A novel mammalian lithium-sensitive enzyme with a dual enzymatic activity, 3'-phosphoadenosine 5'-phosphate phosphatase and inositol-polyphosphate 1-phosphatase.

We report the molecular cloning in Rattus norvegicus of a novel mammalian enzyme (RnPIP), which shows both 3'-phosphoadenosine 5'-phosphate (PAP) phosphatase and inositol-polyphosphate 1-phosphatase activities. This enzyme is the first PAP phosphatase characterized at the molecular level in mammals, and it represents the first member of a novel family of dual specificity enzymes. The phosphatase activity is strictly dependent on Mg2+, and it is inhibited by Ca2+ and Li+ ions. Lithium chloride inhibits the hydrolysis of both PAP and inositol-1,4-bisphosphate at submillimolar concentration; therefore, it is possible that the inhibition of the human homologue of RnPIP by lithium ions is related to the pharmacological action of lithium. We propose that the PAP phosphatase activity of RnPIP is crucial for the function of enzymes sensitive to inhibition by PAP, such as sulfotransferase and RNA processing enzymes. Finally, an unexpected connection between PAP and inositol-1,4-bisphosphate metabolism emerges from this work.     

A novel target of lithium therapy

Phosphatases converting 3'-phosphoadenosine 5'-phosphate (PAP) into adenosine 5'-phosphate are of fundamental importance in living cells as the accumulation of PAP is toxic to several cellular systems. These enzymes are lithium-sensitive and we have characterized a human PAP phosphatase as a potential target of lithium therapy. A cDNA encoding a human enzyme was identified by data base screening, expressed in Escherichia coli and the 33 kDa protein purified to homogeneity. The enzyme exhibits high affinity for PAP (K(m)<1 microM) and is sensitive to subtherapeutic concentrations of lithium (IC(50)=0.3 mM). The human enzyme also hydrolyzes inositol-1, 4-bisphosphate with high affinity (K(m)=0.4 microM), therefore it can be considered as a dual specificity enzyme with high affinity (microM range) for both PAP and inositol-1,4-bisphosphate. Hydrolysis of inositol-1,4-bisphosphate was also inhibited by lithium (IC(50)=0.6 mM). Thus, we present experimental evidence for a novel target of lithium therapy, which could explain some of the side effects of this therapy.     

X-ray structure of yeast Hal2p, a major target of lithium and sodium toxicity, and identification of framework interactions determining cation sensitivity

The product of the yeast HAL2 gene (Hal2p) is an in vivo target of sodium and lithium toxicity and its overexpression improves salt tolerance in yeast and plants. Hal2p is a metabolic phosphatase which catalyses the hydrolysis of 3'-phosphoadenosine-5'-phosphate (PAP) to AMP. It is, the prototype of an evolutionarily conserved family of PAP phosphatases and the engineering of sodium insensitive enzymes of this group may contribute to the generation of salt-tolerant crops. We have solved the crystal structure of Hal2p in complex with magnesium, lithium and the two products of PAP hydrolysis, AMP and Pi, at 1.6 A resolution. A functional screening of random mutations of the HAL2 gene in growing yeast generated forms of the enzyme with reduced cation sensitivity. Analysis of these mutants defined a salt bridge (Glu238 ellipsis Arg152) and a hydrophobic bond (Va170 ellipsis Trp293) as important framework interactions determining cation sensitivity. Hal2p belongs to a larger superfamily of lithium-sensitive phosphatases which includes inositol monophosphatase. The hydrophobic interaction mutated in Hal2p is conserved in this superfamily and its disruption in human inositol monophosphatase also resulted in reduced cation sensitivity.     

Structural enzymology of Li(+)-sensitive/Mg(2+)-dependent phosphatases

Li(+)-sensitive/Mg(2+)-dependent phosphatases have attracted considerable attention since they have been proposed as targets for lithium therapy in the treatment of manic-depressive patients. The members of this enzyme superfamily display low levels of sequence identity while possessing a common fold and active site. Extensive structural and biochemical data demonstrate the direct involvement of two metal ions in catalysis, and show that lithium exerts its inhibitory action by blocking the products at the active site. By exploiting the different inhibitory properties of magnesium and calcium, we have been able to solve the X-ray structures of the Li(+)-sensitive/Mg(2+)-dependent 3'-phosphoadenosine-5'-phosphatase in complex with its substrate and with its products. The structural comparison of these complexes provides a 3D picture of the different stages of the catalytic cycle. This gives new insights into the understanding of the biological function of this group of enzymes and their lithium inhibition, and should assist in the design of improved inhibitors of therapeutic value.     

