Isolation of inositol 1,3,4-trisphosphate 5/6-kinase, cDNA cloning and expression of the recombinant enzyme.
Inositol 1,3,4-trisphosphate 5/6-kinase was purified 12,900-fold from calf brain using chromatography on heparin-agarose and affinity elution with inositol hexakisphosphate. The final preparation contained proteins of 48 and 36-38 kDa. All of these proteins had the same amino-terminal sequence and were enzymatically active. The smaller species represent proteolysis products with carboxyl-terminal truncation. The Km of the enzyme for inositol 1,3,4-trisphosphate was 80 nM with a Vmax of 60 nmol of product/min/mg of protein. The amino acid sequence of the tryptic peptide HSKLLARPAGGLVGERTCNAXP matched the protein sequence encoded by a human expressed sequence tag clone (GB T09063) at 16 of 22 residues. The expressed sequence tag clone was used to screen a human fetal brain cDNA library to obtain a cDNA clone of 1991 base pairs (bp) that predicts a protein of 46 kDa. The clone encodes the amino-terminal amino acid sequence obtained from the purified calf brain preparation, suggesting that it represents its human homologue. The cDNA was expressed as a fusion protein in Escherichia coli and was found to have inositol 1,3,4-trisphosphate 5/6-kinase activity. Remarkably, both the purified calf brain and recombinant proteins produced both inositol 1,3,4,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate as products in a ratio of 2.3-5:1. This finding proves that a single kinase phosphorylates inositol in both the D5 and D6 positions. Northern blot analysis identified a transcript of 3.6 kilobases in all tissues with the highest levels in brain. The composite cDNA isolated contains 3054 bp with a poly(A) tail, suggesting that 500-600 bp of 5' sequence remains to be identified.
Wilson
M P
MP
Division of Hematology-Oncology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Majerus
P W
PW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism*; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis
D018076:DNA, Complementary; D007295:Inositol Phosphates; D011994:Recombinant Proteins; C058097:inositol 1,3,4-trisphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor)