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 regulation of neutral cholesterol ester hydrolase activity by changes in its phosphorylation state was studied in rat liver microsomes. Treatment with cAMP-dependent protein kinase resulted in increased enzyme activity, which was further enhanced by the addition of cAMP and MgATP. Consistent activations were also achieved with MgCl2 and MgATP, the magnesium effect being abolished by ethylenediaminetetraacetic acid and adenosine triphosphate. Cholesterol ester hydrolase was activated twofold by free calcium and Ca2+/calmodulin; this latter effect was blocked by the chelator ethylene-glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid and the calmodulin antagonist trifluoperazine. The phosphatase inhibitors pyrophosphate and glycerophosphate led to marked and dose-dependent increases in esterase activity, whereas okadaic acid elicited no effect. Furthermore, pyrophosphate and okadaic acid did not change the increases in enzyme activity promoted by Ca2+, Ca2+/calmodulin, Mg2+ and MgATP. Cholesterol ester hydrolase was inactivated in a concentration-dependent manner by nonspecific alkaline phosphatases. In cAMP-dependent protein kinase/cAMP- or Ca2+/calmodulin-activated microsomes, a time-dependent loss of activation in cholesteryl oleate hydrolysis was caused by alkaline phosphatase. These findings suggest that microsomal cholesterol ester hydrolase is activated through cAMP and Ca2+/calmodulin phosphorylation, whereas enzyme deactivation is dependent on phosphatase action.
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PMID:Regulation of rat liver microsomal cholesterol ester hydrolase by reversible phosphorylation. 813 99

The effects of chlorpromazine (CPZ) on tooth mineralization were examined using incisor dentine in adult rats and cultured tooth germs of mandibular first molars dissected from mouse embryos. CPZ (10, 50 and 250 mg/kg, s.c.) substantially inhibited dentine mineralization as evaluated by contact microradiographs. Plasma calcium and phosphorus concentrations were not decreased by CPZ (10 and 50 mg/kg). Physicochemical effects were not involved in the action of CPZ on the mineralization. In vitro experiments showed that CPZ (1 and 10 microM) inhibited mineralization and alkaline phosphatase (ALP) activity in the tooth germs. As CPZ has the properties of a calmodulin antagonist, the calmodulin antagonists W-7 and W-5 were also examined. Both inhibited mineralization and ALP activity in tooth germs; W-5 had less effect than W-7. These in vivo and in vitro findings suggest that CPZ inhibited cell-mediated mineralization in dentine without affecting the calcium-regulating system and physicochemical mineral deposition. In addition, calmodulin could be involved in cell-mediated mineralization.
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PMID:In vivo and in vitro study of the effects of chlorpromazine on tooth mineralization in rats and mice. 814 68

The role of the vitamin D-induced calcium binding protein termed calbindin-D (CaBP) in the biological response to 1,25-dihydroxyvitamin D3 was assessed by photoaffinity labeling techniques. The heterobifunctional cross-linking reagent methyl-4-azidobenzoimidate was employed for studies with the 28 KD chick intestinal calbindin-D28K. Calcium-dependent interactions were evident with purified chick intestinal CaBP-immunoglobulins and bovine intestinal alkaline phosphatase; in the absence of Ca2+ there was a greatly diminished crosslinking process. There were also at least two membrane components of chick intestinal brush border membranes, with M(R) = 60,000 and 130,000, which were photoaffinity cross-linked with CaBP in a calcium-dependent manner. Similar interactions were demonstrated following incubations of CaBP with phosphatidylinositol-specific phospholipase C (PI-PLC)-treated supernatant fractions from chick intestinal brush borders. PI-PLC was shown to release 14% of the alkaline phosphatase from chick intestinal brush borders compared to greater than 80% for rabbit and chick kidney BBM preparations. Specific interactions between CaBP and brush border membrane proteins could also be demonstrated in the absence of photoaffinity labeling by Sephadex G-150 chromatography of Triton X-100 solubilized incubations between calbindin-D28K and chick intestinal BBMS, with 17% of the radiolabelled CaBP comigrating with alkaline phosphatase activity. These studies collectively demonstrate that calbindin-D28K undergoes calcium-dependent conformational changes which alter its subsequent interactions with cellular proteins in a way consistent with other calcium-binding proteins such as calmodulin or troponin C.
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PMID:Evidence for calcium mediated conformational changes in calbindin-D28K (the vitamin D-induced calcium binding protein) interactions with chick intestinal brush border membrane alkaline phosphatase as studied via photoaffinity labeling techniques. 836 39