Inositol polyphosphate 1-phosphatase from calf brain. Purification and inhibition by Li+, Ca2+, and Mn2+

We recently identified an enzyme which we have designated inositol polyphosphate 1-phosphatase that hydrolyzes both inositol 1,3,4-trisphosphate (Ins-1,3,4-P3) and inositol 1,4-bisphosphate (Ins-1,4-P2), yielding inositol 3,4-bisphosphate and inositol 4-phosphate, respectively, as products (Inhorn, R. C., Bansal, V.S., and Majerus, P.W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2170-2174). We have now purified the inositol polyphosphate 1-phosphatase 3600-fold from calf brain supernatant. The purified enzyme has an apparent molecular mass of 44,000 daltons as determined by gel filtration and is free of other inositol phosphate phosphatase activities. The enzyme hydrolyzes Ins-1,4-P2 with an apparent Km of approximately 4-5 microM, while it degrades Ins-1,3,4-P3 with an apparent Km of approximately 20 microM. The enzyme hydrolyzes these substrates at approximately the same maximal velocity. Inositol polyphosphate 1-phosphatase shows a sigmoidal dependence upon magnesium ion, with 0.3 mM Mg2+ causing half-maximal stimulation. A Hill plot of the data is linear with a value of n = 1.9, suggesting that the enzyme binds magnesium cooperatively. Calcium and manganese inhibit enzyme activity, with 50% inhibition at approximately 6 microM. Lithium inhibits Ins-1,4-P2 hydrolysis uncompetitively with a Ki of approximately 6 mM. This mechanism of lithium inhibition is similar to that observed for the inositol monophosphate phosphatase (originally designated myo-inositol-1-phosphatase; Hallcher, L.M., and Sherman, W.R. (1980) J. Biol. Chem. 255, 10896-10901), suggesting that these two enzymes are related. Lithium also inhibits Ins-1,3,4-P3 hydrolysis with an estimated Ki of 0.5-1 mM.     

Cloning and characterization of a mammalian lithium-sensitive bisphosphate 3'-nucleotidase inhibited by inositol 1,4-bisphosphate

Discovery of a structurally conserved metal-dependent lithium-inhibited phosphomonoesterase protein family has identified several potential cellular targets of lithium as used to treat manic depression. Here we describe identification of a novel family member using a "computer cloning" strategy. Human and murine cDNA clones encoded proteins sharing 92% identity and were highly expressed in kidney. Native and recombinant protein harbored intrinsic magnesium-dependent bisphosphate nucleotidase activity (BPntase), which removed the 3'-phosphate from 3'-5' bisphosphate nucleosides and 3'-phosphoadenosine 5'-phosphosulfate with Km and Vmax values of 0.5 microM and 40 micromol/min/mg. Lithium uncompetitively inhibited activity with a Ki of 157 microM. Interestingly, BPntase was competitively inhibited by inositol 1,4-bisphosphate with a Ki of 15 microM. Expression of mammalian BPntase complemented defects in hal2/met22 mutant yeast. These data suggest that BPntase's physiologic role in nucleotide metabolism may be regulated by inositol signaling pathways. The presence of high levels of BPntase in the kidney are provocative in light of the roles of bisphosphorylated nucleotides in regulating salt tolerance, sulfur assimilation, detoxification, and lithium toxicity. We propose that inhibition of human BPntase may account for lithium-induced nephrotoxicity, which may be overcome by supplementation of current therapeutic regimes with inhibitors of nucleotide biosynthesis, such as methionine.     

