In brain and many other tissues, Type I inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase is the major isozyme hydrolysing the calcium-mobilizing second messenger InsP3. We recently reported the cloning and expression of dog thyroid InsP3 5-phosphatase. During the course of this cloning, screening of a human brain eDNA library allowed us to isolate a eDNA clone D1 with 91% sequence identity with the thyroid sequence. When clone DI was expressed in Escherichia coli, the fusion protein had InsP3 5-phosphatase activity. Mr estimates of the recombinant enzyme made by immunodetection, activity assay after SDS/PAGE or silver staining were consistent with the calculated molecular mass. In situ hybridization on human cerebellum sections localised the mRNA for this enzyme to the Purkinje cells.
Ins(1 ,4,5)P3 5-phosphatase catalyses the dephosphorylation of Ins(1,4,5)P3 in the 5 position. At 1 pM Ins(1,4,5)P3, 10-15% of total activity of a bovine brain homogenate was measured in the soluble fraction, whereas 85 -90% was in the particulate fraction. Particulate activity could be solubilized by cholate or, to a lower extent, by 2 M KCI. Two soluble enzymes (type I and type 11) could be fractionated by DEAE-Sephacel chromatography. Soluble activities have been further purified by blue-Sepharose, Sephacryl S-200 and phosphocellulose chromatography. Specific activities reached 10-30 pmol . min-' mg protein-' for type I and were 10-20 times lower for type 11. Type I and type 11 Ins(1,4,5)P3 5-phosphatase displayed different K,,, values and molecular masses, as estimated by gel filtration. Type I dephosphorylated both Ins(1,4,5)P3 and Ins(1 ,3,4,5)P4; in contrast, type I1 specifically dephosphorylated Ins(1 ,4,5)P3 but not Ins(1,3,4,5)P4. Type 1 lns(1,4,5)P3 5-phosphatase eluted as a single peak of activity with an apparent molecular mass of 51 kDa when gel filtration was performed in the presence of cholate. This molecular mass is identical to the molecular mass estimated for the particulate Ins(1 ,4,5)P3 5-phosphatase that was solubilized by cholate. K , values for Ins(1,4,5)P3 and Ins(1 ,3,4,5)P4 obtained with type I Ins(1,4,5)P3 5-phosphatase were 11 pM and 1 pM, respectively. Similar values were obtained with particulate Ins(1 ,4,5)P3 5-phosphatase. In conclusion, the catalytic domains of type I and particulate Ins(1 ,4,5)P3 5-phosphatase activity may be very similar, if not identical, but different from type I1 phosphatase.Agonist-stimulated hydrolysis of phosphatidylinositol 4,s-bisphosphate produces two signal molecules, Ins(1 ,4,5)P3 and diacylglycerol. Ins( 1 ,4,5)P3, a second messenger for mobilizing intracellular calcium [l, 21, has been shown to be metabolized to Ins(1,4)P2 or phosphorylated to Ins(1,3,4,5)P4 [3]. These reactions are catalyzed by an Ins(1,4,5)P3 5-phosphomonoesterase or phosphatase and an Ins(1 ,4,5)P3 3-kinase, respectively. These two enzymes represent potential control points at which both Ins(1 ,4,5)P3 and Ins(1,3,4,5)P4 concentrations could be regulated.Ins(1,4,5)P3 5-phosphatase was originally described in human erythrocyte membranes, where its activity is M g Z fdependent and inhibited by 2,3-bisphosphoglycerate [4]. The product of dephosphorylation is Ins(1 ,4)P2. The same enzyme specifically removes the 5 phosphate from Ins( 1,3,4,5)P4 to form lns(1,3,4)P3, the most abundant InsP3 isomer in many cells [S]. The Ins(1,4,5)P3 5-phosphatase is found in the soluble fractions of most tissues, although the membrane-bound activity is often quantitatively more important 16-91. A single enzyme form was purified from the soluble fraction of human platelets by the group of Majerus [lo]. Its apparent molecular mass was 38 kDa as determined by gel filtration. Both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 appeared to be substrates for the platelet Ins(1 ,4,5)P3 5-phosphatase [I I]. In platelets, ...
