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)

The hallmark of biological mineralization is the precise regulation of mineral deposition in space and time. The cells which produce mineralized tissues are themselves controlled by developmental programs and hormonal signals which result in regulation of gene expression and modulation of protein function. These signals are transduced into changes in enzyme levels and/or activity. Upon activation, cellular enzymes then act to synthesize the organic matrix and process it extracellularly, utilize metabolic energy to transport ions from the blood to the matrix, and to initiate the mineralization cascade. The first enzyme activity described in mineralizing tissues was alkaline phosphatase and it is still the best characterized enzyme in the mineralization process. Yet, important questions about the role of this protein remain unanswered, and it continues to occupy a central focus in mineralized tissue investigation. Other phosphatases, including protein tyrosine phosphatases are important in regulating tyrosine kinase mediated signals. Investigators have now begun to look closely at several groups of kinases which are also important for proper mineralization. As peptide hormones are important modulators of mineralized tissues, protein kinase A has always been presumed to play a key role in phosphorylating intracellular proteins. There is also considerable interest in protein kinase C, as well as tyrosine kinases in mineralized tissue signal transduction. Another group of kinases important in mineralized tissues are the enzymes which phosphorylate the matrix phosphoproteins. Of these, casein kinase II appears to be involved in intracellular and extracellular protein phosphorylation. Several enzymes present in the premineralized matrix are thought to be significant in triggering mineralization. Alkaline phosphatase may act at this level, but new data also suggests that metalloproteases and gelatinases, by modifying or digesting matrix components, may be important in the initiation of calcification.
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PMID:Enzymes in mineralizing systems: state of the art. 908 56

Microtubules are important for the growth and maintenance of stable neuronal processes and their organisation is controlled partly by microtubule-associated proteins (MAPs). MAP 1B is the first MAP to be expressed in neurons and plays an important role in neurite outgrowth. MAP 1B is phosphorylated at multiple sites and it is believed that the function of the protein is regulated by its phosphorylation state. We have shown that the monoclonal antibody (mAb) RT97, which recognises phosphorylated epitopes on neurofilament proteins, fetal tau, and on Alzheimer's paired helical filament-tau, also recognises a developmentally regulated phosphorylation epitope on MAP 1B. In the rat cerebellum, Western blot analysis shows that mAb RT97 recognises the upper band of the MAP 1B doublet and that the amount of this epitope peaks very early postnatally and decreases with increasing age so that it is absent in the adult, despite the continued expression of MAP 1B in the adult. We confirmed that mAb RT97 binds to MAP 1B by showing that it recognises MAP 1B immunoprecipitated from postnatal rat cerebellum using polyclonal antibodies to recombinant MAP 1B proteins. We established that the RT97 epitope on MAP 1B is phosphorylated by showing that antibody binding was abolished by alkaline phosphatase treatment of immunoblots. Epitope mapping experiments suggest that the mAb RT97 site on MAP 1B is near the N-terminus of the molecule. Despite our immunoblotting data, immunostaining of sections of postnatal rat cerebellum with mAb RT97 shows a staining pattern typical of neurofilaments with no apparent staining of MAP 1B. For instance, basket cell axons and axons in the granule cell layer and white matter stained, whereas parallel fibres did not. These results suggest that the MAP 1B epitope is masked or lost under the immunocytochemical conditions in which the cerebellar sections are prepared. The upper band of the MAP 1B doublet is believed to be predominantly phosphorylated by proline-directed protein kinases (PDPKs). PDPKs are also good candidates for phosphorylating neurofilament proteins and tau and therefore we postulate that the sites recognised by RT97 on these neuronal cytoskeletal proteins may be phosphorylated by similar kinases. Important goals are to determine the precise location of the RT97 epitope on MAP 1B and the kinase responsible.
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PMID:The neurofilament antibody RT97 recognises a developmentally regulated phosphorylation epitope on microtubule-associated protein 1B. 930 99

