Purification, cloning, and expression of an apyrase from the bed bug Cimex lectularius. A new type of nucleotide-binding enzyme.
An enzyme that hydrolyzes the phosphodiester bonds of nucleoside tri- and diphosphates, but not monophosphates, thus displaying apyrase (EC 3.6.1.5) activity, was purified from salivary glands of the bed bug, Cimex lectularius. The purified C. lectularius apyrase was an acidic protein with a pI of 5.1 and molecular mass of approximately 40 kDa that inhibited ADP-induced platelet aggregation and hydrolyzed platelet agonist ADP with specific activity of 379 units/mg protein. Amplification of C. lectularius cDNA corresponding to the N-terminal sequence of purified apyrase produced a probe that allowed identification of a 1.3 kilobase pair cDNA clone coding for a protein of 364 amino acid residues, the first 35 of which constituted the signal peptide. The processed form of the protein was predicted to have a molecular mass of 37.5 kDa and pI of 4.95. The identity of the product of the cDNA clone with native C. lectularius apyrase was proved by immunological testing and by expressing the gene in a heterologous host. Immune serum made against a synthetic peptide with sequence corresponding to the C-terminal region of the predicted cDNA clone recognized both C. lectularius apyrase fractions eluted from a molecular sieving high pressure liquid chromatography and the apyrase active band from chromatofocusing gels. Furthermore, transfected COS-7 cells secreted a Ca2+-dependent apyrase with a pI of 5.1 and immunoreactive material detected by the anti-apyrase serum. C. lectularius apyrase has no significant sequence similarity to any other known apyrases, but homologous sequences have been found in the genome of the nematode C. elegans and in mouse and human expressed sequence tags from fetal and tumor EST libraries.
Valenzuela
J G
JG
Medical Entomology Section, Laboratory of Parasitic Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0425, USA.
Charlab
R
R
Galperin
M Y
MY
Ribeiro
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001081:Apyrase / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D001511:Bedbugs / Q000201:enzymology*; D019556:COS Cells; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008969:Molecular Sequence Data; D012469:Salivary Glands / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet
D001081:Apyrase