Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Kinetic parameters for the hydrolysis of a number of physiologically important phosphoesters by purified human liver alkaline phosphatase have been determined. The enzyme was studied at pH values of 7.0 to 10.0. The affinity of the enzyme for the compounds was determined by competition experiments and by their direct employment as substrates. Phosphodiesters and phosphonates were not hydrolysed but the latter were inhibitors. Calcium and magnesium ions inhibited the hydrolysis of ATP and PP1 and evidence is presented to show that the metal complexes of these substrates are not hydrolysed by alkaline phosphatase. A calcium-stimulated ATPase activity could not be demonstrated for the purified enzyme or the enzyme in the presence of a calcium-dependent regulator protein. Nevertheless, the influence of magnesium and calcium ions on the ATPase activity of alkaline phosphatase means that precautions must be taken when assaying for Ca2+-ATPase in the presence of alkaline phosphatase. The low substrate Km values and the hydrolysis which occurs at pH 7.4 mean that the enzyme could have a significant phosphohydrolytic role. However, liver cell phosphate concentrations, if accessible to the enzyme, are sufficient to strongly inhibit this activity.
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PMID:Phosphoester specificity of purified human liver alkaline phosphatase. 3 70

A case of adult hypophosphatasia under treatment with a high orthophosphate (P1) intake is described. The patient is a 53-year-old woman. Her symptoms have progressed for seven years, and it has been necessary to perform osteosynthesis of both crura. The diagnosis rests upon a characteristic clinical picture, low serum alkaline phosphatase activity, high urinary excretion of phosphoethanolamine, and an invariably elevated concentration of inorganic pyrophosphate (PP1) in plasma accompanied by a very high excretion of this compound in the urine. An improved technique allowed specific determinations of microquantities of PP1 in biologic materials. The concentrations of PP1 in the plasma and urine remained unchanged when the patient's intake of phosphorus was increased to 1.98 g/day. The PP1/P1 ratio in the urine was 10-20 before treatment. During treatment P1 excretion increased. PP1 excretion did not change, and the ratio decreased to around 7. The renal tubular transport of PP1 probably was saturated, and therefore PP1, which was circulating in abnormally high concentrations in the patient's fluids, could not be removed by loading with P1. Four months of treatment did not benefit the patient.
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PMID:Adult hypophosphatasia. 16 53

A survey of the hydrolytic activity of alkaline phosphatase (EC 3.1.3.1) reveals that PP1, like phosphomonoesters, can serve as substrate in vitro. This pp1-phosphohydrolytic activity can be distinguished from PP1-phosphohydrolytic activities of inorganic pyrophosphatases (EC 3.6.1.1) and glucose-6-phosphatase (EC 3.1.3.9) by several criteria. Discrimination among these hydrolytic enzymes is possible by their dependence on variation of pH and of magnesium to PP1 ratios in the assay solutions. The true substrates and modifiers are not simply PP1 and magnesium, but the equilibrium species in mixtures of these two. The physiological significance of each of the three enzymes is not predictable from their differential efficiency as catalysts of PP1-hydrolysis in vitro.
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PMID:Enzyme catalyzed hydrolysis of inorganic pyrophosphate. Current view of problems in characterization of PP1-phosphohydrolytic activity associated with alkaline phosphatases (EC 3.1.3.1). 23 7

The properties of a highly purified inorganic pyrophosphatase (pyrophosphate phosphohydrolase; EC 3.6.1.1) from pig scapula cartilage were studied. The enzyme had a molecular weight of 66 000 and a pH optimum of 7-8. It was markedly activated by magnesium, but not, or only to a much smaller degree, by other metal ions. PP1 was the only substrate found and had a Km value of 11 muM. The enzyme was not inhibited by phosphate and other inhibitors of alkaline phosphatase such as CN- minus, amino acids and theophylline; it was slightly inhibited by tartrate, formaldehyde and ammonium molybdate and strongly inhibited by F- minus, Ca2+ and other metal ions. The properties of the enzyme in the presence of concentrations of PP1 present in plasma (3.5 muM) were similar to those found at higher (2 mM) concentrations of PP1. The diphosphonates ethane-1-hydroxy-1,1-diphosphonate and dichloromethylenediphosphonate inhibited the enzyme in the presence of low PP1 concentrations. The characteristics of this enzyme are therefore similar to pyrophosphatases from other sources, such as from yeast and erythrocytes, and do not support a specific role of this enzyme in the calcification process.
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PMID:Properties of inorganic pyrophosphatase of pig scapula cartilage. 23 96

