Dependent Phosphatase Activity Research Articles

D-2-Hydroxyglutarate Inhibits Calcineurin Phosphatase Activity to Abolish NF-AT Activation and IL-2 Induction in Stimulated Lymphocytes.

Gliomas expressing mutant isocitrate dehydrogenases excessively synthesize d-2-hydroxyglutarate (D2HG), suppressing immune surveillance. A portion of this D2HG is released from these tumor cells, but the way environmental D2HG inhibits lymphocyte function is undefined. We incubated human PBLs or Jurkat T cells with D2HG at concentrations present within and surrounding gliomas or its obverse l-2-hydroxyglutarate (L2HG) stereoisomer. We quantified each 2HG stereoisomer within washed cells by N-(p-toluenesulfonyl)-l-phenylalanyl chloride derivatization with stable isotope-labeled D2HG and L2HG internal standards, HPLC separation, and mass spectrometry. D2HG was present in quiescent cells and was twice as abundant as L2HG. Extracellular 2HG rapidly increased intracellular levels of the provided stereoisomer by a stereoselective, concentration-dependent process. IL-2 expression, even when elicited by A23187 and PMA, was abolished by D2HG in a concentration-dependent manner, with significant reduction at just twice its basal level. In contrast, L2HG was only moderately inhibitory. IL-2 expression is regulated by increased intracellular Ca2+ that stimulates calcineurin to dephosphorylate cytoplasmic phospho-NF-AT, enabling its nuclear translocation. D2HG abolished stimulated expression of a stably integrated NF-AT-driven luciferase reporter that precisely paralleled its concentration-dependent inhibition of IL-2. D2HG did not affect intracellular Ca2+. Rather, surface plasmon resonance showed D2HG, but not L2HG, bound calcineurin, and D2HG, but not L2HG, inhibited Ca2+-dependent calcineurin phosphatase activity in stimulated Jurkat extracts. Thus, D2HG is a stereoselective calcineurin phosphatase inhibitor that prevents NF-AT dephosphorylation and so abolishes IL-2 transcription in stimulated lymphocytes. This occurs at D2HG concentrations found within and adjacent to gliomas independent of its metabolic or epigenetic transcriptional regulation.

Cardiomyocyte stromal interaction molecule 1 is a key regulator of Ca2+ -dependent kinase and phosphatase activity in the mouse heart.

Stromal interaction molecule 1 (STIM1) is a major regulator of store‐operated calcium entry in non‐excitable cells. Recent studies have suggested that STIM1 plays a role in pathological hypertrophy; however, the physiological role of STIM1 in the heart is not well understood. We have shown that mice with a cardiomyocyte deletion of STIM1 (crSTIM1−/−) develop ER stress, mitochondrial, and metabolic abnormalities, and dilated cardiomyopathy. However, the specific signaling pathways and kinases regulated by STIM1 are largely unknown. Therefore, we used a discovery‐based kinomics approach to identify kinases differentially regulated by STIM1. Twelve‐week male control and crSTIM1−/− mice were injected with saline or phenylephrine (PE, 15 mg/kg, s.c, 15 min), and hearts obtained for analysis of the Serine/threonine kinome. Primary analysis was performed using BioNavigator 6.0 (PamGene), using scoring from the Kinexus PhosphoNET database and GeneGo network modeling, and confirmed using standard immunoblotting. Kinomics revealed significantly lower PKG and protein kinase C (PKC) signaling in the hearts of the crSTIM1−/− in comparison to control hearts, confirmed by immunoblotting for the calcium‐dependent PKC isoform PKCα and its downstream target MARCKS. Similar reductions in crSTIM1−/− hearts were found for the kinases: MEK1/2, AMPK, and PDPK1, and in the activity of the Ca2+‐dependent phosphatase, calcineurin. Electrocardiogram analysis also revealed that crSTIM1−/− mice have significantly lower HR and prolonged QT interval. In conclusion, we have shown several calcium‐dependent kinases and phosphatases are regulated by STIM1 in the adult mouse heart. This has important implications in understanding how STIM1 contributes to the regulation of cardiac physiology and pathophysiology.

Protective effect of taurine on cellular structures in rat hippocampus following ethanol intoxication.

