Rat brain myo-inositol 3-phosphate synthase is a phosphoprotein.

The therapeutic effects of lithium in bipolar disorder are poorly understood. Lithium decreases free inositol levels by inhibiting inositol monophosphatase 1 and myo-inositol 3-phosphate synthase (IPS). In this study, we demonstrate for the first time that IPS can be phosphorylated. This was evident when purified rat IPS was dephosphorylated by lambda protein phosphatase and analyzed by phospho-specific ProQ-Diamond staining and Western blot analysis. These techniques demonstrated a mobility shift consistent with IPS being phosphorylated. Mass spectral analysis revealed that Serine-524 (S524), which resides in the hinge region derived from exon 11 of the gene, is the site for phosphorylation. Further, an antibody generated against a synthetic peptide of IPS containing monophosphorylated-S524, was able to discriminate the phosphorylated and non-phosphorylated forms of IPS. The phosphoprotein is found in the brain and testis, but not in the intestine. The intestinal IPS isoform lacks the peptide bearing S524, and hence, cannot be phosphorylated. Evidences suggest that IPS is monophosphorylated at S524 and that the removal of this phosphate does not alter its enzymatic activity. These observations suggest a novel function for IPS in brain and other tissues. Future studies should resolve the functional role of phospho-IPS in brain inositol signaling.

Biochemical and molecular properties of lithium-sensitive myo-inositol monophosphatase.

Myo-inositol monophosphatase is a pivotal enzyme of the inositol second messenger system which is specifically inhibited by therapeutic levels of lithium salts, implicating inhibition of this enzyme as a potential site of its action in bipolar disease. This enzyme has a native molecular weight of 59,000, and has traditionally been found in the cytosolic fraction, although a membrane-bound form has also been identified. Possessing two identical subunits, this enzyme hydrolyzes those monophosphates which are equatorially located within the inositol ring, and several nucleoside monophosphates phosphorylated at the 2-position. Each subunit of the native enzyme contains an active site with unusually large caverns as revealed by crystallographic studies, which may explain the accommodation of these structurally unrelated substrates. We have suggested that the uncompetitive inhibition of this phosphatase by lithium ions may prevent the formation of an enzyme-bound non-isomeric (meso) intermediate, Mg(2+)-inositol 1,3 or 4,6 cyclic monophosphate when this enzyme hydrolyzes its respective isomeric substrates.

myo-inositol monophosphatase from rat testes: purification and properties.

myo-Inositol monophosphatase (EC 3.1.3.25) has been purified to homogeneity from the high-speed supernatant of rat testes and its properties were investigated. By means of ammonium sulfate precipitation, followed by heating, anion exchange, and gel filtration high-pressure liquid chromatographic techniques, polylysine agarose and phenyl-Sepharose column chromatographic methods, this phosphatase was purified 2563-fold to a specific activity of 7972 mU/mg protein. It showed an apparent native molecular weight of 58,000 as determined by gel filtration chromatography and was composed of two identical subunits of molecular weight of 29,000 as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Among several divalent cations tested for activation of the enzyme, Mg2+ was most effective and optimally active at pH 7.8. The Km values for D- and L-myo-inositol 1-phosphate (which were equal) and 2'-AMP were 0.12 +/- 0.02 and 0.17 +/- 0.03 mM, respectively. Lithium ions inhibited this phosphatase specifically and kinetic studies demonstrated uncompetitive inhibition. Preparations of polyclonal antibodies against the homogeneous enzyme in rabbits cross-reacted with the partially purified enzyme preparations from liver, kidney, heart, and brain show immunological identity. Western blot analysis after SDS-polyacrylamide gel electrophoresis confirmed a major band corresponding to a subunit molecular weight of 29,000. A sensitive enzyme staining method was also developed to localize the site of myo-inositol monophosphatase activity on polyacrylamide gels which helped to differentiate this phosphatase from nonspecific contaminating phosphatases. To explain the unusual stereospecificity of this enzyme on its isomeric substrates, a working model was suggested involving the production of a myo-inositol 1,3-cyclic phosphate intermediate during the course of its reaction.

The identification of a membrane-bound myo-inositol 1-phosphatase in rat brain, liver, and testes.

A membrane-bound myo-inositol 1-phosphatase has been solubilized and partially purified from rat tissues. This particulate enzyme was detected in brain, liver and testis and certain physicochemical and enzymological properties were examined. Previously this major enzyme of the inositol signaling system was considered strictly cytosolic. The ratio of activity in the membrane form was approximately one-eighth of the activity found with the cytosolic fraction. The molecular weight of this phosphatase was found to be 59,000 by gel filtration chromatography and a subunit molecular weight of 29,000 by Western blot analysis, values comparable to the cytosolic form. This phosphatase cleaves both D- and L- myo-inositol 1-phosphates which originate from two different cellular pathways and is inhibited by lithium ions. Polyclonal antibodies were raised against homogeneous testicular cytosolic myo-inositol 1-phosphatase and cross-reacted with this membrane form as determined by western blot analysis showing immunological identity.