Calcium ion plays an important role in the hair cell's mechanoelectrical transduction process; in particular, Ca2+ controls adaptation to protracted mechanical stimuli. Because calmodulin is a ubiquitous intracellular receptor for Ca2+ and has been shown to accumulate at the tips of stereocilia, we determined its concentration and identified the proteins with which it interacts in the hair bundle. By performing quantitative immunoblot analysis on isolated bundles, we ascertained that the average concentration of calmodulin within each stereocilium is approximately 70 microM. Extraction experiments disclosed that, in the presence of 20 microM Ca2+, 50% of the calmodulin is bound to detergent-soluble receptors. To distinguish these receptors, we developed an assay that utilizes calmodulin crosslinked to alkaline phosphatase. This technique is approximately 100-fold more sensitive than calmodulin-binding assays that employ 125I- or biotin-labeled calmodulin. When used with chemiluminescence detection in a blot-overlay assay, the calmodulin-alkaline phosphatase conjugate identified hair-bundle proteins of molecular masses 25, 35, 145, 175, 240, and 350 kDa. We examined the subcellular distribution of these receptors; all but the 240-kDa molecule are soluble in a nonionic detergent. The relatively high concentration of calmodulin and the presence of several calmodulin-binding proteins provide evidence for a role of calmodulin in hair bundles.
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PMID:Calmodulin and calmodulin-binding proteins in hair bundles. 838 44

Interleukin-8 (IL-8) such as LUCT (lung giant cell carcinoma-derived chemotactic protein), NAP (neutrophil activating protein) and MDNCF (monocyte-derived neutrophil chemotactic factor), and formylmethionyl-leucyl-phenylalanine (fMLP) are well-known chemoattractants for human polymorphonuclear leukocytes (PMNs) and are able to stimulate phosphorylation of 64-kd protein (p64) in these leukocytes. To elucidate the molecular mechanism of PMN activation with chemoattractants, we investigated the phosphorylation process of p64 in an intact cell. 32P-Labeled PMNs were stimulated with LUCT/IL-8, fMLP, leukotriene B4, or C5a, and phosphorylated proteins were analyzed by two-dimensional electrophoresis and autoradiography. A marked phosphorylation of p64 was observed after stimulation. A new spot of phosphorylated p64 (pp64) could be detected on the gel stained with Coomassie Brilliant Blue, indicating that the isoelectric point (pI) of p64 shifted from 5.3 to a more acidic pI by the phosphorylation forming pp64. The spot of pp64 was shown to be dephosphorylated to p64 by treatment with calf intestine alkaline phosphatase. Other proteins having molecular masses 82, 66, 58, 55 and 50 kd were also phosphorylated. The fMLP-stimulated phosphorylation was time-dependent and saturated within 5 min. Maximum stimulation was achieved with 10 nM fMLP. Phosphoamino acid analysis revealed phosphorylation of serine residues in pp64. Staurosporine (100 nM) and W-7 (100 microM) significantly inhibited the phosphorylation of p64, but H-7 slightly inhibited it. H-8 and herbimycin A did not effect phosphorylation. Phorbol myristate acetate was found to stimulate significantly. Protein kinase C did not stimulate the phosphorylation. These data suggest that protein kinase C and calmodulin-like protein are indirectly involved in the phosphorylation of p64 during chemoattractant-activation of PMN.
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PMID:Characterization of a 64-kd protein phosphorylated during chemotactic activation with IL-8 and fMLP of human polymorphonuclear leukocytes. I. Phosphorylation of a 64-kd protein and other proteins. 839 62

Nerve growth factor (NGF) treatment of PC12 cells led to the rapid phosphorylation of a calmodulin-binding protein of 100 kDa (CaM-BP100) identified on blot overlays with 125I-labeled CaM. The effect was detected as a retardation in the mobility of the protein by an apparent 10 kDa on SDS gels. The mobility shift was complete within 5 min and was maintained for 24 h in the continued presence of NGF. The protein was present in both the soluble and crude particulate fractions, and the gel mobility shift occurred in both fractions. Epidermal growth factor elicited a similar response, but the mobility shift was reversed within 12 h. The gel retardation was due to phosphorylation of CaM-BP100, as it could be reversed if cytoplasmic extracts were held under dephosphorylating conditions at 37 degrees C for 10 min prior to electrophoresis; dephosphorylation was inhibited by okadaic acid but not vanadate, suggesting the participation of a Ser/Thr phosphatase. Treatment with either acid or alkaline phosphatase also reversed the mobility shift. CaM-BP100 phosphorylation was stimulated by 12-O-tetradecanoylphorbol-13-acetate in intact cells, but the effect of NGF did not involve a protein kinase C-dependent process, because it occurred in PC12 cells depleted of protein kinase C. The phosphorylation event appeared to be due to an NGF-stimulated protein kinase, as mixing extracts from NGF-treated cells with extracts from control cells in the presence of ATP and Mg2+ reconstituted the mobility shift in vitro. CaM-BP100 appears to be a minor cellular phosphoprotein, as 32P labeling of the protein could not be detected in crude cell extracts. These results suggest that receptor tyrosine kinases communicate with at least one component of the Ca2+/calmodulin-signaling pathway early in signal transduction.
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PMID:Rapid and sustained phosphorylation of a calmodulin-binding protein (CaM-BP100) in NGF-treated PC12 cells. 839 55

