The isolation and characterization of cDNA encoding human and rat brain inositol polyphosphate 4-phosphatase.
Inositol polyphosphate 4-phosphatase, an enzyme of the inositol phosphate signaling pathway, catalyzes the hydrolysis of the 4-position phosphate of inositol 3,4-bisphosphate, inositol 1,3,4-trisphosphate, and phosphatidylinositol 3,4-bisphosphate. The amino acid sequences of tryptic and CNBr peptides of the enzyme isolated from rat brain were determined. Degenerate oligonucleotide primers based on this sequence were used to amplify a 74-base pair polymerase chain reaction product. This product was used to isolate a 5607-base pair composite cDNA, which had an open reading frame encoding a protein with 939 amino acids with a predicted molecular mass of 105,588 Da. The rat brain polymerase chain reaction product was used as a probe to isolate a human brain cDNA that predicts a protein with 938 amino acids and a molecular mass of 105,710 Da. Remarkably, the human and rat proteins were 97% identical. Recombinant rat protein expressed in Escherichia coli catalyzed the hydrolysis of all three substrates of the 4-phosphatase. Northern blot hybridization indicates that the 4-phosphatase is widely expressed in rat tissues with the highest levels of expression occurring in brain, heart, and skeletal muscle. Polyclonal antiserum directed against the carboxyl terminus of the 4-phosphatase immunoprecipitated > 95% of the 4-phosphatase activity in crude homogenates of rat brain, heart, skeletal muscle, and spleen, suggesting that this enzyme accounts for the 4-phosphate activity present in rat tissues. This antiserum also immunoprecipitated the 4-phosphatase from human platelet sonicates.
Norris
F A
FA
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Auethavekiat
V
V
Majerus
P W
PW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001792:Blood Platelets / Q000201:enzymology; D015152:Blotting, Northern; D001921:Brain / Q000201:enzymology*; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D007295:Inositol Phosphates / Q000378:metabolism*; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014018:Tissue Distribution
D018076:DNA, Complementary; D007295:Inositol Phosphates; D011994:Recombinant Proteins; C058097:inositol 1,3,4-trisphosphate; D010744:Phosphoric Monoester Hydrolases; C053153:phosphatidylinositol-3,4-bisphosphate 4-phosphatase