Background. Distinct brain structures, in particular, the hippocampus, have a selective sensitivity to acute and chronic alcohol intoxication. Study of the pathogenesis of alcohol-related brain damage with a view to the development of methods of prevention and treatment is an urgent task.Objective.Our main interest was in elucidating the effects of the biologically active substance, such as taurine on cellular structures in the hippocampus following ethanol intoxication.Methods.We applied a histochemical method to estimate the Ca2 + -dependent acid phosphatase activity. The present study was carried out in female albino rats. All experimental animals received an ethanol (15%) diet as their sole source of fluid at various time points.To study the effect of taurine on cellular structures of the rat brain following short-term and chronic alcohol consumption, rats were given daily injections of taurine solution for 7 days. Results. The morphological picture of the hippocampus is characterized by swelling of granular cells in the dentate gyrus that undergo chromatolysis, as well as a decrease in numerical density and a weak expression of processes of pyramidal neurons in the early stages of alcohol consumption. Degenerative changes occur in pyramidal cells in the hippocampus in the middle periods of alcohol consumption. The density of neurons is decreased; pyramidal cells lose their characteristic shape. In the conditions of long-term alcohol consumption, not only changes in the size and number of pyramidal neurons but also in the volume of the hippocampus and its regions are observed. The morphological picture of hippocampal neurons is a morphological proof of the disorders of their metabolism. In rats treated with taurine, there are positive changes in the structural properties of neurons and an increase in phosphatase activity (increased metabolism) in the hippocampus, which determines cell survival and functional recovery of irritated neurons. Conclusion. The results indicate that taurine has a neuroprotective effect on cellular structures in rat hippocampus.

Voltage Dependent Phosphatase Activity is Enhanced by Intracellular Acidification

Voltage sensitive phosphatases (VSPs) are PI(4,5)P2/PI(3,4,5)P3-5- and PI(3,4)P2/PI(3,4,5)P3-3-phosphatases. For non-mammalian VSPs, this activity is regulated by membrane voltage via a voltage sensor domain (VSD). For mammalian VSPs the VSD seems insensitive to voltage changes yet still essential for control of the phosphatase activity. Under physiological conditions, the non-mammalian VSPs strongly deplete PI(4,5)P2 in a voltage dependent manner. The physiological regulator of mammalian VSP activity remains elusive. VSPs are suggested to play a role in fertilization and development, where changes in intracellular pH are known to occur. They are found to be expressed in tissues like kidney, stomach, sperm, and ovary which are known to undergo such pH changes. Therefore, we speculated that intracellular pH might modulate VSP activity.To test this hypothesis we performed whole-cell patch-clamp experiments in CHO cells expressing diverse VSPs and fluorescent PI(4,5)P2 reported domains, the latter allowing for monitoring VSP activity by means of total internal reflection microscopy (TIRF-M). The whole-cell patch-clamp configuration allowed for control not only over membrane voltage but also intracellular pH by dialysing the cell with solutions with the desired pH.We find that acidification of the cytoplasm results in increased PI(4,5)P2 depletion, accompanied by a shift of the apparent voltage dependence towards more negative potentials. An increase in intracellular pH has the opposite effect. The voltage dependence of sensing currents was unaffected by the pH changes, suggesting that alterations of the VSD are not causal for the observed changes in voltage dependent activity. Similar effects were observed in all tested VSPs We conclude that the overall activity of the phosphatase is enhanced under acidic and diminished under alkaline conditions. Kinetic modeling predicts a shift in apparent voltage dependence under these circumstances that is in agreement with the observed shift.In conclusion, we suggest that intracellular pH can play a role in the regulation of the activity of VSPs.This work was supported by a research grant of the University Medical Center Giessen and Marburg (UKGM32/2011MR) to C.R.H and by Deutsche Forschungsgemeinschaft (SFB593 TP A12) to D.O.

Detection of Ca2+-dependent acid phosphatase activity identifies neuronal integrity in damaged rat central nervous system after application of bacterial melanin

The study aims to confirm the neuroregenerative effects of bacterial melanin (BM) on central nervous system injury using a special staining method based on the detection of Ca2+-dependent acid phosphatase activity. Twenty-four rats were randomly assigned to undergo either unilateral destruction of sensorimotor cortex (group I; n = 12) or unilateral rubrospinal tract transection at the cervical level (C3–4) (group II; n = 12). In each group, six rats were randomly selected after surgery to undergo intramuscular injection of BM solution (BM subgroup) and the remaining six rats were intramuscularly injected with saline (saline subgroup). Neurological testing confirmed that BM accelerated the recovery of motor function in rats from both BM and saline subgroups. Two months after surgery, Ca2+-dependent acid phosphatase activity detection in combination with Chilingarian’s calcium adenoside triphosphate method revealed that BM stimulated the sprouting of fibers and dilated the capillaries in the brain and spinal cord. These results suggest that BM can promote the recovery of motor function of rats with central nervous system injury; and detection of Ca2+-dependent acid phosphatase activity is a fast and easy method used to study the regeneration-promoting effects of BM on the injured central nervous system.

