Alkaline Phosphatases: Structure, substrate specificity and functional relatedness to other members of a large superfamily of enzymes

Our knowledge of the structure and function of alkaline phosphatases has increased greatly in recent years. The crystal structure of the human placental isozyme has enabled us to probe salient features of the mammalian enzymes that differ from those of the bacterial enzymes. The availability of knockout mice deficient in each of the murine alkaline phosphatase isozymes has also given deep insights into their in vivo role. This has been particularly true for probing the biological role of bone alkaline phosphatase during skeletal mineralization. Due to space constraints this mini-review focuses exclusively on structural and functional features of mammalian alkaline phosphatases as identified by crystallography and probed by site-directed mutagenesis and kinetic analysis. An emphasis is also placed on the substrate specificity of alkaline phosphatases, their catalytic properties as phosphohydrolases as well as phosphodiesterases and their structural and functional relatedness to a large superfamily of enzymes that includes nucleotide pyrophosphatase/phosphodiesterase

Structure-activity relationships of anthraquinone derivatives derived from bromaminic acid as inhibitors of ectonucleoside triphosphate diphosphohydrolases (E-NTPDases)

Reactive blue 2 (RB-2) had been characterized as a relatively potent ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) inhibitor with some selectivity for NTPDase3. In search for the pharmacophore and to analyze structure-activity relationships we synthesized a series of truncated derivatives and analogs of RB-2, including 1-amino-2-sulfo-4-ar(alk)ylaminoanthraquinones, 1-amino-2-methyl-4-arylaminoanthraquinones, 1-amino-4-bromoanthraquinone 2-sulfonic acid esters and sulfonamides, and bis-(1-amino-4-bromoanthraquinone) sulfonamides, and investigated them in preparations of rat NTPDase1, 2, and 3 using a capillary electrophoresis assay. Several 1-amino-2-sulfo-4-ar(alk)ylaminoanthraquinone derivatives inhibited E-NTPDases in a concentration-dependent manner. The 2-sulfonate group was found to be required for inhibitory activity, since 2-methyl-substituted derivatives were inactive. 1-Amino-2-sulfo-4-p-chloroanilinoanthraquinone (18) was identified as a nonselective competitive blocker of NTPDases1, 2, and 3 (Ki 16–18 μM), while 1-amino-2-sulfo-4-(2-naphthylamino)anthraquinone (21) was a potent inhibitor with preference for NTPDase1 (Ki 0.328 μM) and NTPDase3 (Ki 2.22 μM). Its isomer, 1-amino-2-sulfo-4-(1-naphthylamino)anthraquinone (20), was a potent and selective inhibitor of rat NTPDase3 (Ki 1.5 μM)

Kinetic characterization of an ATP-diphosphohydrolase in alkaline phosphatase-free rat bone plate membranes