The effects of lithium isotopes on the myo-inositol 1-phosphatase reaction in rat brain, liver, and testes.

Enzyme inhibition studies were performed with several lithium isotopes in order to more precisely define how lithium inhibits the enzyme myo-inositol 1-phosphatase. This lithium-induced inhibition is thought to be central to the therapeutic effects of lithium in the treatment of manic-depressive disorder. Naturally occurring lithium (NLi) exists as a combination of isotopes: 6Li and 7Li. Lethality studies were performed comparing 6LiCl, 7LiCl, and NLiCl, did not demonstrate a differential effect as previous studies had suggested. Enzyme inhibition studies were performed with these individual lithium isotopes, and compared to the effects of the naturally occurring combination (NLi) on the inhibition of myo-inositol 1-phosphatase using a partially purified enzyme preparation from rat brain, liver and testes. Identical inhibition was observed with all lithium isotopes and their combinations. In addition, both D- and L-myo-inositol 1-phosphates were used as enzyme substrates and found to be equivalent. These experiments, along with previous work demonstrating lithium acting as an uncompetitive inhibitor in the reaction, and the lack of lithium binding sites on the enzyme, suggests the hypothesis that lithium is possibly inhibiting this reaction by interfering with the formation of a transition cyclic intermediate, myo-inositol 1,3-cyclic phosphate, which may be formed from either the D- or L-substrates. This proposal is in contrast to previous suggestions regarding the inhibitory mechanism of action of lithium on the myo-inositol 1-phosphatase reaction.

The identification of a novel inositol lipid, phosphatidylinositol trisphosphate (PIP3), in rat cerebrum using in vivo techniques.

Rats received intraventricular injections of 20 uCi of [3H]-myo-inositol, and were sacrificed 24 hrs later by high-power head-focused microwave fixation. Two inositol lipid extraction methods were compared: The Hauser and Eichberg method yielded higher recovery of inositol lipids, but a lower inositol phosphate content. The Schacht method yielded reduced radiolabel in the lipid fractions, but increased water soluble phosphates. Both methods extracted a novel inositol lipid (PIP3) which contained inositol tetrakisphosphate (IP4) as its polar head group. This was determined by alkaline hydrolysis and analyzed by high performance liquid chromatography with authentic IP4 standard. Furthermore, preliminary studies of the fatty acid composition indicated a similarity with other inositol lipids. The radiolabel ratio of PIP2:PIP3 was 5:1. In summary, we have isolated a novel inositol phospholipid in rat brain, PIP3, the parent compound for inositol tetrakisphosphate (IP4).

Carbamazepine inhibits electroconvulsive shock-induced inositol trisphosphate (IP3) accumulation in rat cerebral cortex and hippocampus.

Carbamazepine is used to treat manic-depressive disorder, and is also an anticonvulsant. Rats were injected with this drug 90 min prior to this experiment, when mild inhibition of convulsions took place. Intraventricular injections of 14 muCi [3H]myoinositol were made 20-24 hrs prior to the experiment. Ninety min after intraperitoneal injection of carbamazepine or vehicle, rats were given electroconvulsive shock or sham procedure and sacrificed 30 sec later. Incorporation of radiolabel into inositol lipids and inositol phosphates was analyzed in cerebral cortex and hippocampus. Carbamazepine's effects on the brain inositol lipid cycle, studied here for the first time, showed 1) enhanced labeling in the polyphosphoinositides (carbamazepine-ECS groups showed increases of about 40% in PIP2); 2) decreased [H]IP1 levels; and 3) inhibition of ECS-induced [3H]-IP3 accumulation.

Electroconvulsive shock stimulates polyphosphoinositide degradation and inositol trisphosphate accumulation in rat cerebrum: lithium pretreatment does not potentiate these changes.

Using an in vivo model, we explored the acute effects of electroconvulsive shock (ECS) and lithium on rat cerebral polyphosphoinositides and inositol phosphates. ECS was shown to increase the [3H]inositol trisphosphate ([3H]IP3) by 75%, decrease the endogenous mass of phosphatidylinositol 4,5-bisphosphate (PIP2) by 23%, and enhance [3H]myo-inositol labeling into the polyphosphoinositides. In contrast, lithium pretreatment 20-24 h prior to ECS appeared to attenuate the ECS-induced [3H]IP3 increase and the decrease in mass of PIP2; [3H]inositol monophosphate ([3H]IP1) levels demonstrated no differences between the lithium ECS and lithium-alone groups. These results indicate that ECS stimulates the inositol lipid cycle in brain possibly due to neurotransmitter release. Moreover, the effects of lithium suggest other possible sites of action of this cation on inositol lipid metabolism in addition to an inhibition of inositol-1-phosphatase.