Engineered Voltage Sensing Phosphatases: What do they tell us about the Gating Mechanism?

Voltage sensing phosphatases (VSPs) contain a phosphoinositide phosphatase domain (PD), which is under the control of a voltage sensor domain (VSD). Here, the underlying coupling mechanism is not fully understood. Recently, Liu et al. (NSMB 2012) and Hobiger et al. (Biophys J 2012, PLoS one 2013) proposed for Ciona intestinalis (Ci-) VSP an interaction of positively charged amino acid residues in the linker, which connects the PD to the VSD, with the negatively charged residue Asp400 in the ‘TI’ loop of the PD. In light of these findings we revisited the engineered VSPs PTENCiV (Lacroix et al., JBC 2011) and hVSP1CiV (Halaszovich et al., J Lipid Res 2012), chimeras consisting of Ci-VSP's VSD and the PD of PTEN or hVSP1, respectively. In those chimeras Asp400 (Ci-VSP numbering) is not conserved. Nonetheless, they show robust voltage dependent phosphatase activity. Mutations within the linker showed effects mirroring those previously reported for Ci-VSP, suggesting a gating mechanism similar to the one found in wild-type Ci-VSP. We mutated Asp400 in Ci-VSP to Asn or Arg and the corresponding Asn (hVSP1) or Arg (PTEN) in the chimeric enzymes to Asp and measured their voltage dependent enzymatic activity as well as sensing currents to elucidate the importance of Asp400 for VSD-PD-coupling. We propose that the interactions involving Asp400 described by Liu et al. and Hobiger et al. are not a necessary prerequisite for the voltage dependent activation of VSPs, but allow for a more efficient VSD-to-PD coupling. Further work will be required to establish the essential interactions between VSP, linker, and PD. Supported by Deutsche Forschungsgemeinschaft (DFG, grant SFB 593, TP12 to D.O.) and University Medical Center Giessen and Marburg (UKGM, grant 32/2011MR to C.R.H.)

An Active C-Terminally Truncated Form of Ca2+/Calmodulin-Dependent Protein Kinase Phosphatase-N (CaMKP-N/PPM1E)

Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) and its nuclear homolog CaMKP-N (PPM1E) are Ser/Thr protein phosphatases that belong to the PPM family. CaMKP-N is expressed in the brain and undergoes proteolytic processing to yield a C-terminally truncated form. The physiological significance of this processing, however, is not fully understood. Using a wheat-embryo cell-free protein expression system, we prepared human CaMKP-N (hCaMKP-N(WT)) and the truncated form, hCaMKP-N(1–559), to compare their enzymatic properties using a phosphopeptide substrate. The hCaMKP-N(1–559) exhibited a much higher V max value than the hCaMKP-N(WT) did, suggesting that the processing may be a regulatory mechanism to generate a more active species. The active form, hCaMKP-N(1–559), showed Mn2+ or Mg2+-dependent phosphatase activity with a strong preference for phospho-Thr residues and was severely inhibited by NaF, but not by okadaic acid, calyculin A, or 1-amino-8-naphthol-2,4-disulfonic acid, a specific inhibitor of CaMKP. It could bind to postsynaptic density and dephosphorylate the autophosphorylated Ca2+/calmodulin-dependent protein kinase II. Furthermore, it was inactivated by H2O2 treatment, and the inactivation was completely reversed by treatment with DTT, implying that this process is reversibly regulated by oxidation/reduction. The truncated CaMKP-N may play an important physiological role in neuronal cells.