The present study characterizes the inhibitory effects of nodularin, a recently isolated hepatotoxic compound from the cyanobacterium Nodularia spumigena, on type 1 (PP1), type 2A, (PP2A), type 2B (PP2B), and type 2C (PP2C) protein phosphatases. Both PP2A and PP1 were potently inhibited (IC50 = 0.026 and 1.8 nM, respectively) by nodularin, whereas PP2B was inhibited to a lesser extent (IC50 = 8.7 microM). Nodularin had no apparent effect on PP2C, alkaline phosphatase, acid phosphatase, insulin receptor tyrosine kinase, protein kinase A, phosphorylase kinase, or protein kinase C. In a whole-cell extract of T51B liver cells, nodularin inhibited PP1 and PP2A activity with a potency similar to that seen with their purified catalytic subunits. Thus, due to the high specificity of nodularin for PP2A and PP1, this hepatotoxin may prove to be useful as a probe for distinguishing the activity of these protein phosphatases in cell extracts.
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PMID:Cyanobacterial nodularin is a potent inhibitor of type 1 and type 2A protein phosphatases. 165 93

To determine which factors may regulate the DNA binding and transcriptional properties of retinoic acid receptors (RARs and RXRs), we investigated the sensitivity of reporter genes bearing various retinoic acid response elements (RAREs) to protein phosphatases (PPases) inhibition. PPases inhibition by okadaic acid led to an increase of the reporter genes activity in a RARE-dependent and ligand-independent manner and was dependent on the type of response element used. Overexpression of protein phosphatases 2A and 1 (PP2A and PP1) decreased the inducibility of the reporter genes tested. Nuclear extracts from okadaic acid-treated COS cells displayed an 2-5-fold increased level of receptor binding to RAREs in vitro, suggesting that PPases inhibition increased the DNA binding activity of retinoid receptors. Treatment of receptors extracted from COS cells by alkaline phosphatase and partially purified PP1 and PP2A decreased their DNA binding activity, but heterodimers bound to DNA were not sensitive to phosphatase treatment. Reconstitution experiments showed that phosphorylation of both receptors increased the DNA binding activity of RXR/RAR heterodimers. Taken together, these data show that the modulation of the phosphorylation state of RARs and RXRs represents an other level of regulation of the retinoid signaling pathway.
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PMID:Protein phosphatases 1 and 2A regulate the transcriptional and DNA binding activities of retinoic acid receptors. 773 17

The Wcs120 gene encodes a highly abundant protein which appears to play an important role during cold acclimation of wheat. To understand the regulatory mechanism controlling its expression at low temperature, the promoter region has been characterized. Electrophoretic mobility shift assays using short promoter fragments revealed the presence in nuclear extracts from non-acclimated (NA) plants of multiple DNA-binding proteins which interact with several elements. In contrast, no DNA-binding activity was observed in the nuclear extracts from cold-acclimated (CA) plants. In vitro dephosphorylation of these CA nuclear extracts with alkaline phosphatase restored the binding activity. Moreover, okadaic acid (a potent phosphatase inhibitor) markedly stimulated the in vivo accumulation of the WCS120 family of proteins. This suggests that protein phosphatases PP1 and/or PP2A negatively regulate the expression of the Wcs120 gene. In addition, both Ca(2+)-dependent and Ca(2+)-independent kinase activities were found to be significantly higher in the CA nuclear extracts. Western analysis using antibodies directed against protein kinase C (PKC) isoforms showed that a PKCgamma homolog (84 kDa) is selectively translocated into the nucleus in response to low temperature. Taken together, our results suggest that, in vivo, the expression of the Wcs120 gene may be regulated by nuclear factors whose binding activity is modulated by a phosphorylation/dephosphorylation mechanism.
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PMID:Low temperature-stimulated phosphorylation regulates the binding of nuclear factors to the promoter of Wcs120, a cold-specific gene in wheat. 949 Oct 74

