Masaharu Eguchi

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.

Multiple forms of midgut alkaline phosphatase in the silkworm: Separation and comparison of two isoenzymes

Abstract Two forms of alkaline phosphatase in the midgut of the silkworm, Bombyx mori , were separated by several methods, and the properties of these isozymes were compared. Differential centrifugation, ammonium sulphate fractionation, and agar gel electrophoresis suggested that one form, F, is free and the other, S, form is membrane-bound enzyme. The p H optimum for the F form was 10.1, whereas this value was 11.3 in the S form. The Michaelis constants for the F and S forms were 1.0 × 10 −3 M and 2.1 × 10 −3 M respectively. There were also differences between the two isozymes in the time course of enzyme activity and substrate specificity. The physiological significance of these isozymes is discussed.

Changes in esterase zymograms in the silkworm, Bombyx mori L., during development

Abstract By means of thin layer electrophoresis in agar gel, esterases of various tissues of the silkworm were studied. Nine different esterase bands were detected with β-naphthyl acetate as the substrate and naphthanil diazoblue B as coupler. Eight bands migrated towards the anode, while one band migrated towards the cathode. There is a stage and tissue specificity in the patterns observed, as in other organisms. Gradual changes in enzyme activity occurred during development, and there were great shifts from the late period of the fifth instar to the pupal stage. Substrate and inhibition specificities of the blood and intestine esterases were tested by pH indicator method, and the nature of each component was 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.

Purification and properties of a chymotrypsin inhibitor from the silkworm haemolymph

Abstract 1. 1. A chymotrypsin inhibitor of the silkworm haemolymph was purified by ammonium sulphate fractionation, column chromatographies on DEAE-Sephacel, Ultrogel AcA54 and Con A-Sepharose 4B, and chromatofocusing. 2. 2. The purified inhibitor gave a single protein and inhibitor band on polyacrylamide gel electrophoresis. 3. 3. The inhibitor had a mol. wt of approx. 43,000 and an isoelectric point of 5.06, and its inhibitory activity was not affected appreciably by pH 4.5–9.0. 4. 4. The kinetic analysis showed that this inhibitor inhibits chymotrypsin noncompetitively. 5. 5. The inhibitor suppressed not only α-chymotrypsin but also main digestive fluid protease-6B3 from the silkworm and fungal protease from Aspergillus melleus . 6. 6. However, no inhibition of trypsin was detected.

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.

Multiple forms of midgut alkaline phosphatase in the silkworm: New band formation and the relationship between the midgut and digestive fluid enzymes

Abstract In agar gel electrophoresis of extracts from the midgut a new alkaline phosphatase band appeared between the F (fast) and S (slow migrating) bands after treatment with detergents or lipase. The new band was also formed by the addition of digestive fluid to the extract from midgut tissue, and a main component in digestive fluid had an electrophoretic mobility similar to that of the new band on the alkaline phosphatase zymogram. On incubation of extracts from the midgut with digestive fluid the activity of the F form phosphatase decreased markedly, while that of the S form tended to increase. Enzyme activities in the digestive fluid of three different strains of larvae were compared. Alkaline phosphatase activity in the digestive fluid of F + S − was remarkably lower than that in the F − S + or F + S + strain. Results obtained suggest that there is a close relationship between the S isozyme in the midgut tissue and digestive fluid phosphatase.

Genetic variants of protease inhibitors against fungal protease and alpha-chymotrypsin from hemolymph of the silkworm, Bombyx mori

Many electrophoretic variants of hemolymph inhibitors of proteases from Aspergillus mellus and pancreatic α-chymotrypsin were found using 126 silkworm strains. Six inhibitors of the fungal protease were detected and eight of chymotryspin; the distribution of inhibitors among Japanese, Chinese, and European races was investigated. Comparison of electrophoretic patterns from F1 hybrids and parents showed that the offspring produce inhibitors of both parental types. Segregation in F2 and backcrossing suggest that the expression of each inhibitor is controlled in most cases by a pair of alleles which are responsible for strong and null bands. Two bands of fungal protease inhibitors C and D were controlled by codominant alleles. These results suggest that polymorphism of hemolymph protease inhibitors in the silkworm would be a useful experimental system for the study of the genetic control of protease inhibitors.

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.

Comparison of five chymotrypsin inhibitors from the silkworm haemolymph

Abstract 1. 1. Using two silkworm strains selected from various electrophoretic variants, five haemolymph chymotrypsin inhibitors were partially purified and their properties were compared. 2. 2. Five inhibitor fractions separated on DEAE-Sephacel and Ultrogel AcA54 columns corresponded to the electrophoretic bands; these were classified into two groups, comparatively high, 39,000 (c and d) and low molecular weights, 5500–8500 (a, e and g). 3. 3. Inhibitor a was extremely heat stable, e and g were also stable considerably, but c and d were labile. 4. 4. Ouchterlonys double diffusion test showed that the inhibitors c and d are immunologically identical whereas no precipitate was observed between inhibitors a, e, g and anti-d serum.