Inorganic pyrophosphatase in the roundworm Ascaris and its role in the development and molting process of the larval stage parasites.
Inorganic pyrophosphatase (PPase) is an important enzyme that catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into ortho-phosphate (Pi). We report here the molecular cloning and characterization of a gene encoding the soluble PPase of the roundworm Ascaris suum. The predicted A. suum PPase consists of 360 amino acids with a molecular mass of 40.6 kDa and a pI of 7.1. Amino acid sequence alignment and phylogenetic analysis indicates that the gene encodes a functional Family I soluble PPase containing features identical to those of prokaryotic, plant and animal/fungal soluble PPases. The Escherichia coli-expressed recombinant enzyme has a specific activity of 937 micro mol Pi.min-1.mg-1 protein corresponding to a kcat value of 638 s-1 at 55 degrees C. Its activity was strongly dependent on Mg2+ and was inhibited by Ca2+. Native PPases were expressed in all developmental stages of A. suum. A homolog was also detected in the most closely related human and dog roundworms A. lumbricoides and Toxocara canis, respectively. The enzyme was intensely localized in the body wall, gut epithelium, ovary and uterus of adult female worms. We observed that native PPase activity together with development and molting in vitro of A. suum L3 to L4 were efficiently inhibited in a dose-dependent manner by imidodiphosphate and sodium fluoride, which are potent inhibitor of both soluble- and membrane-bound H+-PPases. The studies provide evidence that the PPases are novel enzymes in the roundworm Ascaris, and may have crucial role in the development and molting process.
Islam
M Khyrul
MK
Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural Research Organization, 3-1-5, Kannondai, Tsukuba, Ibaraki, Japan.
Miyoshi
Takeharu
T
Kasuga-Aoki
Harue
H
Isobe
Takashi
T
Arakawa
Takeshi
T
Matsumoto
Yasunobu
Y
Tsuji
Naotoshi
N
D000595:Amino Acid Sequence; D000818:Animals; D017165:Ascaris suum / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development*; D002648:Child; D004791:Enzyme Inhibitors / Q000378:metabolism; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D018978:Molting / Q000502:physiology*; D010802:Phylogeny; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012969:Sodium Fluoride / Q000378:metabolism
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D012969:Sodium Fluoride; D011755:Pyrophosphatases