title: Cloning and expression of vacuolar proton-pumping ATPase subunits in the follicular epithelium of the bullfrog endolymphatic sac.
abstract: In an investigation aimed at clarifying the mechanism of crystal dissolution of the calcium carbonate lattice in otoconia (the mineral particles embedded in the otolithic membrane) of the endolymphatic sac (ELS) of the bullfrog, cDNAs encoding the A- and E-subunits of bullfrog vacuolar proton-pumping ATPase (V-ATPase) were cloned and sequenced. The cDNA of the A-subunit consisted of an 11-bp 5'-untranslated region (UTR), a 1,854-bp open reading frame (ORF) encoding a protein comprising 617 amino acids with a calculated molecular mass of 68,168 Da, and a 248-bp 3'-UTR followed by a poly(A) tail. The cDNA of the E-subunit consisted of a 72-bp 5'-UTR, a 681-bp ORF encoding a protein of 226 amino acids with a calculated molecular mass of 26,020 Da, and a 799-bp 3'-UTR followed by a poly(A) tail. Western blot and immunofluorescence analyses using specific anti-peptide antisera against the V-ATPase A- and E-subunits revealed that these subunits were present in the ELS, urinary bladder, skin, testes, and kidneys. In the ELS, positive cells were scattered in the follicular epithelium which, as revealed by electron microscopy, corresponds to the location of mitochondria-rich cells. These findings suggest that V-ATPase, including the A- and E-subunits, exists in mitochondria-rich cells of the ELS, which might be involved in dissolution of the calcium carbonate crystals in the lumen of the ELS.
authors.affiliation: Department of Biology, Faculty of Science, Shizuoka University, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Yajima
authors.forename: Makoto
authors.initials: M
authors.lastname: Kubota
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakakura
authors.forename: Takahiro
authors.initials: T
authors.lastname: Hasegawa
authors.forename: Nobuto
authors.initials: N
authors.lastname: Katagiri
authors.forename: Hideaki
authors.initials: H
authors.lastname: Tomura
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Sasayama
authors.forename: Masakazu
authors.initials: M
authors.lastname: Suzuki
authors.forename: Shigeyasu
authors.initials: S
authors.lastname: Tanaka
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry*; D004712:Endolymphatic Sac / Q000201:enzymology* / Q000648:ultrastructure; D004848:Epithelium / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D011892:Rana catesbeiana / Q000235:genetics / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D025262:Vacuolar Proton-Translocating ATPases