Purification and properties of inositol-1,4-bisphosphate 4-phosphohydrolase from rat brain

Inositol-1,4-bisphosphate 4-phosphohydrolase (inositol-1,4-bisphosphatase) was highly purified from a soluble fraction of rat brain. On SDS-polyacrylamide gel electrophoresis, the purified enzyme gave a single protein band and its molecular weight was estimated to be 42000. The isoelectric point of the enzyme was 4.3. The enzyme specifically hydrolyzed the 4-phosphomonoester linkage of inositol 1,4-bisphosphate. The Km value for inositol 1,4-bisphosphate was 30 microM, and it required Mg2+ for activity. Ca2+ was a competitive inhibitor with a Ki value of 60 microM as regards the Mg2+ binding. Li+, which is known to be a strong inhibitor of inositol 1-phosphatase (EC 3.1.3.25), inhibited the enzyme activity and caused 50% inhibition at a concentration of 1 mM (IC50 = 1 mM). Li+ was an uncompetitive inhibitor of substrate binding with a Ki value of 0.6 mM. These inhibitory parameters of Li+ were quite similar to those for inositol 1-phosphatase (IC50 = 1 mM, Ki = 0.3 mM). Thus, the effect of Li+ on decreasing the free inositol level with a subsequent decrease in agonist-sensitive phosphoinositides, is caused by its inhibition of multiple enzymes involved in conversion of inositol 1,4-bisphosphate to inositol.     

NADP(H) phosphatase activities of archaeal inositol monophosphatase and eubacterial 3'-phosphoadenosine 5'-phosphate phosphatase

NADP(H) phosphatase has not been identified in eubacteria and eukaryotes. In archaea, MJ0917 of hyperthermophilic Methanococcus jannaschii is a fusion protein comprising NAD kinase and an inositol monophosphatase homologue that exhibits high NADP(H) phosphatase activity (S. Kawai, C. Fukuda, T. Mukai, and K. Murata, J. Biol. Chem. 280:39200-39207, 2005). In this study, we showed that the other archaeal inositol monophosphatases, MJ0109 of M. jannaschii and AF2372 of hyperthermophilic Archaeoglobus fulgidus, exhibit NADP(H) phosphatase activity in addition to the already-known inositol monophosphatase and fructose-1,6-bisphosphatase activities. Kinetic values for NADP+ and NADPH of MJ0109 and AF2372 were comparable to those for inositol monophosphate and fructose-1,6-bisphosphate. This implies that the physiological role of the two enzymes is that of an NADP(H) phosphatase. Further, the two enzymes showed inositol polyphosphate 1-phosphatase activity but not 3'-phosphoadenosine 5'-phosphate phosphatase activity. The inositol polyphosphate 1-phosphatase activity of archaeal inositol monophosphatase was considered to be compatible with the similar tertiary structures of inositol monophosphatase, fructose-1,6-bisphosphatase, inositol polyphosphate 1-phosphatase, and 3'-phosphoadenosine 5'-phosphate phosphatase. Based on this fact, we found that 3'-phosphoadenosine 5'-phosphate phosphatase (CysQ) of Escherichia coli exhibited NADP(H) phosphatase and fructose-1,6-bisphosphatase activities, although inositol monophosphatase (SuhB) and fructose-1,6-bisphosphatase (Fbp) of E. coli did not exhibit any NADP(H) phosphatase activity. However, the kinetic values of CysQ and the known phenotype of the cysQ mutant indicated that CysQ functions physiologically as 3'-phosphoadenosine 5'-phosphate phosphatase rather than as NADP(H) phosphatase.     

Enzymic dephosphorylation of D-myo-inositol 1,4-bisphosphate in rat brain.

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,4-bisphosphate [Ins(1,4)P2] phosphatase activities were measured in both 180,000 g (60 min) particulate and supernatant fractions of rat brain homogenates. Although Ins(1,4,5)P3 was mostly hydrolysed by a particulate phosphatase [Erneux, Delvaux, Moreau & Dumont (1986) Biochem. Biophys. Res. Commun. 134, 351-358], Ins(1,4)P2 phosphatase was predominantly soluble. The latter enzyme was Mg2+-dependent and sensitive to thiol-blocking agents (e.g. p-hydroxymercuribenzoate). In contrast with Ins(1,4,5)P3 phosphatase activity measured in the soluble fraction, Ins(1,4)P2 phosphatase was insensitive to 0.001-1 mM-2,3-bisphosphoglycerate. Lithium salts, widely used in psychiatric treatment, inhibited both Ins(1,4)P2 and Ins(1)P1 phosphatase activities of the crude soluble fraction. In particular, 50% inhibition of phosphatase activity, with 2 microM-Ins(1,4)P2 as substrate, was achieved at 3-5 mM-LiCl. At these concentrations, LiCl did not change Ins(1,4,5)P3 phosphatase activity measured in the same fraction with 1-4 microM-Ins(1,4,5)P3 as substrate. Chromatography of the soluble fraction of a rat brain homogenate on DEAE-cellulose resolved three phosphatase activities. These forms, peaks I, II and III, dephosphorylated Ins(1,4,5)P3, Ins(1)P1 and Ins(1,4)P2 respectively. If LiCl (10 mM) was included in the assay mixture, it inhibited both peak-II Ins(1)P1 phosphatase and peak-III Ins(1,4)P2 phosphatase, suggesting the existence of at least two Li+-sensitive phosphatases.     

