Genetically defined membrane-bound and soluble alkaline phosphatases of the silkworm: their discrete localization and properties.
In the midgut tissue of the silkworm, Bombyx mori, alkaline phosphatase isozymes, membrane-bound (m-ALP) and soluble (s-ALP) forms are controlled by non-allelic genes on the same chromosome. We purified and characterized both ALPs to elucidate their possible functions and to compare with mammalian ALPs. Both forms were found to be similar Mr = 68,000 in gel permeation chromatography and as a single subunit as a monomer in SDS-polyacrylamide gel electrophoresis with Mr = 58,000 for m-ALP and Mr = 61,000 for s-ALP. The pH optima of ALPs were 10.9 (m-ALP) and 9.8 (s-ALP), and the former was extremely stable even in pH 10-12 which accords with the physiological milieu in Bombyx midgut lumen. Both ALPs had similar substrate specificities. L-cysteine inhibited strongly both ALPs, but inhibitory effects of L-phenylalanine, L-homoarginine, and L-leucine were undetectable for s-ALP and very weak for m-ALP. The antibody raised against purified m-ALP recognized m-ALP but not purified s-ALP and vice versa. Rocket-immunoassay showed that m-ALP was distributed in similar levels along the length of midgut except for the most anterior portion. Seventy percent of s-ALP activity existed in the last one-third of midgut. Immunohistochemical study revealed that the m-ALP was localized at the brush border of columnar cells in the middle and posterior midgut epithelia. In contrast, the s-ALP was localized at the apical surface of goblet cells through the length of midgut. We detected ATPase activity in the purified s-ALP preparation; Mg2+ was essential for the ATPase activity and the activity also increased with KHCO3 but not with KCl. The solubilization test of m- ALP with various agents was attempted and the relationship between m-ALP and the digestive fluid-ALP was discussed.
Eguchi
M
M
Department of Applied Biology, Kyoto Institute of Technology, Japan.
Azuma
M
M
Yamamoto
H
H
Takeda
S
S
D000469:Alkaline Phosphatase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D012831:Bombyx / Q000201:enzymology* / Q000235:genetics; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D005260:Female; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008970:Molecular Weight
D007527:Isoenzymes; D000469:Alkaline Phosphatase