The activities of enzymes involved in lipid metabolism--phospholipase A2 (PLA2) and phosphatidylethanolamine N-methyltransferase (PE N-MTase)--were found to be differently affected by pre-incubation of rod outer segments (ROS) under protein phosphorylating or dephosphorylating conditions. Exposure to cAMP-dependent protein kinase (PKA), under dark or light conditions, produced a significant increase in PE N-MTase activity, whereas PLA2 activity decreased. Under standard protein kinase C (PKC) phosphorylating conditions in light, PE N-MTase activity was stimulated and PLA2 activity was not affected. When the assays were performed in the dark, both enzymatic activities were unaffected when compared to the corresponding controls. Incubation of ROS membranes in light in the presence of PKC activators phorbol 12,13-dibutyrate (PDBu) and dioctanoylglycerol (DOG) resulted in the same pattern of changes in enzyme activities as described for standard PKC phosphorylating condition. Pre-incubation of membranes with the PKC inhibitor H-7 reduced the stimulation of PDBu on PE N-MTase activity, and had no effect on PLA2 activity in ROS membranes incubated with the phorbol ester. Pre-treatment of isolated ROS with alkaline phosphatase resulted in decreased PE N-MTase activity and produced a significant stimulation of PLA2 activity under dark as well as under light conditions when compared to the corresponding controls. These findings suggest that ROS protein phosphorylation and dephosphorylation modulates PE N-MTase and PLA2 activities in isolated ROS, and that these activities are independently and specifically modulated by particular kinases. Furthermore, dephosphorylation of ROS proteins has the opposite effect to that produced by protein phosphorylation on the enzymes studied.
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PMID:Phosphorylation of rod outer segment proteins modulates phosphatidylethanolamine N-methyltransferase and phospholipase A2 activities in photoreceptor membranes. 985 16

Theophylline is an effective bronchodilatator used in the treatment of asthma which requires frequent control because of its narrow therapeutic index. Over the past decade much attention has been dedicated to the peculiar properties of the inner water pools of AOT (sodium 2-bishexyl-ethyl sulfosuccinate) microemulsions as enzyme microreactors, yet few analytical applications of the latter have been reported. We developed an original assay based on the uncompetitive inhibition by theophylline of the reaction catalyzed by alkaline phosphatase from bovine liver (E.C. 3.1.3.1) of the ELF-97 fluorogenic substrate in borate buffer 20 mM (pH 8.6)/AOT/iso-octane-ethyl acetate (95:5) at a temperature of 37 degrees C. Optimal activity of endogenous plasmatic alkaline phosphatase isoenzymes approximately pH 10.5, interfering activity of the serum are avoided. The assay is multiple point rate, monitoring the appearance of the photostable fluorescence emission of the reaction product (510-530 nm) out of the water pool. The influence of several parameters such as the amount of buffer (W(o)), the amount of alkaline phosphatase, sample volume (10-30 microl) [corrected], optimal run time (1-7 min) and the use of phosphorylating acceptor (2A2MP) are discussed. The method was compared to HPLC UV and TDx methods.
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PMID:Fluorimetric determination of theophylline in serum by inhibition of bovine alkaline phosphatase in AOT based water/in oil microemulsion. 991 59

