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)

Differentiating chick limb-bud mesenchymal cells plated in micromass culture form a cartilage matrix that can be mineralized in the presence of 4 mM inorganic phosphate (Pi), and 1 mM calcium. Previous studies showed that when beta-glycerophosphate (beta GP) is used in place of Pi, the mineral crystals formed are larger and differ in distribution. The present study shows that the difference in distribution is not associated with alterations in cell proliferation, protein synthesis, or with collagen, proteoglycan core protein, or alkaline phosphatase gene expression. Cultures with 2.5, 5, and 10 mM beta GP did show different levels of alkaline phosphatase activity, and in the presence of low (0.3 mM) Ca had different Pi contents (4, 6 and 9 mM, respectively), indicating that the increase in CaxP product may in part be responsible for the altered pattern of mineralization. However, cultures with beta GP in which alkaline phosphatase activity was inhibited with levamisole still had an altered mineral distribution as revealed by Fourier transform-infrared (FT-IR) microspectroscopy. The presence of a casein kinase II-like activity in the mineralizing cultures, the ability of specific inhibitors of this enzyme to block mineralization, and the known ability of beta GP to block phosphoprotein phosphatase activity suggests that altered patterns of matrix protein phosphorylation may influence mineral deposition in these cultures.
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PMID:The mechanism of beta-glycerophosphate action in mineralizing chick limb-bud mesenchymal cell cultures. 891 77

In this paper, specific PHO13 alkaline phosphatase from Saccharomyces cerevisiae was demonstrated to possess phosphoprotein phosphatase activity on the phosphoseryl proteins histone II-A and casein. The enzyme is a monomeric protein with molecular mass of 60 kDa and hydrolyzes p-nitrophenyl phosphate with maximal activity at pH 8.2 with strong dependence on Mg2+ ions and an apparent Km of 3.6 x 10(-5) M. No other substrates tested except phosphorylated histone II-A and casein were hydrolyzed at any significant rate. These data suggest that the physiological role of the p-nitrophenyl phosphate-specific phosphatase may involve participation in reversible protein phosphorylation. 1988 Federation of European Microbiological Societies.
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PMID:A specific alkaline phosphatase from Saccharomyces cerevisiae with protein phosphatase activity. 956 42

A chimeric protein containing the uncleaved signal sequence of plasminogen activators inhibitor-2 (PAI2) fused to alkaline phosphatase (AP) interferes with Escherichia coli protein export and arrests growth. Suppressors of this toxicity include secG mutations that define the Thr-41-Leu-42-Phe-43 (TLF) domain of SecG. These mutations slow down the export of PAI2-AP. Another construct encoding a truncated PAI2 signal sequence (hB-AP) is also toxic. Most suppressors exert their effect on both chimeric proteins. We describe here five secG suppressors that only suppress the toxicity of hB-AP and selectively slow down its export. These mutations do not alter the TLF domain: three encode truncated SecG, whereas two introduce Arg residues in the transmembrane domains of SecG. The shortest truncated protein only contains 13 residues of SecG, suggesting that the mutation is equivalent to a null allele. Indeed, a secG disruption selectively suppresses the toxicity of hB-AP. However, the missense mutations are not null alleles. They allow SecG binding to SecYE, although with reduced affinity. Furthermore, these mutated SecG are functional, as they facilitate the export of endogenous proteins. Thus, SecG participates in signal sequence recognition, and both transmembrane domains of SecG contribute to ensure normal signal sequence recognition by the translocase.
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PMID:Both transmembrane domains of SecG contribute to signal sequence recognition by the Escherichia coli protein export machinery. 1106 81

