Trehalase transformation in silkworm midgut during metamorphosis
The particulate trehalase from silkworm larval midgut was effectively solubilized by repeated freezing and thawing, and by incubation with snake venom and non-ionic detergents (Lubrol PX and WX and Triton X-100). With solubilization the activity was enhanced and the activation behaviour was dependent upon the developmental stage of silkworms, being highest (up to about 3-fold) at the spinning stage.
When chromatographed on DEAE-cellulose columns separately, the enzyme solubilized by freezing and thawing and the soluble trehalases from feeding larval midgut were respectively eluted as single peaks, P I and P II. However, both P I and P II trehalases were demonstrated after solubilization of the particulate fraction from feeding larvae with Triton X-100, or after treatment of the midgut of spinning larvae by freezing and thawing.
The apparent molecular weights of P I and P II trehalases as estimated by Sephadex G-200 chromatography were about 70,000 and 140,000, respectively. The optimum pH was 6.0 for P I and about 5.0 for P II trehalase. TheKm values were about 1.0 mM for P I trehalase and 0.30 mM for P II trehalase.
These results suggest that in feeding larval midgut there are two types of trehalase which are distinguishable from each other by intracellular localization, protein nature and kinetic properties. Furthermore, when the midgut undergoes metamorphosis, the P I enzyme found predominantly in feeding stages seems to be transformed to the P II enzyme via an intermediate form (Ppt-P II) with transitional properties.
KeywordsEnzyme Activation Behaviour Single Peak Protein Nature Particulate Fraction
Unable to display preview. Download preview PDF.
- Andrews, P.: Estimation of the molecular weight of proteins by Sephadex G-200. Biochem. J.91, 222–233 (1964)Google Scholar
- Andrews, P.: The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J.96, 595–606 (1965)Google Scholar
- Dahlman, D.L.: Trehalase activity in tobacco hornworm tissue. Ann. ent. Soc. Amer.63, 1563–1565 (1970)Google Scholar
- Dahlman, D.L.: Age-dependent trehalase activity in adult tobacco hornworm tissues. Insect Biochem.2, 143–149 (1972)Google Scholar
- Droste, H.J., Zebe, E.: Carbohydrasen und Kohlenhydratverdauung im Darmtrakt vonLocusta migratoria. J. Insect Physiol.20, 1639–1657 (1974)Google Scholar
- Fischl, J., Ishay, J., Talmor, N.: Trehalase: Extraction from the midgut of larvae of the oriental hornet and its use in microdetermination of trehalose. Insect Biochem.6, 53–58 (1976)Google Scholar
- Fogal, W.H., Kwain, M.-J.: Metabolism and excretion of nitrogen during metamorphosis and egg production in the sawflyNeodiprion sertifer. J. Insect Physiol.20, 1287–1301 (1974)Google Scholar
- Friedman, S.: Multiple forms of trehalase inPhormia regina. Partial purification, tissue specificities, and some kinetic properties of adult enzymes. Insect Biochem.5, 151–164 (1975)Google Scholar
- Friedman, S., Alexander, S.: Multiple forms of trehalase inPhormia regina. Biochem. biophys. Res. Commun.42, 818–823 (1971)Google Scholar
- Fuwa, H., Tanaka, M., Nishio, H.: Inhibition by trytophan of color development in colorimetric determination of glucose by the glucose oxidase-peroxidase method. Denpunkagaku20, 172–176 (1973) [In Japanese with English summary]Google Scholar
- Gilby, A.R., Wyatt, S.S., Wyatt, G.R.: Trehalases from the cockroach,Blaberus discoidalis: Activation, solubilization and properties of the muscle enzyme and some properties of the intestinal enzyme. Acta biochim. pol.14, 83–100 (1967)Google Scholar
- Gussin, A.E.S., Wyatt, G.R.: Membrane-bound trehalase from cecropia silkmoth muscle. Arch. Biochem. Biophys.112, 626–634 (1965)Google Scholar
- Huber, R.E., Lefebvre, Y.A.: The purification and some properties of soluble trehalase and sucrase fromDrosophila melanogaster. Canad. J. Biochem.49, 1155–1164 (1971)Google Scholar
- Huggett, A.S.G., Nixon, D.A.: Enzymatic determination of blood glucose. Biochem. J.66, 12 (Proc. Biochem. Soc.) (1957)Google Scholar
- Judy, K.J., Gilbert, L.I.: Morphology of the alimentary canal during metamorphosis ofHyalophora cecropia (Lepidoptera: Saturniidae). Ann. ent. Soc. Amer.62, 1438–1446 (1969)Google Scholar
- Laurent, T.C., Killander, J.: A theory of gel filtration and its experimental verification. J. Chromatography14, 317–330 (1964)Google Scholar
- Lefebvre, Y.A., Huber, R.E.: Solubilization, purification, and some properties of trehalase from honeybee (Apis mellifera). Arch. Biochem. Biophys.140, 514–518 (1970)Google Scholar
- Nelson, N.: A photometric adaptation of the Somogyi method for determination of glucose. J. biol. Chem.153, 375–380 (1944)Google Scholar
- Talbot, B.G., Huber, R.E.: Partial purification, stabilization and characterization of adult honey bee midgut trehalase and a new trahalase specific disc gel stain method. Insect Biochem.5, 337–347 (1975)Google Scholar
- Talbot, B.G., Huber, R.E.: An electrophoretic and pH comparison of the soluble trehalases of several insect species. Comp. Biochem. Physiol.53B, 367–369 (1976)Google Scholar
- Talbot, B.G., Muir, J.G., Huber, R.E.: Properties of a free and a solubilized form of bound α,α-trehalase purified from honey bee thorax. Canad. J. Biochem.53, 1106–1117 (1975)Google Scholar
- Tsujita, M.: Histological and cytological studies of the midgut epithelial cell in the silkworm. Bull. seric. Exp. Sta. Tokyo11, 211–293 (1943) [In Japanese]Google Scholar
- Waku, Y., Sumimoto, Y.: Metamorphosis of midgut epithelial cells in the silkworm (Bombyx mori) L. with special regard to the calcium salt deposits in the cytoplasm-I. Light microscopy. Tissue & Cell3, 127–136 (1971)Google Scholar
- Wyatt, G.R.: The biochemistry of sugars and polysaccharides in insects. Adv. Insect Physiol.4, 287–360 (1967)Google Scholar
- Wyatt, G.R.: Biochemistry of insect metamorphosis. In: Metamorphosis, a problem in developmental biology (Etkin, W., Gilbert, L.I., eds.), pp. 143–184. New York: Appleton-Century-Crofts 1968Google Scholar
- Yamashita, O., Hasegawa, K., Seki, M.: Effect of the diapause hormone on trehalase activity in pupal ovaries of the silkworm,Bombyx mori L. Gen. comp. Endocr.18, 515–523 (1972)Google Scholar
- Yamashita, O., Sumida, M., Hasegawa, K.: Developmental changes in midgut trehalase activity and its localization in the silkworm,Bombyx mori. J. Insect Physiol.20, 1079–1085 (1974)Google Scholar
- Yanagawa, H.: Purification and properties of trehalases from larval muscle and midgut of the silkworm,Bombyx mori. Insect Biochem.1, 102–112 (1971)Google Scholar