We examined how cholecalciferol (vitamin D) nutrition affected serum 25-hydroxycholecalciferol (25(OH)D) and 1, 25-dihydroxycholecalciferol (1,25(OH)(2)D). Rats were fed conventional diet (vitamin D, 4.5 IU/g, or 7 nmol/d) or the same diet plus 18 nmol/d of extra vitamin D for 3 wk. The extra vitamin D resulted in greater serum 25(OH)D (51 +/- 3, vs. control of 21 +/- 2 nmol/L), and kidney mRNA for vitamin D receptor [VDR mRNA] (P = 0. 026) and lower serum 1,25(OH)(2)D (72 +/- 16 vs. control of 161 +/- 10 pmol/L, P = 0.001), and parathyroid hormone (PTH) (89 +/- 4 vs. control of 160 +/- 15 ng/L, P = 0.001). Kidney VDR mRNA relative to GAPDH mRNA correlated inversely with serum 1,25(OH)(2)D (r = -0.714, P = 0.006). There were no differences in serum calcium, phosphate, alkaline phosphatase, or weight gain. Experiment 2 compared groups supplemented with 0.2, 2 or 20 nmol/d of vitamin D orally, or 20 nmol/d dermally to see how vitamin D nutrition influenced the response of 1,25(OH)(2)D to changes in diet calcium. Vitamin D did not affect urinary calcium or pyridinoline excretion, serum calcium, phosphate, vitamin D binding protein or alkaline phosphatase. In groups given 20 nmol/d of vitamin D, renal mitochondrial 25(OH)D-1alpha-hydroxylase was lower (P < 0.01) and 25(OH)D-24-hydroxylase was higher (P < 0.05). Higher 25(OH)D concentration was related to proportionally lower 1,25(OH)(2)D at every calcium intake, indicating greater tissue sensitivity to 1, 25(OH)(2)D. We conclude suppression of 1,25(OH)(2)D and PTH, and higher renal VDR mRNA and 24-hydroxylase did not involve higher free 1,25(OH)(2)D concentration or a first pass effect at the gut. Thus, 25(OH)D or a metabolite other than 1,25(OH)(2)D is a physiological, transcriptionally and biochemically active, noncalcemic vitamin D metabolite.
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PMID:Improved cholecalciferol nutrition in rats is noncalcemic, suppresses parathyroid hormone and increases responsiveness to 1, 25-dihydroxycholecalciferol. 1070 88

We investigated whether the assembly/disassembly of the 26S proteasome is regulated by phosphorylation/dephosphorylation. The regulatory complex disassembled from the 26S proteasome was capable of phosphorylating the p45/Sug1/Rpt6 subunit, suggesting that the protein kinase is activated upon dissociation of the 26S proteasome or that the phosphorylation site of p45 becomes susceptible to the protein kinase. In addition, the p45-phosphorylated regulatory complex was found to be incorporated into the 26S proteasome. When the 26S proteasome was treated with alkaline phosphatase, it was dissociated into the 20S proteasome and the regulatory complex. Furthermore, the p45 subunit and the C3/alpha2 subunit were cross-linked with DTBP, whereas these subunits were not cross-linked by dephosphorylating the 26S proteasome. These results indicate that the 26S proteasome is disassembled into the constituent subcomplexes by dephosphorylation and that it is assembled by phosphorylation of p45 by a protein kinase, which is tightly associated with the regulatory complex. It was also revealed that the p45 subunit is directly associated with the 20S proteasome alpha-subunit C3 in a phosphorylation-dependent manner.
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PMID:Assembly of the 26S proteasome is regulated by phosphorylation of the p45/Rpt6 ATPase subunit. 1114 24

An enzyme-linked immunosorbent assay (ELISA) for the measurement of p21-activated kinase (PAK) activity is described. The development of this method takes advantage of the fact that a phospho-epitope-specific antibody against the regulatory autophosphorylation site sequence of PAK was successfully produced, and after being phosphorylated by PAK, a cross-linked peptide containing the autophosphorylation site of PAK could be recognized on immunoblot by this antibody. This procedure involves coating the cross-linked peptide on microtiter plates, phosphorylating the cross-linked peptide by adding active PAK plus ATP.Mg(2+), and detecting peptide phosphorylation using the phospho-epitope-specific antibody and secondary antibody conjugated with alkaline phosphatase followed by reaction with p-nitrophenyl phosphate (for colorimetric detection) or fluorescein diphosphate (for fluorimetric detection). The PAK activity detected by this method was linearly proportional to the amount of kinase used in the reaction and to the duration of the kinase reaction. Furthermore, fluorimetric detection proved more sensitive than colorimetric detection in terms of both detection limit and signal magnitude. Kinase inhibitor assay revealed that the IC(50) value of staurosporine obtained by this ELISA was very close to that obtained in radioassay. Besides staurosporine, the inhibitory activity of several kinase inhibitors was also tested by the PAK ELISA. The results taken together demonstrate the feasibility and efficacy of this solid phase method for the measurement of PAK activity in a non-radioactive way. Development of this method can be helpful in further high-throughput screening of potential inhibitors of this kinase.
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PMID:Enzyme-linked immunosorbent assay for the determination of p21-activated kinase activity. 1117 26

