Abstract
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.
Highlights
The phosphatidylinositol signaling pathway involves a complex scheme in which cells use a series of kinases and phosphatases to interconvert the six known inositol lipids and the more than 20 inositol phosphates that exist in eukaryotic cells (1)
Inositol 1,3,4-trisphosphate (Ins(1,3,4)P3)1 is at a branch point in inositol phosphate metabolism
Because the Ins(1,3,4)P3 5/6-kinase enzyme is at a branch point in metabolism leading to multiple different end products, it is likely to be regulated by its various end products
Summary
Materials—Heparin-agarose type I, bacterial ATP grade I, pepstatin A, soybean trypsin inhibitor type IIS, leupeptin, bestatin, diisopropyl fluorophosphate, benzamidine, phenylmethylsulfonyl fluoride, Coomassie Blue R250, isopropyl-1-thio--D-galactopyranoside, calmodulin, calmodulin-agarose, phytic acid (inositol hexaphosphoric acid), CHAPS, dithiothreitol, and ammonium sulfate were obtained from Sigma. Lysyl endopeptidase was purchased from Wako Chemicals (Dallas, TX). Calpain inhibitors I and II were from Calbiochem. Frozen calf brains were supplied by By Prod Corporation (St. Louis, MO). A Mono Q HR 5/5 fast protein liquid chromatography column was purchased from Pharmacia Biotech. Inc. Inositol 1,3,4-trisphosphate, D-[inositol-1-3H(N)], 21 Ci/ mmol, inositol 1,4,5-trisphosphate, D-[inositol-1-3H(N)], 15–30 Ci/mmol, and Colony/Plaque Screen filters were obtained from DuPont NEN. Escherichia coli expressing recombinant inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) 3-kinase were generously provided by S. Dowex AG 1-X8 (formate form), protein assay dye reagent concentrate, and low molecular weight markers were obtained from Bio-Rad. Dowex AG 1-X8 (formate form), protein assay dye reagent concentrate, and low molecular weight markers were obtained from Bio-Rad. [32P]
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