The Arabidopsis HAL2-like gene family includes a novel sodium-sensitive phosphatase

The yeast HAL2 gene encodes a lithium- and sodium-sensitive phosphatase that hydrolyses 3'-phosphoadenosine-5'-phosphate (PAP). Salt toxicity in yeast results from Hal2 inhibition and accumulation of PAP, which inhibits sulphate assimilation and RNA processing. We have investigated whether the model plant Arabidopsis thaliana contains sodium-sensitive PAP phosphatases. The Arabidopsis HAL2-like gene family is composed of three members: AtAHL and AtSAL2, characterized in the present work, and the previously identified AtSAL1. The AtAHL and AtSAL2 cDNAs complement the auxotrophy for methionine of the yeast hal2 mutant and the recombinant proteins catalyse the conversion of PAP to AMP in a Mg(2+)-dependent reaction sensitive to inhibition by Ca2+ and Li+. The PAP phosphatase activity of AtAHL is sensitive to physiological concentrations of Na+, whereas the activities of AtSAL1 and AtSAL2 are not. Another important difference is that AtAHL is very specific for PAP while AtSAL1 and AtSAL2 also act as inositol polyphosphate 1-phosphatases. AtAHL constitutes a novel type of sodium-sensitive PAP phosphatase which could act co-ordinately with plant sulphotransferases and serve as target of salt toxicity in plants.     

Evidence for a SAL1-PAP chloroplast retrograde pathway that functions in drought and high light signaling in Arabidopsis

Compartmentation of the eukaryotic cell requires a complex set of subcellular messages, including multiple retrograde signals from the chloroplast and mitochondria to the nucleus, to regulate gene expression. Here, we propose that one such signal is a phosphonucleotide (3'-phosphoadenosine 5'-phosphate [PAP]), which accumulates in Arabidopsis thaliana in response to drought and high light (HL) stress and that the enzyme SAL1 regulates its levels by dephosphorylating PAP to AMP. SAL1 accumulates in chloroplasts and mitochondria but not in the cytosol. sal1 mutants accumulate 20-fold more PAP without a marked change in inositol phosphate levels, demonstrating that PAP is a primary in vivo substrate. Significantly, transgenic targeting of SAL1 to either the nucleus or chloroplast of sal1 mutants lowers the total PAP levels and expression of the HL-inducible ASCORBATE PEROXIDASE2 gene. This indicates that PAP must be able to move between cellular compartments. The mode of action for PAP could be inhibition of 5' to 3' exoribonucleases (XRNs), as SAL1 and the nuclear XRNs modulate the expression of a similar subset of HL and drought-inducible genes, sal1 mutants accumulate XRN substrates, and PAP can inhibit yeast (Saccharomyces cerevisiae) XRNs. We propose a SAL1-PAP retrograde pathway that can alter nuclear gene expression during HL and drought stress.     