Membranas de placa óssea de rato foram preparadas a partir de implante de pó de osso desmineralizado no tecido subcutâneo de ratos Wistar e purificadas em coluna de Sepharose 4B (Cell. Mol. Biol. 32: 55, 1986). A remoção da fosfatase alcalina das membranas foi feita com fosfolipase C específica para fosfatidilinositol (Biochim. Biophys. Acta 1368: 108, 1998) e a atividade de nucleotídeo fosfatase determinada a 37°C, em tampão Hepes 50 mM, pH 7,5, contendo MgCl22 mM, teofilina 5 mM, em um volume final de 1 mL. Durante o processo de purificação das membranas livres de fosfatase alcalina, a razão entre as velocidades de hidrólise do ATP e do ADP permaneceu constante (da ordem 2,4) sugerindo que elas correspondem a uma mesma enzima. O mesmo perfil de pH foi observado para a hidrólise do ATP e ADP e o pH ótimo aparente é 7,5. Estudos de competição de substratos mostrou que, independentemente da relação entre as concentrações de ATP e ADP presentes no meio reacional, as atividades específicas sempre similares sugerem inequivocamente que a hidrólise do ATP e ADP representam reações competitivas para um único sítio na enzima. Uma significativa inibição da hidrólise do ATP e ADP pela enzima foi observada na presença de azida de sódio, um inibidor de apirases, em concentrações que variaram entre 2,5 mM (ADP) e 7,5 mM (ATP). Os valores dos parâmetros cinéticos calculados para a hidrólise dos diferentes substratos pela enzima mostram que a atividade NTFase é quase duas vezes maior que a correspondente NDFase enquanto a NMFase é nula ou desprezível. A preparação não está contaminada por fosfatases não específicas, ATP-pirofosfatase (EC 3.6.1.8), pirofosfatase inorgânica (EC 3.6.1.1), fosfodiesterases, adenilato quinase e proteína fosfatases. Em condições saturantes de ATP e magnésio, a atividade das membranas livres de fosfatase alcalina não é inibida pela oligomicina, ouabaína, bafilomicina A1 tapsigargina, omeprazol, ácido etacrínico e AP5A. Entretanto, a velocidade de hidrólise do ATP e ADP é reduzida cerca de 30% e 40%, respectivamente, na presença de suramina 1 mM, um antagonista do receptor de purina P2. Na ausência de íons magnésio ou cálcio, a hidrólise do ATP e ADP é desprezível enquanto na presença de íons cálcio ou magnésio a estimulação da atividade da enzima por tais íons é similar e cada íon pode substituir o outro durante o ciclo catalítico da enzima. A hidrólise do ATP e ADP diminuiu consideravelmente na presença de Polidocanol 1%, Lubrol WX 1,35%, Lubrol PX 1%, CHAPS 2,5%, C12E8 1%, Triton X-114 1%, Triton X-100 0,2%, n-octil-β-D-glicosídeo 30 mM, polioxietileno-5-decil éter 0,5%. Os resultados mais promissores foram obtidos com a adição conjunta de digitonina e dimiristoil L-α-fosfatidilcolina (DMPC). Dentre todos os detergentes utilizados, a digitonina, na proporção de 5:1 (mg/mL) foi mais eficaz na solubilização. A ATPase existente na membrana de placa óssea de rato e livre de fosfatase alcalina pode ser considerada uma ATP-difosfohidrolase por apresentar especificidade para nucleotídeos di e trifosfatos; não hidrolisar nucleotídeos monofosfato e fosfomonoésteres não nucleotídeos; ser ativada por íons cálcio ou magnésio; não ser inibida pelos inibidores de V-, Ca2+-, H+-, e Na+ ou K+-ATPases; ser inibida por altas concentrações de azida de sódio; apresentar a mesma faixa de pH para NTF e NDF; ser facilmente inativada por detergentes; apresentar valores similares de Km (100 µM) para o ATP e ADP.Rat osseous plate membranes obtained fourteen days after the implantation of demineralized bone particles in the subcutaneous tissue of Wistar rats, were prepared and purified on Sepharose 4B column (Cell. Mol. Biol. 32:55-62). Alkaline phosphatase-free membranes were prepared treating the rat osseous plate membranes with phosphatidylinositol-specific phospholipase C from B. thuringiensis (Biochim. Biophys. Acta 1368: 108, 1998). Nucleotide phosphatase activity was assayed at 37°C, in Hepes buffer 50 mM, pH 7,5, containing MgCl2 mM and theophylline 5 mM, in a final volume of 1 mL. The ATPase present in the membranes of osseous plate hydrolyzed nucleotide triphosphates (NTP) at a rate 2-fold higher than that of nucleotide diphosphates (NDP). However, the hydrolysis of nucleotide monophosphates was negligible. The apparent optimum pH of hydrolysis for NTP and NDP was 7.5. The enzyme hydrolyzed ATP following Michaelian kinetics, with V= 1,278.7 U/mg e Km = 83.3 µM. For ADP, it was also observed a single family of hydrolyzing sites (n= 1,0) with V= 473.9 U/mg e Km= 150.6 µM. In the absence of magnesium ions, the hydrolysis of ATP or ADP was negligible. A systematic study of the stimulation of the enzyme by calcium and magnesium ions suggested that any of these ions can replace the other during the catalytic cycle ofthe enzyme. Theophylline, a phosphodiesterase inhibitor, showed a negligible inhibition of the hydrolysis of ATP and ADP at concentrations up to 7 mM, but sodium azide was a effective inhibitor of ATP (7.5 mM) and ADP (2.5 mM) hydrolysis. On the other hand, the classical V-, P- and F-type ATPase inhibitors had no effect on the hydrolysis of ATP and ADP by the alkaline-free rat osseous plate membranes. A systematic study including a series of other inhibitors excluded the presence of contaminations by non specific phosphatases, inorganic pyrophosphatase, phosphodiesterase and protein phosphatase that could participate on the hydrolysis of NDP and NTP. AP5A had no effect on the hydrolysis of ATP and ADP by the enzyme, but suramin inhibited 30 and 40% the hydrolysis of ATP and ADP, respectively. The solubilization of the alkaline phosphatase-free rat osseous plate membrane ATPase with detergents commonly used to solubilize membrane proteins resulted in a significant depletion ofthe enzyme activity. The kinetic similarities of the alkaline phosphatase-free rat osseous plate membrane ATPase with respect to the hydrolysis of NTP and NDP suggested that it apparently functions as an ATP-diphosphohydrolase

Sulphated cholecystokinin-8 promotes CD36- mediated fatty acid uptake into primary mouse duodenal enterocytes