Sustained activation of poly(ADP-ribose) polymerase-1 (PARP-1) and extracellular signal-regulated kinases 1/2 (ERK1/2) both promote neuronal death. Here we identify a direct link between these two cell death pathways. In a rat model of hypoglycemic brain injury, neuronal PARP-1 activation and subsequent neuronal death were blocked by the ERK1/2 inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059). In neuron cultures, PARP-1-mediated neuronal death induced by N-methyl-d-aspartate, peroxynitrite, or DNA alkylation was similarly blocked by ERK1/2 pathway inhibitors. These inhibitors also blocked PARP-1 activation and PARP-1-mediated death in astrocytes. siRNA down-regulation of ERK2 expression in astrocytes also blocked PARP-1 activation and cell death. Direct effects of ERK1/2 on PARP-1 were evaluated by using isolated recombinant enzymes. The activity of recombinant human PARP-1 was reduced by incubation with alkaline phosphatase and restored by incubation with active ERK1 or ERK2. Putative ERK1/2 phosphorylation sites on PARP-1 were identified by mass spectrometry. Using site-directed mutagenesis, these sites were replaced with alanine (S372A and T373A) to block phosphorylation, or with glutamate (S372E and T373E) to mimic constitutive phosphorylation. Transfection of PARP-1 deficient mouse embryonic fibroblasts with the mutant PARP-1 species showed that the S372A and T373A mutations impaired PARP-1 activation, whereas the S372E and T373E mutations increased PARP-1 activity and eliminated the effect of ERK1/2 inhibitors on PARP-1 activation. These results suggest that PARP1 phosphorylation by ERK1/2 is required for maximal PARP-1 activation after DNA damage.
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PMID:Direct phosphorylation and regulation of poly(ADP-ribose) polymerase-1 by extracellular signal-regulated kinases 1/2. 1662 22

A soybean histone-type protein kinase was used to prepare (32)P-labeled histone H1 as substrate for purification and characterization of a phosphoprotein phosphatase (EC 3.1.3.16) from soybean hypocotyls. The phosphatase has been purified 169-fold by ammonium sulfate fractionation, ethanol precipitation, and chromatography on Sephadex G-150, DEAE-Sephadex A-25 and Sephadex G-100. The activity of the phosphoprotein phosphatase is distinct from that of acid and alkaline phosphatases (EC 3.1.3.1) as well as from that of nucleotidases. The final enzyme preparation does not contain histone protease activity, although it can be detected during the early stages of purification. The protease(s) apparently can attack phosphorylated histone H1, indicating that phosphorylation does not protect the protein against proteolytic degradation.The amounts of (32)P released from [(32)P]histone H1 are proportionally recovered as [(32)P]Pi, indicating that the dephosphorylation is due to the action of phosphoprotein phosphatase. The enzyme shows maximal activity at pH 7 to 8 and has a specific activity of 19 nanomoles of [(32)P]Pi released from [(32)P]histone H1 per minute per milligram of protein. The apparent K(m) for phosphohistone H1 is 4.0 +/- 0.4 micromolar. The estimated molecular weight of the enzyme is approximately 30,000 by gel filtration. The enzyme activity does not depend upon the addition of reducing agent and metal ion. Zn(2+), Co(2+), NaF, pyrophosphate, or ATP at 1 millimolar, however, inhibits the enzyme activity by about 70%. The enzyme activity is unaffected by cyclic nucleotides and plant growth substances but is inhibited by polyamines. All the phosphorylated histone species and protamine, not low molecular weight phosphoesters, act as competitive inhibitors for the dephosphorylation of [(32)P]histone H1.Besides its action on phosphohistone H1, the soybean enzyme also catalyzes the dephosphorylation of other phosphohistone species (H2A, H2B, H3, and H4), degraded phosphohistone H1, and possibly phosphorylated casein and phosvitin. All these results indicate that the enzyme is a nonspecific phosphoprotein phosphatase.
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PMID:Phosphoprotein Phosphatase of Soybean Hypocotyls: PURIFICATION, PROPERTIES, AND SUBSTRATE SPECIFICITIES . 1666 38