A plant 3'-phosphoesterase involved in the repair of DNA strand breaks generated by oxidative damage

Two novel, structurally and functionally distinct phosphatases have been identified through the functional complementation, by maize cDNAs, of an Escherichia coli diphosphonucleoside phosphatase mutant strain. The first, ZmDP1, is a classical Mg(2+)-dependent and Li(+)-sensitive diphosphonucleoside phosphatase that dephosphorylates both 3'-phosphoadenosine 5'-phosphate (3'-PAP) and 2'-PAP without any discrimination between the 3'- and 2'-positions. The other, ZmDP2, is a distinct phosphatase that also catalyzes diphosphonucleoside dephosphorylation, but with a 12-fold lower Li(+) sensitivity, a strong preference for 3'-PAP, and the unique ability to utilize double-stranded DNA molecules with 3'-phosphate- or 3'-phosphoglycolate-blocking groups as substrates. Importantly, ZmDP2, but not ZmDP1, conferred resistance to a DNA repairdeficient E. coli strain against oxidative DNA-damaging agents generating 3'-phosphate- or 3'-phosphoglycolate-blocked single strand breaks. ZmDP2 shares a partial amino acid sequence similarity with a recently identified human polynucleotide kinase 3'-phosphatase that is thought to be involved in DNA repair, but is devoid of 5'-kinase activity. ZmDP2 is the first DNA 3'-phosphoesterase thus far identified in plants capable of converting 3'-blocked termini into priming sites for reparative DNA polymerization.     

Purification and properties of inositol-1,4-bisphosphatase from bovine brain.

Inositol-1,4-bisphosphatase has been purified 13,000-fold from bovine brain supernatant. The enzyme is monomeric, with an apparent subunit Mr of 40,000. Maximal hydrolytic rates were observed in Tris buffer, pH 7.8, in the presence of 9 mM-Mg2+. The enzyme acted as a 1-phosphatase, hydrolysing both inositol 1,4-bisphosphate [Ins(1,4)P2] (Km 0.04 mM) and inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] (Km 0.5 mM) to inositol 4-phosphate and inositol 3,4-bisphosphate respectively. Li+ inhibited the hydrolysis of both substrates in an uncompetitive manner, with apparent Ki values of 9.63 mM and 0.46 mM for Ins(1,4)P2 and Ins(1,3,4)P3 respectively.     

Dephosphorylation of myo-inositol 1,4,5-trisphosphate and myo-inositol 1,3,4-triphosphate.

We have augmented our previous studies [Storey, Shears, Kirk & Michell (1984) Nature (London) 312, 374-376] on the subcellular location and properties of Ins(1,4,5)P3 (inositol 1,4,5-trisphosphate) phosphatases in rat liver and human erythrocytes. We also investigate Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate) metabolism by rat liver. Membrane-bound and cytosolic Ins(1,4,5)P3 phosphatases both attack the 5-phosphate. The membrane-bound enzyme is located on the inner face of the plasma membrane, and there is little or no activity associated with Golgi apparatus. Cytosolic Ins(1,4,5)P3 5-phosphatase (Mr 77,000) was separated by gel filtration from Ins(1,4)P2 (inositol 1,4-bisphosphate) and inositol 1-phosphate phosphatases (Mr 54,000). Ins(1,4,5)P3 5-phosphatase activity in hepatocytes was unaffected by treatment of the cells with insulin, vasopressin, glucagon or dibutyryl cyclic AMP. Ins(1,4,5)P3 5-phosphatase activity in cell homogenates was unaffected by changes in [Ca2+] from 0.1 to 2 microM. After centrifugation of a liver homogenate at 100,000 g, Ins(1,3,4)P3 phosphatase activity was largely confined to the supernatant. The sum of the activities in the supernatant and the pellet exceeded that in the original homogenate. When these fractions were recombined, Ins(1,3,4)P3 phosphatase activity was restored to that observed in unfractionated homogenate. Ins(1,3,4)P3 was produced from Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate) and was metabolized to a novel InsP2 that was the 3,4-isomer. Ins(1,3,4)P3 phosphatase activity was not changed by 50 mM-Li+ or 0.07 mM-Ins(1,4)P2 alone, but when added together these agents inhibited Ins(1,3,4)P3 metabolism. In Li+-treated and vasopressin-stimulated hepatocytes, Ins(1,4)P2 may reach concentrations sufficient to inhibit Ins(1,3,4)P3 metabolism, with little effect on Ins(1,4,5)P3 hydrolysis.     

Influence of the phosphorylation state of neurofilament proteins on the interactions between purified filaments in vitro.

