wt until 16 days after pupation (day 16), then decreased rapidly to

Carbonic anhydrase in the face fly, Musca autumnalis (Degeer) (Diptera: Muscidae)

Abstract Carbonic anhydrase was detected in the face fly, Musca autumnalis , and the development profile of the enzyme showed that maximum enzymatic activity occurred in the post-feeding larva. By comparison, little carbonic anhydrase activity was observed in post-feeding larvae of the house fly, Musca domestica . The enzyme was localized in the haemolymph, salivary glands, midgut and brain of post-feeding face fly larvae using an electrometric assay and Hanssons histochemical technique. No enzymatic activity was detected in the anterior Malpighian tubules, posterior Malpighian tubules, fat body, tracheae and carcass. These findings suggest that carbonic anhydrase has no function in the formation of the calcospherites located in the anterior Malpighian tubules of post-feeding larvae, but may be important in the ecdysial elimination of minerals during pupariation.

The Localization, Purification and Partial Characterization of Carbonic Anhydrase in the Face Fly, Musca autumnalis

Several authors have associated carbonic anhydrase with biomineralization.* Musca autumnalis is one of a few Diptera that have a calcified puparium. Face fly larvae store large quantities of minerals, primarily calcium phosphate, as calcopherites in the anterior malpighian tubules during de~elopment .~ During pupariation and concurrently with ecdysis, the salt in the anterior malpighian tubules is mobilized and incorporated into the puparium. This process has been termed ecdysial elimination. A developmental profile of carbonic anhydrase activity in Musca autumnalis shows that peak activity occurs in the post-feeding larva; this period of high enzymatic activity is short, lasting approximately 24 hours, and is concomitant with ecdysial elimination (FIG. 1). By Comparison, the post-feeding larva of the house fly, Musca domestica, a closely related species with uncalcified sclerotized puparia, shows no similar peak and has only low levels of carbonic anhydrase activity. We localized carbonic anhydrase activity in post-feeding face fly larvae using two independent methodologies. The first was an electrometric assay of the crude supernatants of dissected tissue homogenates (TABLE 1). The second, Hansson’s histochemical m e t h ~ d , ~ gave results similar to those obtained by electrometric assay. Based upon carbonic anhydrase staining the midgut appeared to be functionally differentiated into two distinct regions. In the first region the precipitate was uniformly distributed in the midgut cells. In the second region the basal cytoplasm of the midgut cells was lightly stained while intense staining occurred along the basal border of the cell membranes. Carbonic anhydrase was purified from post-feeding face fly larvae using affinity chromatography, chromatofocusing (PH 8.0-5.0), and gel filtration chromatography on Sephadex G-75-40. The enzyme appears to be similar to other animal carbonic anhydrases. It is monomeric with a molecular weight of approximately 31,000 as estimated by gel filtration chromatography and SDS polyacrylamide gel fly.

Isolation and partial characterization of carbonic anhydrase from erythrocytes of Meleagris gallopavo

SummaryA soluble enzyme with carbonic anhydrase activity has been isolated from domestic turkey(Meleagris gallopavo) erythrocytes and purified by chloroformethanol precipitation, ammonium sulfate fractionation and gel filtration on a Sephadex G-75 column. Analytical polyacrylamide gel disc electrophoresis showed one major and two minor bands. The specific activity for the CO2 hydration reaction was approximately 2000 Wilbur-Anderson units/mg protein at 0°C. The presence of the reducing agents 2-mercaptoethanol or dithioerythritol was required throughout the procedure. Upon removal of the 2-mercaptoethanol by dialysis the activity was lost but could be restored by addition of the reducing agent. The enzymatic activity was inhibited by acetazolamide,p-chloromercuribenzoate ando-iodosobenzoate. Esterase activity was detected withp-nitrophenylacetate as the substrate. The molecular weight of the enzyme was determined as 31,000 by gel filtration and 34,000 ± 2000 with analytical ultracentrifugation. Atomic absorption spectroscopy indicated the presence of zinc in the ratio of one mole of zinc per one mole of enzyme.

Isolation, purification and characterization of an insect carbonic anhydrase

Abstract Carbonic anhydrase (carbonate hydro-lyase; EC 4.2.1.1) was purified and partially characterized from post-feeding larvae of the face fly, Musca autumnalis DeGeer. Analytical polyacrylamide gel electrophoresis (PAGE) of the enzyme purified by affinity chromatography showed the presence of three bands. Band-B appeared as a minor component which was lost during subsequent purification steps. Band-A and band-C appeared as major proteins and were separated using PAGE. Isoelectric points of 6.4 and 5.4 were estimated for band-A and band-C, respectively, by chromatofocusing. SDS-PAGE of the purified proteins produced single bands each having a mol. wt of approx. 32,000. The native molecular weight of these bands, estimated by gel filtration chromatography, was 31,000. The absence of inhibition by o -iodosobenzoate and p -chloromercuribenzoate suggested that reduced sulfhydryl groups were not essential to maintain high activity. A K m of 13.1 mM was determined for the dehydration reaction and is of the same order of magnitude as K m s reported for the high activity forms studied in mammals. The amino acid composition of the enzyme was determined by reversed phase HPLC. Zn content was determined by atomic absorption spectroscopy.