Endogenous dephosphorylation of the light-harvesting chlorophyll-protein complex of photosystem II in pea (Pisum sativum, L. cv Progress 9) thylakoids drives the state 2 to state 1 transition; the responsible enzyme is a thylakoid-bound, fluoride-sensitive phosphatase with a pH optimum of 8.0 (Bennett J [1980] Eur J Biochem 104: 85-89). An enzyme with these characteristics was isolated from well-washed thylakoids. Its molecular mass was estimated at 51.5 kD, and this monomer was catalytically active, although the activity was labile. The active site could be labeled with orthophosphate at pH 5.0. High levels of alkaline phosphatase activity were obtained with the assay substrate, 4-methylumbelliferyl phosphate (350 micromoles per minute per milligram purified enzyme). The isolated enzyme functioned as a phosphoprotein phosphatase toward phosphorylated histone III-S and phosphorylated, photosystem II-enriched particles from pea, with typical activities in the range of 200 to 600 picomoles per minute per milligram enzyme. These activities all had a pH optimum of 8.0 and were fluoride sensitive. The enzyme required magnesium ion for maximal activity but was not dependent on this ion. Evidence supporting a putative function for this phosphatase in dephosphorylation of thylakoid proteins came from the inhibition of this process by a polyclonal antibody preparation raised against the partially purified enzyme.
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PMID:Isolation and characterization of an alkaline phosphatase from pea thylakoids. 1666 67

Protein phosphorylation and dephosphorylation are important regulators of cellular and extracellular events. The purpose of this study was to define how these events regulate cartilage matrix calcification in a cell culture system that mimics endochondral ossification. The presence of casein kinase II (CK2), an enzyme known to phosphorylate matrix proteins, was confirmed by immunohistochemistry. The importance of phosphoprotein phosphorylation and dephosphorylation was examined by comparing effects of inhibiting CK2 or phosphoprotein phosphatases on mineral accretion relative to untreated mineralizing controls. Specific inhibitors were added to differentiating chick limb-bud mesenchymal cell micromass cultures during the development of a mineralized matrix at the times of cell differentiation, proliferation, formation of the mineralized matrix, or proliferation of the mineral crystals. The mineralizing media for these cultures contained 4 mM inorganic phosphate and no organic-phosphate esters; control cultures had 1 mM inorganic phosphate. Mineralization was monitored based on (45)Ca uptake and infrared characterization of the mineral; cell viability was assessed by three independent methods. Treatments that caused cell toxicity were excluded from the analysis. Inhibition of CK2 activity with apigenin or CK2 inhibitor II reduced the rate of mineral deposition, but did not block mineral accretion. Effects were greatest during the time of mineralized matrix formation. Inhibition of phosphoprotein phosphatase activities with okadaic acid, calyculin A, and microcystin-LR, at early time points also markedly inhibited mineral accretion. Inhibition after mineralization had commenced increased the mineral yield. Levamisole, an alkaline phosphatase inhibitor, had no effect on mineral accretion in this system, suggesting the involvement of other phosphatases. Adding additional inorganic phosphate to the inhibited cultures after mineralization had started, but not earlier, reversed the inhibition indicating that the phosphatases were, in part, providing a source of inorganic phosphate. To characterize the roles of specific phosphoproteins blocking studies were performed. Blocking with anti-osteopontin antibody confirmed osteopontin's previously reported role as a mineralization inhibitor. Blocking antibodies to bone sialoprotein added from day 9 or on days 9 and 11 retarded mineralization, supporting its role as a mineralization nucleator. Antibodies to osteonectin slightly stimulated early mineralization, but had no effect after the time that initial mineral deposition occurs. Taken together, the results of this study demonstrate the importance of the phosphorylation state of extracellular matrix proteins in regulating mineralization in this culture system.
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PMID:Modulation of extracellular matrix protein phosphorylation alters mineralization in differentiating chick limb-bud mesenchymal cell micromass cultures. 1839 25