Bacterial Melanin Favors Regeneration after Motor Tract and Peripheral Nerve Damage

Experiments were performed on 48 albino rats. All experimental animals were initially trained to a balancing instrumental conditioned reflex (ICR). Unilateral bulbar pyramidotomy performed in 24 rats caused contralateral hemiparesis. On the next day following the operation 12 rats (first group) were injected intramuscularly with bacterial melanin (BM) solution. Recovery periods of ICR and paralyzed hindlimb movements were registered for melanin injected rats ( n = 12) and for operated rats, not treated with melanin ( n = 12, second group). In rats injected with bacterial melanin the posttraumatic recovery is shorter than in animals not treated with melanin. Morphohistochemical examination was carried out to confirm the results of behavioral and electrophysiological experiments. Medulla slices were prepared to trace the regeneration of nerve fibers. Examination of transection area revealed that bacterial melanin increases vascularization, dilates the capillaries in nervous tissue and stimulates the process of sprouting. Ischiadic nerve transection was performed in third and fourth groups of rats (12 rats in each group). Third group animals were injected with BM on the next day of surgery. ICR was used to assess the recovery of movements after nerve damage. Method for Ca 2+ -dependent acidic phosphatase activity measurement was used to examine sections of nerve fibers and to trace the recovery of the nerve and limb movements after its injury. Acceleration of the instrumental conditioned reflex recovery and data from morphohistochemical study showed that bacterial melanin has neuroprotective action and facilitates recovery of limb movements after peripheral nerve or motor tract lesions.

Structural and Biochemical Characterization of Human Mitochondrial Branched-chain α-Ketoacid Dehydrogenase Phosphatase

The branched-chain α-ketoacid dehydrogenase phosphatase (BDP) component of the human branched-chain α-ketoacid dehydrogenase complex (BCKDC) has been expressed in Escherichia coli and purified in the soluble form. The monomeric BDP shows a strict dependence on Mn(2+) ions for phosphatase activity, whereas Mg(2+) and Ca(2+) ions do not support catalysis. Metal binding constants for BDP, determined by competition isothermal titration calorimetry, are 2.4 nm and 10 μm for Mn(2+) and Mg(2+) ions, respectively. Using the phosphorylated decarboxylase component (p-E1b) of BCKDC as a substrate, BDP shows a specific activity of 68 nmol/min/mg. The Ca(2+)-independent binding of BDP to the 24-meric transacylase (dihydrolipoyl transacylase; E2b) core of BCKDC results in a 3-fold increase in the dephosphorylation rate of p-E1b. However, the lipoyl prosthetic group on E2b is not essential for BDP binding or E2b-stimulated phosphatase activity. Acidic residues in the C-terminal linker of the E2b lipoyl domain are essential for the interaction between BDP and E2b. The BDP structure was determined by x-ray crystallography to 2.4 Å resolution. The BDP structure is dominated by a central β-sandwich. There are two protrusions forming a narrow cleft ∼10 Å wide, which constitutes the active site. The carboxylate moieties of acidic residues Asp-109, Asp-207, Asp-298, and Asp-337 in the active-site cleft participate in binding two metal ions. Substitutions of these residues with alanine nullify BDP phosphatase activity. Alteration of the nearby Arg-104 increases the K(m) for p-E1b peptide by 60-fold, suggesting that this residue is critical for the recognition of the native p-E1b protein.

Nuclear Envelope Phosphatase 1-Regulatory Subunit 1 (Formerly TMEM188) Is the Metazoan Spo7p Ortholog and Functions in the Lipin Activation Pathway

Lipin-1 catalyzes the formation of diacylglycerol from phosphatidic acid. Lipin-1 mutations cause lipodystrophy in mice and acute myopathy in humans. It is heavily phosphorylated, and the yeast ortholog Pah1p becomes membrane-associated and active upon dephosphorylation by the Nem1p-Spo7p membrane complex. A mammalian ortholog of Nem1p is the C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1, formerly "dullard"), but its Spo7p-like partner is unknown, and the need for its existence is debated. Here, we identify the metazoan ortholog of Spo7p, TMEM188, renamed nuclear envelope phosphatase 1-regulatory subunit 1 (NEP1-R1). CTDNEP1 and NEP1-R1 together complement a nem1Δspo7Δ strain to block endoplasmic reticulum proliferation and restore triacylglycerol levels and lipid droplet number. The two human orthologs are in a complex in cells, and the amount of CTDNEP1 is increased in the presence of NEP1-R1. In the Caenorhabditis elegans embryo, expression of nematode CTDNEP1 and NEP1-R1, as well as lipin-1, is required for normal nuclear membrane breakdown after zygote formation. The expression pattern of NEP1-R1 and CTDNEP1 in human and mouse tissues closely mirrors that of lipin-1. CTDNEP1 can dephosphorylate lipins-1a, -1b, and -2 in human cells only in the presence of NEP1-R1. The nuclear fraction of lipin-1b is increased when CTDNEP1 and NEP1-R1 are co-expressed. Therefore, NEP1-R1 is functionally conserved from yeast to humans and functions in the lipin activation pathway.

