Akiko Iwamoto

Alkaline proteases in the midgut tissue and digestive fluid of the silkworm, Bombyx mori

Abstract In larvae of Bombyx mori proteolytic activity was found in the midgut tissue and digestive fluid. The pH-activity curves of both proteases were very similar and optimal activity was about pH 11.2. The reduction in activity at 50°C, for 10 min was about 80% in digestive fluid protease, and about 40% in tissue protease. HgCl2 and DFP strongly inhibited protease activity, and the influence was greater in digestive fluid than in the midgut tissue. Most of the tissue proteases was found to be membrane bound enzyme from results of differential centrifugation and column experiments.

Interrelation of proteases from the midgut lumen, epithelia and peritrophic membrane of the silkworm, bombyx moki L.

Abstract 1. 1. Caseinolytic enzymes of the midgut epithelia, peritrophic membrane and digestive fluid of the silkworm were studied in their activity, intracellular distribution, elution pattern and immunological properties. 2. 2. Most of the proteases from the midgut and peritrophic membrane were recovered in the particulate tractions by differential centrifugation. 3. 3. Similar elution profiles were observed in three different sources of protease with some differences. 4. 4. One of three peaks of digestive fluid protease was produced by solubilization of the midgut protease with Lubrol WX. 5. 5. From results obtained possible mechanisms of the transport and conversion of proteases in the alimentary canal were discussed.

Properties of protease inhibitors from the haemolymph of silkworms, Bombyx mori, Antheraea pernyi and Philosamia cynthia ricini.

1. Effects of inhibitors in the haemolymph from three silkworms on proteases from the alimentary canal of respective insects were studied as well as those on bovine trypsin and alpha-chymotrypsin. 2. In Bombyx haemolymph, three inhibitor fractions were separated by gel filtration on Sephadex G-75. These fractions differed in the specificity of inhibition for different proteases and in thermal stability. 3. In Antheraea haemolymph, a relatively different elution profile was observed compared to that of Bombyx. Philosamia haemolymph showed the intermediate type of elution pattern. 4. Inhibitors from the haemolymph of Antheraea and Philosamia were heat stable. 5. Distinct electrophoretic patterns of inhibitors were observed in three silkworms.

Comparison of three alkaline proteases from digestive fluid of the silkworm, Bombyx mori L.

1. Digestive fluid proteases of the silkworm, 6B1-3, were separated, partially purified and their properties were compared. 2. These proteases were different in the substrate specificity, effect of inhibitors, Km and influence of Mn2+. 3. Hydrolyzing ability for natural substrates was comparatively high in 6B1, whereas the hydrolysis of synthetic substrates of trypsin by 6B1 was lower than that by 6B2 or 3. 4. The protease activity was sensitive to DFP and PMSF. The soybean trypsin inhibitor differentially affected three proteases. Silkworm haemolymph strongly inhibited the protease activity of 6B2 and 3, but scarcely affected 6B1.

Proteolytic enzyme in the midgut of the pharate adult of the silkworm, Bombyx mori

Abstract The protease activity in the midgut of the silkworm, Bombyx mori, increased in the pharate adult period, reached a peak just before emergence of the moth, and decreased markedly thereafter. Optimal activity of the enzyme was at about pH9. Casein was rapidly hydrolysed at comparatively low concentrations, and the activity increased linearly within 2 hr. By agar gel electrophoresis of extracts from the midgut of the pharate adult from about 100 strains of silkworms, five different positively migrating bands were observed. Most of the proteolytic activities were detected in the contents of the midgut of the pharate adult, not midgut tissue, and the electrophoretic pattern of this enzyme was somewhat different from that of the cocoon-digesting enzyme. The protease activity in the pharate adult midgut was inhibited by the haemolymph of larva or pharate adult. The relationship between protease in the midgut of the pharate adult and the cocoon-digesting enzyme is discussed.

Hydrolysis of solubilized fibroin and silk proteins in the midgut of the pharate adult of Bombyx mori

Abstract Midgut protease in the pharate adult hydrolysed native silk proteins and solubilized fibroin by ethylenediamine cupric hydroxide or lithium bromide. By agar gel electrophoresis one to three protease bands moving toward the anode were detected, and the number of bands and the electrophoretic mobility were different among the various strains. Optimal activity of the enzyme was at about pH 8·3. The protease activity was found to decrease in higher concentrations of the substrates. One peak of protease activity was seen in Sepharose 6B chromatography, and the elution pattern and peak position of the enzyme were very similar to those of protease activity with casein. In DEAE-cellulose chromatography, the peak of activity for casein overlapped but did not coincide with a broad peak of protease hydrolysing native silk proteins. The results obtained support the assumption that the midgut protease in the pharate adult is one of the sources of the cocoon-digesting enzyme.

Rôle of the midgut, crop, and maxillae of Bombyx mori in the production of cocoon-digesting enzyme

Abstract The role of the midgut, crop, and maxillae in the production and utilization of the cocoon-digesting enzyme was investigated in the silkworm, Bombyx mori . About a sixtyfold purified preparation of midgut protease was obtained by ammonium sulphate precipitation and column chromatography. Immunological studies by the agar diffusion method of Ouchterlony revealed that the crop and midgut proteases of the pharate adult are antigenically identical whereas that of the maxillary protease is different. From the results of extirpation experiments and previous studies it was shown that the midgut, crop, and maxillae play important roles in the escape of moths from their cocoons.