1. The aim of this study was to characterize further the two main metabolic pathways of regulation of the Na+-Ca2+ exchanger in squid axons induced by its two naturally ocurring high-energy compounds: ATP and phosphoarginine (Pa). [Na+]o-dependent Ca2+ efflux (forward Na+o-Ca2+i exchange) and [Ca2+]o-dependent Ca2+ efflux (Ca2+o-Ca2+i exchange) were measured in internally dialysed squid axons at 16-17 C. 2. Measurements of changes in the apparent affinity of the Na+-Ca2+ exchanger for transporting (Na+o, Na+i, Ca2+o, Ca2+i) and regulatory (Ca2+i) ions induced by ATP and Pa show marked differences for the two substrates: (i) ATP strongly alters the affinity for Na+o and Na+i, while Pa does not, and (ii) in the absence of Na+i, ATP has no stimulatiory effect; on the other hand, Pa causes a dramatic increase in Na+o-Ca2+i exchange with little activation of Ca2+o-Ca2+i exchange. 3. The MgATP analogue chromium-ATP (CrATP) completely inhibits MgATP stimulation of the Na+-Ca2+ exchanger. Nevertheless, even with the effects of the nucleotide blocked, Pa exhibits its usual activation of the [Na+]o-dependent Ca2+ efflux. 4. None of the classical serine-threonine-tyrosine kinase inhibitors, nor the PP1 and PP2 phosphatase inhibitors, affects either the ATP or the Pa effect. However, intracellular microinjections of an exogenous phosphatase (alkaline phosphatase) completely reverses the stimulation of the Na+-Ca2+ exchange induced by ATP and Pa. 5. Prolonged intracellular dialysis with highly permeable porous capillaries (18 kDa molecular weight cut-off), which normally induces a complete run-down of the MgATP effect, does not alter the Pa stimulation of the exchanger, even after 6 h of continuous dialysis. 6. We conclude that the ATP and Pa modulation of Na+-Ca2+ exchange in an invertebrate nerve fibre are two genuinely different mechanisms, which affect the carrier properties in very different ways. An interesting similarity between ATP and Pa is that a phosphorylation-dephosphorylation process seems to be a common feature of these two regulation modes.
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PMID:Differential up-regulation of Na+-Ca2+ exchange by phosphoarginine and ATP in dialysed squid axons. 950 35

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel activity declines rapidly when excised from transfected Chinese hamster ovary (CHO) or human airway cells because of membrane-associated phosphatase activity. In the present study, we found that CFTR channels usually remained active in patches excised from baby hamster kidney (BHK) cells overexpressing CFTR. Those patches with stable channel activity were used to investigate the regulation of CFTR by exogenous protein phosphatases (PP). Adding PP2A, PP2C, or alkaline phosphatase to excised patches reduced CFTR channel activity by > 90% but did not abolish it completely. PP2B caused weak deactivation, whereas PP1 had no detectable effect on open probability (Po). Interestingly, the time course of deactivation by PP2C was identical to that of the spontaneous rundown observed in some patches after excision. PP2C and PP2A had distinct effects on channel gating Po declined during exposure to exogenous PP2C (and during spontaneous rundown, when it was observed) without any change in mean burst duration. By contrast, deactivation by exogenous PP2A was associated with a dramatic shortening of burst duration similar to that reported previously in patches from cardiac cells during deactivation of CFTR by endogenous phosphatases. Rundown of CFTR-mediated current across intact T84 epithelial cell monolayers was insensitive to toxic levels of the PP2A inhibitor calyculin A. These results demonstrate that exogenous PP2C is a potent regulator of CFTR activity, that its effects on single-channel gating are distinct from those of PP2A but similar to those of endogenous phosphatases in CHO, BHK, and T84 epithelial cells, and that multiple protein phosphatases may be required for complete deactivation of CFTR channels.
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PMID:Differential regulation of single CFTR channels by PP2C, PP2A, and other phosphatases. 961 28

The purpose of this study was to relate dose-dependent hepatotoxicity stemming from prolonged exposure to sublethal concentrations of the cyclic heptapeptide microcystin-LR (Mcyst) to hepatic Mcyst concentrations and protein phosphatase activity. Mcyst is a potent inhibitor of protein phosphatase types 1 and 2A (PP1 and PP2A). Twenty male Sprague-Dawley rats were infused continuously with 0, 3, 6, or 9 micrograms Mcyst/day for 28 days using intraperitoneal mini-osmotic pumps containing highly purified toxin or saline. At the end of 28 days, dose-dependent increases in several serum biochemical tests including sorbitol dehydrogenase, aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase, and bile acids had occurred. Serum albumin decreased in a dose-dependent fashion. Liver activity of both PP1 and PP2A decreased in a dose-dependent manner, but with a relatively greater effect on PP2A than PP1. Liver cytosol Mcyst concentrations, measured by direct competitive ELISA, also increased in a dose-dependent manner, although at a higher rate than would be predicted from the incremental increase in dose given. This disproportional increase is suggestive of the bioaccumulation of Mcyst with increasing dose. Histopathological abnormalities included hepatocellular apoptosis and cytosolic vacuolation of principally zone 3 hepatocytes. Immunohistochemical stains revealed Mcyst predominantly within pericanalicular regions of zone 3 hepatocytes. It was concluded that prolonged exposure to sublethal concentrations of Mcyst results in multiple dose-dependent hepatotoxic effects that correspond to decreased hepatic serine/threonine protein phosphatase activity and increasing cytosolic Mcyst concentrations. The disproportional increase of hepatic Mcyst concentrations observed may suggest the bioaccumulation of toxin and an increasing relative risk of hepatotoxicity with increasing dose.
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PMID:Prolonged sublethal exposure to the protein phosphatase inhibitor microcystin-LR results in multiple dose-dependent hepatotoxic effects. 972 Jan 45


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