Regucalcin and cell regulation: role as a suppressor protein in signal transduction

Regucalcin was discovered in 1978 as a calcium-binding protein that does not contain EF-hand motif of calcium-binding domain (Yamaguchi and Yamamoto Chem Pharm Bull 26:1915-1918, 1978). The name regucalcin was proposed for this calcium-binding protein, which can regulate various Ca(2+)-dependent enzyme activations in liver cells. The regucalcin gene is localized on the chromosome X, and the organization of the regucalcin gene consists of seven exons and six introns. AP-1, NF1-A1, and RGPR-p117 bind to the promoter region of the rat regucalcin gene and enhance transcription activity of regucalcin gene expression that is mediated through calcium signaling. Regucalcin plays a pivotal role in the keep of intracellular calcium ion (Ca(2+)) homeostasis due to activating Ca(2+) pump enzymes in the plasma membrane (basolateral membrane), microsomes (endoplasmic reticulum), mitochondria, and nuclei of many cell types. Regucalcin has a suppressive effect on calcium signaling from the cytoplasm to the nucleus in the proliferative cells. Regucalcin has also been demonstrated to transport to the nucleus, and it can inhibit Ca(2+)-dependent protein kinase and protein phosphatase activities, Ca(2+)-activated deoxyribonucleic acid (DNA) fragmentation, and DNA and ribonucleic acid (RNA) synthesis in the nucleus. Overexpression of regucalcin suppresses cell death and apoptosis in the cloned rat hepatoma cells induced by various signaling factors. Regucalcin can inhibit the enhancement of cell proliferation due to hormonal stimulation. Regucalcin plays an important role as a regulatory protein in cell signaling system, and it is proposed to play a pivotal role in keep of cell homeostasis and function.

DevT (Alr4674), resembling a Ser/Thr protein phosphatase, is essential for heterocyst function in the cyanobacterium Anabaena sp. PCC 7120

Heterocyst-forming cyanobacteria are able to perform oxygenic photosynthesis and nitrogen fixation simultaneously in the same filament, by restricting the highly O(2)-sensitive nitrogenase to specialized cells, the heterocysts. A remarkable change in morphology and metabolism accompanies the differentiation of heterocysts, which only occurs when no source of combined nitrogen is available. In this study, we characterized DevT (Alr4674), a putative protein phosphatase from Anabaena PCC 7120. Mutants defective in devT are able to form morphologically mature heterocysts, which however cannot fix N(2), and the mutant cannot survive without a source of combined nitrogen. DevT shows homology to phosphatases of the PPP family and displays a Mn(2+)-dependent phosphatase activity that can be inhibited by phosphatase inhibitors and oxidizing conditions. DevT is constitutively expressed in both vegetative cells and heterocysts, and is not regulated by NtcA. The heterocyst regulator HetR may exert a certain inhibition on the expression of devT. Under diazotrophic growth conditions, DevT protein accumulates specifically in mature heterocysts. Therefore DevT plays a still unknown role in a late essential step of heterocyst differentiation.

Protective Effects of Hypothalamic Proline-Rich Peptide and Cobra Venom Naja Naja Oxiana on Dynamics of Vestibular Compensation Following Unilateral Labyrinthectomy

We tested the action of proline-rich peptide (PRP-1) and cobra venom Naja Naja Oxiana (NOX) on Deiters' nucleus neurons at 3rd, 15th and 35th days after unilateral labyrinthectomy (UL). Early and late tetanic, post-tetanic potentiation and depression of Deiters'neurons to bilateral high frequency stimulation of hypothalamic supraoptic and paraventricualar nuclei was studied. The analysis of spike activity was carried out by mean of on-line selection and special program. The complex averaged peri-event time and frequency histograms shows the increase of inhibitory and excitatory reactions of Deiters' neurons at early stage of vestibular compensation following PRP-1 and NOX injection, reaching the norm at the end of tests. In histochemical study the changes in Ca(2+)-dependent acidic phosphatase (AP) activity in neurons was discovered. It was shown that in UL animals the total disappearance or delay of decolorizing of Deiters' neurons lead to neurodegenerative pattern as cellular "shade". AP activity after UL and PRP-1 injection exerts more effective recovery of neurons in comparison with events, observed after the administration of NOX. The data of this study indicate that PRP-1 and NOX are protectors, which may successfully recover the disturbed vestibular functions.