Dephosphorylation of 1D-myo-inositol 1,4-bisphosphate [Ins(1,4)P2] in rat liver is catalysed by a cytosolic phosphatase that removes the 1-phosphate group. The Km for Ins(1,4)P2 is approx. 17 microM. Li+ (100 mM) causes 50% inhibition of Ins(1,4)P2 phosphatase activity when activity is measured at the very low substrate concentration of 10 nM, but on raising the substrate concentration to 100 microM there is a greater than 10-fold increase in sensitivity to Li+, suggesting that Li+ acts mainly, but not entirely, as an uncompetitive inhibitor of Ins(1,4)P2 phosphatase. In addition, rat liver cytosol shows Li+-sensitive phosphatase activity against 1D-myo-inositol 1-,3- and 4-monophosphates. The Ins(1,4)P2 1-phosphatase and inositol monophosphatase activities all share an apparent Mr of 47 x 10(3), as determined by gel-filtration chromatography. However, the Ins(1,4)P2 1-phosphatase is more sensitive to inactivation by heat, and can be separated from inositol monophosphatase activity by anion-exchange chromatography. We conclude that rat liver cytosol contains an Ins(1,4)P2 1-phosphatase that is distinct from, but in many ways similar to, inositol monophosphatase.     

A single mutation converts the nucleotide specificity of phenol sulfotransferase from PAP to AMP

Sulfotransferases (STs) catalyze all the known biological sulfonations, in which a sulfuryl group from a common sulfonate donor such as 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is transferred to a nucleophilic acceptor. In addition to PAPS, phenol sulfotransferase (PST), a member of the ST family, utilizes other nucleotides as substrates with much less catalytic efficiency [Lin, E. S., and Yang, Y. S. (2000) Biochem. Biophys. Res. Commun. 271, 818-822]. Six amino acid residues of PST have been chosen for mutagenesis studies on the basis of a model of PST and its sequence alignment with those of available cytosolic and membrane-anchored STs. Systematic analyses of the mutants reveal that Ser134 is important for the regulation of nucleotide specificity between 3'-phosphoadenosine 5'-phosphate (PAP) and adenosine 5'-monophosphate (AMP). Kinetic studies also indicate that Ser134 plays a key role in nucleotide binding (K(m)) but not in catalysis (kcat). Consequently, the catalytic efficiency (kcat/K(m)) of PST can be altered by 5 orders of magnitude with a mutation of Ser134. Moreover, the change in nucleotide specificity from PAP to AMP can be achieved by mutation of Ser134 to any of the following residues: Glu, Gln, Arg, and His. Roles of Lys44, Arg126, and Arg253, which interact directly with the 5'- and 3'-phosphate of PAP, were also investigated by mutagenesis and kinetic experiments. On the basis of these findings, we suggest that Ser134 is the key residue that enables PST to discriminate PAP from AMP.     

Formation and metabolism of inositol 1,3,4,5-tetrakisphosphate in liver

The inositol lipid pools of isolated rat hepatocytes were labeled with [3H]myo-inositol, stimulated maximally with vasopressin and the relative contents of [3H]inositol phosphates were measured by high performance liquid chromatography. Inositol 1,4,5-trisphosphate accumulated rapidly (peak 20 s), while inositol 1,3,4-trisphosphate and a novel inositol phosphate (ascribed to inositol 1,3,4,5-tetrakisphosphate) accumulated at a slower rate over 2 min. Incubation of hepatocytes with 10 mM Li+ prior to vasopressin addition selectively augmented the levels of inositol monophosphate, inositol 1,4-bisphosphate, and inositol 1,3,4-trisphosphate. A kinase was partially purified from liver and brain cortex which catalyzed an ATP-dependent phosphorylation of [3H]inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate. Incubation of purified [3H]inositol 1,3,4,5-tetrakisphosphate with diluted liver homogenate produced initially inositol 1,3,4-trisphosphate and subsequently inositol 1,3-bisphosphate, the formation of which could be inhibited by Li+. The data demonstrate that the most probable pathway for the formation of inositol 1,3,4,5-tetrakisphosphate is by 3-phosphorylation of inositol 1,4,5-trisphosphate by a soluble mammalian kinase. Degradation of both compounds occurs first by a Li+-insensitive 5-phosphatase and subsequently by a Li+-sensitive 4-phosphatase. The prolonged accumulation of both inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in vasopressin-stimulated hepatocytes suggest that they have separate second messenger roles, perhaps both relating to Ca2+-signalling events.     