Cholecystokinin (CCK) is an archetypal incretin hormone secreted by intestinal enteroendocrine cells (EEC) in response to ingested nutrients. The aim of this study was to determine whether CCK modulates enterocyte fatty acid uptake by primary mouse duodenal cells. Exposure of primary mouse duodenal cells to 10 pM sulfated CCK-8 caused a two fold increase in dodecanoic acid fatty acid (FA) uptake. The selective CCK A receptor antagonist loxiglumide (100 μM) completely abolished the CCK-8 induced FA uptake. The CD36 fatty acid translocase-specific inhibitor sulfo-N-succinimidyl oleate (1 μM) also completely inhibited CCK-8 induced FA uptake, as did treatment with 200 μM phloretin. Together these data show CCK induces FA uptake into duodenal enterocytes; this action involves the CCK-RA receptor and is carrier mediated by CD36

Kinetic characterization of a membrane-specific ATPase from rat osseous plate and its possible significance on endochondral ossification

Treatment with phosphatidylinositol-specific phospholipase C of rat osseous plate membranes released up to 90-95% of alkaline phosphatase, but a specific ATPase activity (optimum pH = 7.5) remained bound to the membrane. The hydrolysis of ATP by this ATPase was negligible in the absence of magnesium or calcium ions. However, at millimolar concentrations of magnesium and calcium ions, the membrane-specific ATPase activity increased to about 560-600 U/mg, exhibiting two classes of ATP-hydrolysing sites, and site-site interactions. GTP, UTP, ITP, and CTP were also hydrolyzed by the membrane-specific ATPase. Oligomycin, ouabain, bafilomycin A(1), thapsigargin, omeprazole, ethacrynic acid and EDTA slightly affected membrane-specific ATPase activity while vanadate produced a 18% inhibition. The membrane-specific ATPase activity was insensitive to theophylline, but was inhibited 40% by levamisole. These data suggested that the membrane-specific ATPase activity present in osseous plate membranes, and alkaline phosphatase, were different proteins. (C) 1998 Elsevier B.V. B.V

Duodenal enteroendocrine I-cells contain mRNA transcripts encoding key endocannabinoid and fatty acid receptors

Enteroendocrine cells have a critical role in regulation of appetite and energy balance. I-cells are a subtype of enteroendocrine cells localized in duodenum that release cholecystokinin in response to ingested fat and amino-acids. Despite their potentially pivotal role in nutrient sensing and feeding behaviour, native I-cells have previously been difficult to isolate and study. Here we describe a robust protocol for the isolation and characterization of native duodenal I-cells and additionally, using semi-quantitative RT-PCR we determined that mouse duodenal I-cells contain mRNA transcripts encoding key fatty acid and endocannabinoid receptors including the long chain fatty acid receptors GPR40/FFAR1, GPR120/O3FAR1; short chain fatty acid receptors GPR41/FFAR3 and GPR43/FFAR2; the oleoylethanolamide receptor GPR119 and the classic endocannabinoid receptor CB1. These data suggest that I-cells sense a wide range of gut lumen nutrients and also have the capacity to respond to signals of fatty-acid derivatives or endocannabinoid peptides

Duodenal CCK cells from male mice express multiple hormones including ghrelin

AbstractEnteroendocrine (EEC) cells have a pivotal role in intestinal nutrient sensing and release hormones that orchestrate food digestion and regulate appetite. EEC cells are found scattered throughout the intestine and have typically been classified based on the primary hormone they contain. I cells represent a subset of EEC cells that secrete cholecystokinin (CCK) and are mainly localized to the duodenum. Recent studies have shown that I cells express mRNAs encoding several gut hormones. In this study, we investigated the hormonal profile of murine fluorescence-activated cell sorting-sorted duodenal I cells using semiquantitative RT-PCR, liquid chromatography tandem mass spectrometry, and immunostaining methods. We report that I cells are enriched in mRNA transcripts encoding CCK and also other key gut hormones, including neurotensin, glucose-dependent insulinotropic peptide (GIP), secretin, peptide YY, proglucagon, and ghrelin (Ghrl). Furthermore, liquid chromatography tandem mass spectrometry analysis of fluorescence-activated cell sorting-purified I cells and immunostaining confirmed the presence of these gut hormones in duodenal I cells. Immunostaining highlighted that subsets of I cells in both crypts and villi coexpress differential amounts of CCK, Ghrl, GIP, or peptide YY, indicating that a proportion of I cells contain several hormones during maturation and when fully differentiated. Our results reveal that although I cells express several key gut hormones, including GIP or proglucagon, and thus have a considerable overlap with classically defined K and L cells, approximately half express Ghrl, suggesting a potentially important subset of duodenal EEC cells that require further consideration.</jats:p