Myxococcus xanthus Pph2 Is a Manganese-dependent Protein Phosphatase Involved in Energy Metabolism

The multicellular behavior of the myxobacterium Myxococcus xanthus requires the participation of an elevated number of signal-transduction mechanisms to coordinate the cell movements and the sequential changes in gene expression patterns that lead to the morphogenetic and differentiation events. These signal-transduction mechanisms are mainly based on two-component systems and on the reversible phosphorylation of protein targets mediated by eukaryotic-like protein kinases and phosphatases. Among all these factors, protein phosphatases are the elements that remain less characterized. Hence, we have studied in this work the physiological role and biochemical activity of the protein phosphatase of the family PPP (phosphoprotein phosphatases) designated as Pph2, which is forming part of the same operon as the two-component system phoPR1. We have demonstrated that this operon is induced upon starvation in response to the depletion of the cell energy levels. The increase in the expression of the operon contributes to an efficient use of the scarce energy resources available for developing cells to ensure the completion of the life cycle. In fact, a Deltapph2 mutant is defective in aggregation, sporulation yield, morphology of the myxospores, and germination efficiency. The yeast two-hybrid technology has shown that Pph2 interacts with the gene products of MXAN_1875 and 5630, which encode a hypothetical protein and a glutamine synthetase, respectively. Because Pph2 exhibits Ser/Thr, and to some extent Tyr, Mn(2+)-dependent protein phosphatase activity, it is expected that this function is accomplished by dephosphorylation of the specific protein substrates.

Mutual Regulation of ntcA and hetR during Heterocyst Differentiation Requires Two Similar PP2C-Type Protein Phosphatases, PrpJ1 and PrpJ2, in Anabaena sp. Strain PCC 7120

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 can form heterocysts for N(2) fixation. Initiation of heterocyst differentiation depends on mutual regulation of ntcA and hetR. Control of hetR expression by NtcA is partially mediated by nrrA, but other factors must be involved in this regulation. Anabaena has two closely related PP2C-type protein phosphatases, PrpJ1 (formerly PrpJ) and PrpJ2; PrpJ1 is involved in heterocyst maturation. In this study, we show that PrpJ2, like PrpJ1, has Mn(2+)-dependent phosphatase activity. We further demonstrate that whereas prpJ2 is dispensable for cell growth under different nitrogen regimens tested, a double mutant with both prpJ1 and prpJ2 disrupted did not initiate heterocyst differentiation. Ectopic expression of hetR in the double mutant could rescue the failure to initiate heterocyst development, but the heterocysts formed, like those of a prpJ1 single mutant, were not mature. The expression of prpJ2 was enhanced during heterocyst development, and the upregulation of the gene was directly under the control of NtcA. Upregulation of both ntcA and hetR was affected in the double mutant. We propose that PrpJ1 and PrpJ2 together are required for mutual regulation of ntcA and hetR and are thus involved in regulation of the initiation of heterocyst differentiation.

Application of a histochemical method for detection of Ca2+ dependent acid phosphatase activity for studies of morpho-functional state of the cell structures in the rat brain

Application of a histochemical method for detection of Ca2+ dependent acid phosphatase activity for studies of morpho-functional state of the cell structures in the rat brain