Syringetin (3,5,7,4'-tetrahydroxy-3',5'dimethoxyflavone), a flavonoid derivative, is present in grape and wine. By means of alkaline phosphatase (ALP) activity, osteocalcin, and type I collagen ELISA, we have shown that syringetin exhibits a significant induction of differentiation in MC3T3-E1 mouse calvaria osteoblasts and human fetal osteoblastic 1.19 cell line human osteoblasts. ALP and osteocalcin are phenotypic markers for early-stage differentiated osteoblasts and terminally differentiated osteoblasts, respectively. Our results indicate that syringetin stimulates osteoblast differentiation at various stages, from maturation to terminally differentiated osteoblasts. Induction of differentiation by syringetin is associated with increased bone morphogenetic protein-2 (BMP-2) production. The BMP-2 antagonist noggin blocked syringetin-mediated ALP activity and osteocalcin secretion enhancement, indicating that BMP-2 production is required in syringetin-mediated osteoblast maturation and differentiation. Induction of differentiation by syringetin is associated with increased activation of SMAD1/5/8 and extracellular signal-regulated kinase 1/2 (ERK1/2). Cotreatment of ERK1/2 inhibitor 2'-amino-3'-methoxyflavone inhibited syringetin-mediated ALP upregulation and osteocalcin production. In conclusion, syringetin increased BMP-2 synthesis, and subsequently activated SMAD1/5/8 and ERK1/2, and this effect may contribute to its action on the induction of osteoblast maturation and differentiation, followed by an increase of bone mass.
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PMID:Syringetin, a flavonoid derivative in grape and wine, induces human osteoblast differentiation through bone morphogenetic protein-2/extracellular signal-regulated kinase 1/2 pathway. 1978 98

BACE-1 has been shown to be an attractive therapeutic target in Alzheimer's disease (AD). Using a 1,4-dihydropyridine (DHP) scaffold, we synthesized new inhibitors of BACE-1 by modifying the known BACE inhibitor 2 containing a hydroxyethylamine (HEA) motif. Using structure-based drug design based on computer-aided molecular docking, the isophthalamide ring of 2 was replaced with a 1,4-dihydropyridine ring as a brain-targeting strategy. Several of the new dihydropyridine derivatives were synthesized and their BACE-1-inhibitory activities were evaluated using a cell-based, reporter gene assay system that measures the cleavage of alkaline phosphatase (AP)-APP fusion protein by BACE-1. Most of the 1,4-DHP analogs showed BACE-1-inhibitory activities with IC50 values in the range 8-30 microM, suggesting that the 1,4-DHP skeleton may be utilized to develop brain-targeting BACE-1 inhibitors.
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PMID:Design and synthesis of 1,4-dihydropyridine derivatives as BACE-1 inhibitors. 2030 26

The process of vascular calcification shares many similarities with that of physiological skeletal mineralization, and involves the deposition of hydroxyapatite crystals in arteries. However, the cellular mechanisms responsible have yet to be fully explained. Bone morphogenetic protein (BMP-9) has been shown to exert direct effects on both bone development and vascular function. In the present study, we have investigated the role of BMP-9 in vascular smooth muscle cell (VSMC) calcification. Vessel calcification in chronic kidney disease (CKD) begins pre-dialysis, with factors specific to the dialysis milieu triggering accelerated calcification. Intriguingly, BMP-9 was markedly elevated in serum from CKD children on dialysis. Furthermore, in vitro studies revealed that BMP-9 treatment causes a significant increase in VSMC calcium content, alkaline phosphatase (ALP) activity and mRNA expression of osteogenic markers. BMP-9-induced calcium deposition was significantly reduced following treatment with the ALP inhibitor 2,5-Dimethoxy-N-(quinolin-3-yl) benzenesulfonamide confirming the mediatory role of ALP in this process. The inhibition of ALK1 signalling using a soluble chimeric protein significantly reduced calcium deposition and ALP activity, confirming that BMP-9 is a physiological ALK1 ligand. Signal transduction studies revealed that BMP-9 induced Smad2, Smad3 and Smad1/5/8 phosphorylation. As these Smad proteins directly bind to Smad4 to activate target genes, siRNA studies were subsequently undertaken to examine the functional role of Smad4 in VSMC calcification. Smad4-siRNA transfection induced a significant reduction in ALP activity and calcium deposition. These novel data demonstrate that BMP-9 induces VSMC osteogenic differentiation and calcification via ALK1, Smad and ALP dependent mechanisms. This may identify new potential therapeutic strategies for clinical intervention.
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PMID:BMP-9 regulates the osteoblastic differentiation and calcification of vascular smooth muscle cells through an ALK1 mediated pathway. 2529 51


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