Dentine phosphoprotein (DPP), a major non-collagenous acidic protein of dentine, undergoes altered phosphorylation in vivo in the presence of high fluoride concentrations. This has major implications for the altered mineralization patterns found during fluorosis. In dentine, casein kinase II is involved in phosphorylating DPP, and alkaline phosphatase (ALP) is ascribed roles in the dephosphorylation of DPP, increasing the inorganic phosphate at the mineralization front and the removal of pyrophosphate. Here the influence of fluoride in vitro on the activity of purified casein kinase II and ALP and its relation to altered patterns of mineralization were examined. Kinetic analysis showed that casein kinase II activity was completely inhibited at 0.04 M NaF. Vmax when compared to the control assay was significantly decreased (P < 0.0001) between concentrations 4 x 10(-4)-4 x 10(-8) M NaF. Significant changes to the Km (P < 0.0001) were also observed. ALP activity was inhibited by NaF (0.09-9 x 10(-8) M), with Vmax significantly decreased (P < 0.0001) at 0.09 M NaF. Alterations in the activity of these enzymes in the presence of fluoride may in part explain the decreased phosphorylation observed in DPP isolated from fluorotic dentine and may aid understanding of the altered matrix mediated mineralization patterns found during fluorosis.
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PMID:Fluoride alters casein kinase II and alkaline phosphatase activity in vitro with potential implications for dentine mineralization. 1126 68

PtdIns phosphate kinases (PIPkins), which generate PtdInsP(2) isomers, have been classified into three subfamilies that differ in their substrate specificities. We demonstrate here that the previously identified AtPIP5K1 gene from Arabidopsis thaliana encodes a PIPkin with dual substrate specificity in vitro, capable of phosphorylating PtdIns3P and PtdIns4P to PtdIns(3,4)P(2) and PtdIns(4,5)P(2) respectively. We also show that recombinant AtPIP5K1 is phosphorylated by protein kinase A and a soluble protein kinase from A. thaliana. Phosphorylation of AtPIP5K1 by protein kinase A is accompanied by a 40% inhibition of its catalytic activity. Full activity is recovered by treating phosphorylated AtPIP5K1 with alkaline phosphatase.
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PMID:AtPIP5K1, an Arabidopsis thaliana phosphatidylinositol phosphate kinase, synthesizes PtdIns(3,4)P(2) and PtdIns(4,5)P(2) in vitro and is inhibited by phosphorylation. 1167 32

We previously reported that insulin resistance in skeletal muscle of obese individuals was associated with decreases in insulin signal transduction and tyrosine kinase activity of the insulin receptor. Herein is reviewed the recently published data supporting the hypothesis that protein kinase C (PKC) phosphorylates the insulin receptor on serine/threonine residues to decrease tyrosine kinase activity and cause insulin resistance. Treatment of insulin receptors from obese subjects with alkaline phosphatase restored tyrosine kinase activity, suggesting that the reduced activity was a result of hyperphosphorylation of the receptor. Incubating human muscle fiber strips with PKC inhibitors restored insulin action in muscle of obese patients, while activating PKC with a phorbol ester caused insulin resistance in muscle from lean control patients. The beta isoform of PKC was elevated in muscle of obese, insulin-resistant patients. These data are consistent with the hypothesis that elevated PKC activity may cause insulin resistance by phosphorylating the insulin receptor to decrease tyrosine kinase activity.
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PMID:Mechanisms of muscle insulin resistance in obese individuals. 1191 30


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