Rv2131c from Mycobacterium tuberculosis is a CysQ 3'-phosphoadenosine-5'-phosphatase

Mycobacterium tuberculosis ( Mtb) produces a number of sulfur-containing metabolites that contribute to its pathogenesis and ability to survive in the host. These metabolites are products of the sulfate assimilation pathway. CysQ, a 3'-phosphoadenosine-5'-phosphatase, is considered an important regulator of this pathway in plants, yeast, and other bacteria. By controlling the pools of 3'-phosphoadenosine 5'-phosphate (PAP) and 3'-phosphoadenosine 5'-phosphosulfate (PAPS), CysQ has the potential to modulate flux in the biosynthesis of essential sulfur-containing metabolites. Bioinformatic analysis of the Mtb genome suggests the presence of a CysQ homologue encoded by the gene Rv2131c. However, a recent biochemical study assigned the protein's function as a class IV fructose-1,6-bisphosphatase. In the present study, we expressed Rv2131c heterologously and found that the protein dephosphorylates PAP in a magnesium-dependent manner, with optimal activity observed between pH 8.5 and pH 9.5 using 0.5 mM MgCl 2. A sensitive electrospray ionization mass spectrometry-based assay was used to extract the kinetic parameters for PAP, revealing a K m (8.1 +/- 3.1 microM) and k cat (5.4 +/- 1.1 s (-1)) comparable to those reported for other CysQ enzymes. The second-order rate constant for PAP was determined to be over 3 orders of magnitude greater than those determined for myo-inositol 1-phosphate (IMP) and fructose 1,6-bisphosphate (FBP), previously considered to be the primary substrates of this enzyme. Moreover, the ability of the Rv2131c-encoded enzyme to dephosphorylate PAP and PAPS in vivo was confirmed by functional complementation of an Escherichia coli Delta cysQ mutant. Taken together, these studies indicate that Rv2131c encodes a CysQ enzyme that may play a role in mycobacterial sulfur metabolism.     

Effect of pertussis toxin and neomycin on G-protein-regulated polyphosphoinositide phosphodiesterase. A comparison between HL60 membranes and permeabilized HL60 cells.

Dictyostelium discoideum homogenates contain phosphatase activity which rapidly dephosphorylates Ins(1,4,5)P3 (D-myo-inositol 1,4,5-trisphosphate) to Ins (myo-inositol). When assayed in Mg2+, Ins(1,4,5)P3 is dephosphorylated by the soluble Dictyostelium cell fraction to 20% Ins(1,4)P2 (D-myo-inositol 1,4-bisphosphate) and 80% Ins(4,5)P2 (D-myo-inositol 4,5-bisphosphate). In the particulate fraction Ins(1,4,5)P3 5-phosphatase is relatively more active than the Ins(1,4,5)P3 1-phosphatase. CaCl2 can replace MgCl2 only for the Ins(1,4,5)P3 5-phosphatase activity. Ins(1,4)P2 and Ins(4,5)P2 are both further dephosphorylated to Ins4P (D-myo-inositol 4-monophosphate), and ultimately to Ins. Li+ ions inhibit Ins(1,4,5)P3 1-phosphatase, Ins(1,4)P2 1-phosphatase, Ins4P phosphatase and L-Ins1P (L-myo-inositol 1-monophosphate) phosphatase activities; Ins(1,4,5)P3 1-phosphatase is 10-fold more sensitive to Li+ (half-maximal inhibition at about 0.25 mM) than are the other phosphatases (half-maximal inhibition at about 2.5 mM). Ins(1,4,5)P3 5-phosphatase activity is potently inhibited by 2,3-bisphosphoglycerate (half-maximal inhibition at 3 microM). Furthermore, 2,3-bisphosphoglycerate also inhibits dephosphorylation of Ins(4,5)P2. These characteristics point to a number of similarities between Dictyostelium phospho-inositol phosphatases and those from higher organisms. The presence of an hitherto undescribed Ins(1,4,5)P3 1-phosphatase, however, causes the formation of a different inositol bisphosphatase isomer [Ins(4,5)P2] from that found in higher organisms [Ins(1,4)P2]. The high sensitivity of some of these phosphatases for Li+ suggests that they may be the targets for Li+ during the alteration of cell pattern by Li+ in Dictyostelium.