Carbachol, through a muscarinic receptor, thyrotropin-releasing hormone (TRH), prostaglandin F2 alpha (PGF2 alpha), bradykinin, and adenosine triphosphate (ATP) increased the apparent [Ca2+]i (intracellular free Ca2(+)-concentration) of dog thyrocytes in primary culture. The [Ca2+]i measured by the Quin-2 technique rose immediately after the addition of the agonists and reached a maximal value after less than 30 seconds. Afterwards, the [Ca2+]i declined to a plateau higher than the basal level when the cells were triggered with carbachol. By contrast, in most experiments with PGF2 alpha and in the case of bradykinin, TRH, and ATP, the [Ca2+]i returned to the basal value. If the extracellular Ca2+ was chelated by excess of EGTA, the addition of all agents caused a sharp reduced transient rise in the [Ca2+]i followed by a decline of the [Ca2+]i often below the basal level (especially in the case of carbachol). It is suggested that the first transient phase of these responses is due at least in part to the mobilisation of Ca2+ from intracellular stores whereas the second sustained phase of the response to carbachol mainly originates from an increased Ca2+ influx into the thyrocytes. Carbachol, bradykinin, TRH, PGF2 alpha, and ATP also increased generation of inositol phosphates in dog thyrocytes. This effect was sustained when the cells were triggered with carbachol and was more transient with bradykinin, TRH, PGF2 alpha, or ATP. All these agents and the phorbdester TPA as well as forskolin enhanced to various extent the thyrocyte H2O2 generation. This enhancement was severely reduced in the absence of extracellular Ca2+ and was mimicked by Ca2+ ionophores in the presence of extracellular Ca2+ especially in synergy with protein kinase C activators. These data suggest that the dog thyrocyte H2O2 generation, the limiting step of the thyroid hormone synthesis, is modulated by carbachol, TRH, PGF2 alpha, bradykinin, and ATP through their action on the Ca2(+)-phosphatidylinositol cascade.
In brain and many other tissues, type I inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase is the major isoenzyme hydrolysing the calcium-mobilizing second messenger InsP3. This protein has been purified to apparent homogeneity from a crude soluble fraction of bovine brain, yielding a single major protein band with a molecular mass of 43 kDa after SDS/PAGE. This material was used to determine internal microsequences. A partial DNA sequence has been amplified by PCR by using degenerate primers deduced from two protein sequences (FKAKKYKKV and DENYKSQE). A cDNA clone (BVCT) was isolated by screening a dog thyroid cDNA library. The encoded protein of 412 amino acids has a calculated molecular mass of 47,681 Da. Peptide sequences generated from the bovine brain enzyme were found to be 96% conserved compared with the dog thyroid protein. When clone BVCT was expressed in Escherichia coli, the recombinant protein was shown to hydrolyse both InsP3 and inositol 1,3,4,5-tetrakisphosphate, with apparent Km values of 28 and 3 microM respectively. Enzyme activity was inhibited by EDTA and 2,3-bisphosphoglycerate, both inhibitors of native InsP3 5-phosphatase, but not by EGTA and LiCl, as previously shown for the bovine brain enzyme. Our data show the cloning of type I InsP3 5-phosphatase which, interestingly, does not share any significant sequence identity with the previously cloned type III isoenzyme.
In bovine brain, two soluble inositol-1,4,5-trisphosphate (InsP,) 5-phosphatases, which catalyse the dephosphorylation of InsP, to inositol 1 ,4-bisphosphate, have been separated by DEAE-Sephacel. Type I, i.e. the first eluted enzyme, is the main soluble form and is reminiscent of the membranebound enzyme by multiple criteria. Type I was purified to apparent homogeneity by a method involving chromatography on DEAE-Sephacel, Blue-Sepharose, Sephacryl S-200, phosphocellulose, and Cia HPLC. A single protein band of 42-43 kDa was identified by SDS/PAGE, corresponding to the peak of maximal activity. InsP, 5-phosphatase was purified to apparent homogeneity to a final yield of 45 -50 pg protein. The minimal estimate value of the VmaX for InsP, 5-phosphatase was in the range 20-35 pmol . min-' . mg protein-'.
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