Osmotic Stress and Viscous Retardation of the Na,K-ATPase Ion Pump

The transport function of the Na pump (Na,K-ATPase) in cellular ion homeostasis involves both nucleotide binding reactions in the cytoplasm and alternating aqueous exposure of inward- and outward-facing ion binding sites. An osmotically active, nonpenetrating polymer (poly(ethyleneglycol); PEG) and a modifier of the aqueous viscosity (glycerol) were used to probe the overall and partial enzymatic reactions of membranous Na,K-ATPase from shark salt glands. Both inhibit the steady-state Na,K-ATPase as well as Na-ATPase activity, whereas the K+-dependent phosphatase activity is little affected by up to 50% of either. Both Na,K-ATPase and Na-ATPase activities are inversely proportional to the viscosity of glycerol solutions in which the membranes are suspended, in accordance with Kramers’ theory for strong coupling of fluctuations at the active site to solvent mobility in the aqueous environment. PEG decreases the affinity for Tl+ (a congener for K+), whereas glycerol increases that for the nucleotides ATP and ADP in the presence of NaCl but has little effect on the affinity for Tl+. From the dependence on osmotic stress induced by PEG, the aqueous activation volume for the Na,K-ATPase reaction is estimated to be ∼5–6nm3 (i.e., ∼180 water molecules), approximately half this for Na-ATPase, and essentially zero for p-nitrophenol phosphatase. The change in aqueous hydrated volume associated with the binding of Tl+ is in the region of 9nm3. Analysis of 15 crystal structures of the homologous Ca-ATPase reveals an increase in PEG-inaccessible water space of ∼22nm3 between the E1-nucleotide bound forms and the E2-thapsigargin forms, showing that the experimental activation volumes for Na,K-ATPase are of a magnitude comparable to the overall change in hydration between the major E1 and E2 conformations of the Ca-ATPase.

The Saccharomyces cerevisiae PHM8 Gene Encodes a Soluble Magnesium-dependent Lysophosphatidic Acid Phosphatase

Phosphate is the essential macronutrient required for the growth of all organisms. In Saccharomyces cerevisiae, phosphatases are up-regulated, and the level of lysophosphatidic acid (LPA) is drastically decreased under phosphate-starved conditions. The reduction in the LPA level is attributed to PHM8, a gene of unknown function. phm8Delta yeast showed a decreased LPA-hydrolyzing activity under phosphate-limiting conditions. Overexpression of PHM8 in yeast resulted in an increase in the LPA phosphatase activity in vivo. In vitro assays of the purified recombinant Phm8p revealed magnesium-dependent LPA phosphatase activity, with maximal activity at pH 6.5. The purified Phm8p did not hydrolyze any lipid phosphates other than LPA. In silico analysis suggest that Phm8p is a soluble protein with no transmembrane domain. Site-directed mutational studies revealed that aspartate residues in a DXDXT motif are important for the catalysis. These findings indicated that LPA plays a direct role in phosphate starvation. This is the first report of the identification and characterization of magnesium-dependent soluble LPA phosphatase.

Progress in deciphering the biological role of the human soluble epoxide hydrolase phosphatase activity

The soluble epoxide hydrolase (sEH) is a unique enzyme in that it has two enzymatic activities on two separate domains: a C‐terminal epoxide hydrolase (Cterm‐EH), and a N‐terminal phosphatase (Nterm‐phos). The Cterm‐EH has been reported to hydrolyze epoxy‐fatty acids, especially epoxy‐eicosatrienoic acids (EETs) that are endogenous regulators of blood pressure and inflammation. Thus, Cterm‐EH inhibition is novel pharmacological approach to treat hypertension, pain and inflammation. The N‐terminal domain function is still not well known. It was thought to only help stabilize the C‐terminal domain. However, we recently described its magnesium dependent phosphatase activity on some lipid phosphates. We will report our progress on determining the substrate selectivity of the Nterm‐phos, as well as novel as specific inhibitors for this activity. The results will be discussed in light of possible interaction between the two domains, and biological roles of both sEH activities. This work was supported in part by NIEHS Grant R37 ES02710, NIEHS SBRP Grant P42 ES04699, and NIEHS CEHS Grant P30 ES05707.

Colorimetric determination of pure Mg 2+-dependent phosphatidate phosphatase activity

The malachite green-molybdate reagent was used for a colorimetric assay of pure Mg2 +-dependent phosphatidate phosphatase activity. This enzyme plays a major role in fat metabolism. Enzyme activity was linear with time and protein concentration, and with the concentration of water-soluble dioctanoyl phosphatidate. The colorimetric assay was used to examine enzyme inhibition by phenylglyoxal, propranolol, and dimethyl sulfoxide. Pure enzyme and a water-soluble phosphatidate substrate were required for the assay, which should be applicable to a well-defined large-scale screen of Mg2 +-dependent phosphatidate phosphatise inhibitors (or activators).