We have characterized a Na+/H+ exchanger in the membrane of isolated zymogen granules (ZG) from rat exocrine pancreas and investigated its role in secretagogue-induced enzyme secretion. ZG Na+/H+ exchanger activity was estimated by measuring Na+ or Li+ influx and consequent osmotic swelling and lysis of ZG incubated in Na- or Li-acetate. Alternatively, intragranule pH was investigated by measuring absorbance changes in ZG which had been preloaded with the weak base acridine orange. Na+- or Li+-dependent ZG lysis was enhanced by increasing inward to outward directed H+ gradients. Na+-dependent ZG lysis was not prevented by an inside-positive K+ diffusion potential generated by valinomycin which argues against parallel operation of separate electrogenic Na+ and H+ permeabilities and for coupled Na+/H+ exchange through an electroneutral carrier. Na+- and Li+-dependent ZG lysis was inhibited by EIPA (EC50 approximately 25 microM) and benzamil (EC50 approximately 100 microM), but only weakly by amiloride. Similarly, absorbance changes due to release of acridine orange from acidic granules into the medium were obtained with Na+ and Li+ salts only, and were inhibited by EIPA, suggesting the presence of a Na+/H+ exchanger in the membrane. Na+ dependent lysis of ZG was inhibited by 0.5 mm MgATP and MgATP-gamma-S by about 60% and 35%, respectively. Inhibition by MgATP was prevented by incubation of ZG with alkaline phosphatase (100 U/ml), or by the calmodulin antagonists calmidazolium (0.75 microM), trifluoperazine (100 microM) and W-7 (500 microM), suggesting that the ZG Na+/H+ exchanger is regulated by a ZG membrane-bound calmodulin-dependent protein kinase. Na+ dependence of secretagogue (CCK-OP)-stimulated amylase secretion was investigated in digitonin permeabilized rat pancreatic acini and was higher in acini incubated in Na+ containing buffer (30 mm NaCl/105 mm KCl buffer; 6.4 +/- 0.4% of total amylase above basal) compared to buffer without Na+ (0 mm NaCl/135 mm KCl buffer; 4.7 +/- 0.4% of total amylase above basal, P < 0.03). EIPA (50 microM) reduced CCK-OP-induced amylase secretion in Na+ containing buffer from 7.5 +/- 0.6% to 4.1 +/- 0.8% (P < 0.02). In the absence of Na+ in the buffer, CCK-OP-stimulated amylase release was not inhibited by 50 microM EIPA. The data suggest that an amiloride insensitive, EIPA inhibitable Na+/H+ exchanger is present in ZG membranes, which is stimulated by calmodulin antagonists and could be involved in secretagogue-induced enzyme secretion from rat pancreatic acini.
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PMID:Evidence for involvement of a zymogen granule Na+/H+ exchanger in enzyme secretion from rat pancreatic acinar cells. 867 81

We have found that modification of rat PC12 cells with pertussis toxin resulted in an approximately 50% inhibition of a protein phosphatase 2A-like phosphatase. Protein phosphatase 2A (PP2A) is a major cellular serine/threonine-specific protein phosphatase. Treatment of extracts from pertussis toxin-modified PC12 cells with either immobilized alkaline phosphatase or Ca2+ reversed this inhibition. Reactivation of the PP2A-like phosphatase in Ca2+ appears to result from the dephosphorylation of a protein by the Ca2+/calmodulin-dependent protein phosphatase calcineurin. The PP2A-like phosphatase in extracts from pertussis toxin-modified PC12 cells eluted from a Mono Q column at a higher ionic strength than did the PP2A-like phosphatase in extracts from control cells. After incubation in Ca2+, the PP2A-like phosphatase in extracts from pertussis toxin-modified cells eluted from a Mono Q column at the same ionic strength as did the PP2A-like phosphatase in extracts from control cells. These results indicate that the effect of pertussis toxin on this PP2A-like activity results from the phosphorylation of either one of the subunits of the PP2A-like phosphatase or a protein that when phosphorylated binds to and inhibits this phosphatase. Pertussis toxin modification did not result in the phosphorylation of the catalytic subunit of PP2A. Because phosphorylation regulates the activities of many enzymes and cell surface receptors, a pertussis toxin-induced decrease in PP2A activity could alter signaling pathways and other cellular processes in which G proteins are not directly involved.
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PMID:Pertussis toxin modification of PC12 cells inhibits a protein phosphatase 2A-like phosphatase. 964 72