Structure and composition of urate storage granules from the fat body of Manduca sexta

Uric acid granules were isolated and purified from fat body of fifth instar tobacco hornworms, Manduca sexta (L.). Analysis by light microscopy indicated that preparations were homogeneous and all granules displayed birefringent properties. Scanning electron microscopy confirmed the homogeneity of the preparations and showed near spherical or biconcave shaped granules, many with hollow centers. The granules, 0.5–2.0 μm in dia, consisted of a tight array of concentrically whorled fibers about 0.02 μm in dia and 0.5–1.0 μm in length. Biochemical analyses revealed that the granules contained 75–78% uric acid, 4–5% protein and 0.5% carbohydrate. Elemental analysis indicated the presence of potassium (6.7%) with lesser amounts of sodium (0.5%) and that the granules contained 6–7% water. SDS-polyacrylamide gel electrophoresis indicated the presence of numerous peptides ranging in molecular weight from 20,000 to > 200,000. Amino acid analysis showed an abundance of aspartic acid, glutamic acid and apolar amino acids. Transmission electron microscopic analysis revealed that staining of individual fibers with uranyl acetate and lead citrate was restricted to the outer surfaces. These findings suggest that the granules are composed of tightly coiled fibers of crystalline uric acid/urates and that each fiber is enveloped with protein.

Xanthine dehydrogenase in the fat body of Manduca sexta: Purification, characterization, subcellular localization and levels during the last larval instar

Xanthine dehydrogenase was isolated and purified from the fat body of fifth instar tobacco hornworms, Manduca sexta (L.) by ammonium sulfate precipitation, Sephacryl S-400 gel filtration, QAE-Sephadex ion exchange chromatography and reactive blue-2 Sepharose affinity chromatography. The preparation appeared homogeneous following analysis by polyacrylamide disc gel electrophoresis, sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and rocket and crossed immunoelectrophoresis using rabbit antiserum against purified xanthine dehydrogenase. The molecular weight of native xanthine dehydrogenase was estimated at about 300,000 while SDS-PAGE indicated that the enzyme consisted of two 158,000 mol. wt subunits. Kinetic analyses revealed high affinities for xanthine and hypoxanthine as substrates. Electron acceptors (cofactors) included NAD+, as well as the dyes, p-iodonitrotetrazolium violet and nitro blue tetrazolium. A broad pH optimum was observed between pH 7 and 10, and xanthine dehydrogenase was competitively inhibited by allopurinol and irreversibly inhibited by the sulfhydryl inhibitor, p-hydroxymercuribenzoate. The levels of xanthine dehydrogenase in fat body extracts from fifth instar larvae, as determined by both enzyme assay and immunoelectrophoresis, decreased from days 1 to 5. Ultra-structural analysis of day 3 larval fat body cells by immunocytochemical techniques, using colloidal gold staining, revealed that xanthine dehydrogenase was specifically located either within urate storage vacuoles or near the basal lamina of the cells.

The phase transition in phospholipid vesicles probed by the positron annihilation lifetime technique

Abstract We have measured the phase transition in phosphatidyl choline multilamellar vesicles composed of saturated carbon chains. At the fluid-order phase transition temperature, the positron lifetime increases and the intensity decreases for the long-lived (ortho-) positronium. These data are interpreted in terms of the free volume model for positron annihilation.

The Application of Perturbed Directional Correlation of Gamma Rays to the Study of Protein-Metal Interactions

Several branches of nuclear spectroscopy—magnetic resonance and the Mossbauer effect in particular—have been the subject of considerable interest to obtain structural and motional information about biological macromolecules. Recently, a third technique— perturbed gamma-gamma directional correlation—is being tested for the study of rotational correlation times, internal rotations and conformational changes in biological macromolecules. When two gamma rays from a radioactive nucleus are emitted in a cascade and detected with a coincidence spectrometer, the coincidence counting rate depends strongly on the angle between their directions of propagation. This directional correlation can be perturbed by the interaction of the nucleus with fields existing in macromolecules. A study of such perturbed directional correlation (PDC) has the potential to provide information on protein-metal interactions. The use of a radioactive nucleus as a rotational tracer to label macromolecules offers the possibility of obtaining information on protein structure with the sensitivity and instrumental simplicity of radioactive tracer techniques. Work to date has centered on measurement of internal electric field gradients of metalloproteins, molecular correlation times and the correlation time of water associated with proteins. This paper is a brief review of the theory and experimental technique of perturbed directional correlation of gamma rays in comparison to Mossbauer spectroscopy and the methods of magnetic resonance. Particular emphasis will be placed on the isotopes which can be used in PDC especially those which may occupy sites in biological macromolecules.