Summary
Genetically defined alkaline phosphatase (ALP) isozymes from the larval midgut tissues ofBombyx mori were purified and characterized. The membrane-bound form (m-ALP) was solubilized with 1% Triton X-100, then purified by DEAE-Sephacel, Con A-Sepharose 4B and Ultrogel AcA 34 column chromatography. The soluble form (s-ALP) was purified by DEAE-Sephacel, epoxy Toyopearl coupled with phosphonic acid and Ultrogel AcA 34 column chromatography. About 840- and 650-fold purification were achieved for m-ALP and s-ALP, respectively, and both ALPs were homogeneous as judged by poly-acrylamide gel electrophoresis. Both forms were found to be similar (MW=68 000 in gel permeation chromatography, and a single subunit as a monomer in denaturing SDS-polyacrylamide gels with MW=58 000 for m-ALP and MW=61 000 for s-ALP). The pH optima of ALPs were shown to lie at 10.9 (m-ALP) and 9.8 (s-ALP), the former being extremely stable even in pH 10–12 which accords with the physiological milieu inBombyx midgut lumen. The Km values of the m-ALP and s-ALP forp-nitrophenyl phosphate were 1.99 and 1.49 mM, respectively. Both ALPs had a similar substrate specificity.l-Cysteine strongly inhibited both ALPs, but inhibitory effects ofl-phenylalanine,l-homoarginine andl-leucine were undetectable for s-ALP and very weak for m-ALP. A comparison of enzymatic properties on two ALPs suggested that each isozyme plays different roles.
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Abbreviations
- m-ALP :
-
membrane-bound alkaline phosphatase
- s-ALP :
-
soluble alkaline phosphatase
References
Bensadoun A, Weinstein D (1976) Assay of proteins in the presence of interfering materials. Anal Biochem 70:241–250
Besman M, Coleman JE (1985) Isozymes of bovine intestinal alkaline phosphatase. J Biol Chem 260:11190–11193
Cathala G, Brunel C, Chappelet-Tordo D, Lazdunski M (1975) Bovine kidney alkaline phosphatase: purification, subunit structure, and metalloenzyme properties. J Biol Chem 250:6040–6045
Davis BJ (1964) Disc electrophoresis. II. Method and application to human serum protein. Ann NY Acad Sci 131:404–427
Dow JAT (1984) Extremely high pH in biological systems: a model for carbonate transport. Am J Physiol 246:R633-R635
Eguchi M (1975) Alkaline phosphatase isozymes in the alimentary canal of the silkworm. In: Markert CL (ed) Isozymes. I. Molecular structure. Academic Press, New York, pp 315–332
Eguchi M, Yamashita Y (1977) Genetic study on alkaline phosphatases of the midgut tissue and digestive fluid of the silkworm,Bombyx mori L. J Seric Sci Jpn 46:515–520
Eguchi M, Sawaki M, Suzuki Y (1972a) Multiple forms of midgut alkaline phosphatase in the silkworm: separation and comparison of two isozymes. Insect Biochem 2:167–174
Eguchi M, Sawaki M, Suzuki Y (1972b) Multiple forms of midgut alkaline phosphatase in the silkworm: new band formation and the relationship between the midgut and digestive fluid enzymes. Insect Biochem 2:297–304
Eguchi M, Kuriyama K, Daimon H (1986) High alkalinity and function of proteases of digestive juice from the silkworm,Bombyx mori. J Seric Sci Jpn 55:46–53
Ehle H, Bublitz R, Horn A (1985) Intralumental alkaline phosphatase of the calf intestine. Biomed Biochim Acta 44:223–233
Ey PL, Ferber E (1977) Calf thymus alkaline phosphatase. I. Properties of the membrane-bound enzyme. Biochim Biophys Acta 480:403–416
Giordana, B, Sacchi F (1978) Cellular ionic concentrations in the midgut of two larvae of lepidoptera in vivo and in vitro. Comp Biochem Physiol 59A:17–20
Hanna SD, Mircheff AK, Wright EM (1979) Alkaline phosphatase of basal lateral and brush border plasma membranes from intestinal epithelium. J Supramol Struct 11:451–466
Harper RA, Armstrong FB (1972) Alkaline phosphatase ofDrosophila melanogaster. I. Partial purification and characterization. Biochem Genet 6:75–82
Harper RA, Armstrong FB (1973) Alkaline phosphatase ofDrosophila melanogaster. II. Biochemical comparison among four allelic forms. Biochem Genet 10:29–38
Holmgren PÅ, Stigbrand T (1976) Purification and partial characterization of two genetic variants of placental alkaline phosphatase. Biochem Genet 14:777–789
Horie Y (1958) Physiological studies on the alimentary canal of the silkworm,Bombyx mori. I. Alkaline phosphatase of the midgut. Bull Seric Exp Stn Jpn 15:275–292
Hua JC, Berger J, Pan YCE, Hulmes JD, Udenfriend S (1986) Partial sequencing of human adult, human fetal, and bovine intestinal alkaline phosphatases: comparison with the human placental and liver isozymes. Proc Natl Acad Sci USA 83:2368–2372
Komoda T, Sakagishi Y, Sekine T (1981) Multiple forms of human intestinal alkaline phosphatase: chemical and enzymatic properties, and circulating clearances of the fast- and slow-moving enzymes. Clin Chim Acta 117:167–187
Landt M, Boltz SC, Butler LG (1978) Alkaline phosphatase: affinity chromatography and inhibition by phosphonic acids. Biochem 17:915–919
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
McComb RB, Bowers GN, Posen S (1979) Alkaline phosphatase. Plenum Press, New York
Nakamura T (1940) The phosphorus metabolism during the growth of the animal. The behavior of various phosphates and phosphoric acid compounds ofBombyx mori L. during growth. Mitt Med Akad Kioto 28:335–416
Sridhara S, Bhat JV (1963) Alkaline and acid phosphatases of the silkworm,Bombyx mori L. J Insect Physiol 9:693–701
Sugai E (1957) Alkaline phosphatase in the midgut of the silkworm larva,Bombyx mori. J Seric Sci Jpn 26:35–39
Takahashi Y (1955) Measurement of inorganic and creatine phosphates, and phosphoamidase, creatine phosphokinase activities in the sperm of the pig. J Jpn Biochem Soc 26:690–698
Wieczorek H, Wolfersberger MG, Cioffi M, Harvey WR (1986) Cation-stimulated ATPase activity in purified plasma membranes from tobacco hornworm midgut. Biochim Biophys Acta 857:271–281
Wolfersberger MG (1984) Enzymology of plasma membranes of insect intestinal cells. Am Zool 24:187–197
Yedlin ST, Young GP, Seetharam B, Seetharam S, Alpers DH (1981) Characterization and comparison of soluble and membranous forms of intestinal alkaline phosphatase from the suckling rat. J Biol Chem 256:5620–5626
Yoshitake N, Eguchi M, Akiyama M (1966) Genetic control on the alkaline phosphatase of the midgut in the silkworm,Bombyx mori L. J Seric Sci Jpn 35:1–7
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Okada, N., Azuma, M. & Eguchi, M. Alkaline phosphatase isozymes in the midgut of silkworm: purification of high pH-stable microvillus and labile cytosolic enzymes. J Comp Physiol B 159, 123–130 (1989). https://doi.org/10.1007/BF00691732
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DOI: https://doi.org/10.1007/BF00691732