The effects of prostaglandin E2 (PGE2) on the proliferation and differentiation of osteoblastic cells were studied in osteoblast-like cells isolated from adult rat calvaria. Treatment of the cells with PGE2 within the concentration range 10(-8)-10(-5) M resulted in a dose-dependent increase in alkaline phosphatase (ALP) activity, [3H]proline incorporation into collagenase-digestible protein, and mineralized bone nodule (BN) formation, as well as a dose-dependent decrease in [3H]thymidine incorporation into the cells. PGE2 also caused a dose-dependent increase in the intracellular cyclic adenosine monophosphate (cAMP) content, with a maximal effective concentration of 10(-5) M; this effect of PGE2 was mimicked by forskolin, an adenylate cyclase activator. The treatment of adult calvarial cells with forskolin decreased BN formation, ALP activity, and collagen synthesis. These results suggested that cAMP does not have a stimulatory, but rather a suppressive, effect on the differentiation of adult rat calvarial cells. A time-course study of cAMP accumulation showed that both PGE2- and forskolin-induced cAMP reached a maximum at 5 min after the treatment, but the former rapidly returned to the basal level by 40 min, while the latter declined slowly and was still at 70% of the maximal level at 60 min, suggesting that PGE2 activates phosphodiesterase as well as adenylate cyclase. The presence of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, reduced the rate of degradation of cAMP formed after PGE2 treatment, suggesting the involvement of calmodulin in the activation of phosphodiesterase. However, PGE2 also caused the production of inositol 1,4,5-triphosphate (IP3) and an elevation of the intracellular Ca2+ concentration ([Ca2+]i), both of which peaked at 15 s and returned to the basal level within 1 min. Submaximal responses of the IP3 production and the [Ca2+]i elevation to PGE2 were obtained at 10(-5) M. W-7 decreased both basal and PGE2-induced ALP activity, collagen synthesis and BN formation, indicating the involvement of Ca2+/calmodulin-dependent protein kinase in the PGE2-induced differentiation of calvarial cells. From these results, we concluded that PGE2 inhibits the proliferation and stimulates the differentiation of calvarial osteoblasts by elevating the [Ca2+]i through the activation of a phosphoinositide turnover, but not via an activation of adenylate cyclase. We also found that BN formation varies, depending on the time of PGE2 addition, suggesting that responsiveness of the cells to PGE2 may change during the culture period.
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PMID:Prostaglandin E2 stimulates the formation of mineralized bone nodules by a cAMP-independent mechanism in the culture of adult rat calvarial osteoblasts. 1008 22

Although the role of intraneuronal neurofilamentous aggregates in the pathogenesis of ALS is unknown, their presence forms a key neuropathological hallmark of the disease process. Conversely, the experimental induction of neurofilamentous aggregates in either neurotoxic or transgenic mice gives rise to motor system degeneration. To determine whether alterations in the physiochemical properties of NF are present in sporadic ALS, we purified NF subunit proteins from cervical spinal cord of ALS and age-matched control patients. The cytoskeleton-enriched, Triton X-100 insoluble fraction was further separated into individual NF subunits using hydroxyapatite HPLC. We observed no differences between control and ALS in the characteristics of NFH, including migration patterns on 2D-IEF, sensitivity to E. coli, alkaline phosphatase mediated dephosphorylation, peptide mapping, or proteolysis (calpain, calpain/calmodulin mediated, phosphorylated or dephosphorylated NFH). NFL showed no differences in 2D-IEF migration patterns, peptide mapping, or the extent of NFL nitrotyrosine immunoreactivity in either the Triton soluble or insoluble fractions. The latter observation demonstrated that NFL nitration is a ubiquitous occurrence in neurons and suggests that NFL might function as a sink for free reactive nitrating species. In contrast to the lack of differences in the post-translational processing of NF in ALS, we did observe a selective suppression of NFL steady state mRNA levels in the limb innervating lateral motor neuron column of ALS. This occurred in the absence of modifications in NFH, NFM or neuronal nitric oxide synthase (Type I NOS; nNOS) steady state mRNA levels. Coupled with previous observations of nNOS immunoreactivity co-localizing with NF aggregates in ALS motor neurons, this suggests activation of the nNOS enzyme complex in ALS, which would be predicted to contribute directly to the generation of reactive nitrating species. Given this, the isolated suppression of NFL steady state mRNA levels in ALS may indicate that ALS motor neurons are at an intrinsic deficit in the ability to buffer free reactive nitrating species.
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PMID:Neurofilament metabolism in sporadic amyotrophic lateral sclerosis. 1054 27


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