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 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

A low molecular weight mitogen (LMP) from Streptococcus pyogenes strain NY 5 was successively purified by adsorption on phenylsepharose, chromatography on Resource S and Superdex G 30 and finally by affinity chromatography on antiphosphothreonine agarose. The N-terminal protein sequence of the mitogen was determined. The occurrence of phosphoamino acids was investigated by immunoassay using monoclonal antibodies. The LMP is a threonine-phosphorylated protein different of HPR protein of PTS-system, its mitogenic activity was lost after treatment with streptococcal protein phosphatase or alkaline phosphatase. The inactivated LMP was activated by phosphorylation with phosphokinase and ATP. The active LMP was also inactivated in streptococcal cultures secreting acid protein phosphatase during the phase of phosphate limitation.
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PMID:Influence of the phosphorylation state on the biological activity of a low-molecular mitogen from group A streptococci. 972 1

NS1, the 83-kDa major nonstructural protein of minute virus of mice (MVM), is a multifunctional nuclear phosphoprotein which is required in a variety of steps during progeny virus production, early as well as late during infection. NS1 is the initiator protein for viral DNA replication. It binds specifically to target DNA motifs; has site-specific single-strand nickase, intrinsic ATPase, and helicase activities; trans regulates viral and cellular promoters; and exerts cytotoxic stress on the host cell. To investigate whether these multiple activities of NS1 depend on posttranslational modifications, in particular phosphorylation, we expressed His-tagged NS1 in HeLa cells by using recombinant vaccinia viruses, dephosphorylated it at serine and threonine residues with calf intestine alkaline phosphatase, and compared the biochemical activities of the purified un(der)phosphorylated (NS1(O)) and the native (NS1(P)) polypeptides. Biochemical analyses of replicative functions of NS1(O) revealed a severe reduction of intrinsic helicase activity and, to a minor extent, of ATPase and nickase activities, whereas its affinity for the target DNA sequence [ACCA]2-3 was enhanced compared to that of NS1(P). In the presence of endogenous protein kinases found in replication extracts, NS1(O) showed all functions necessary for resolution and replication of the 3' dimer bridge, indicating reactivation of NS1(O) by rephosphorylation. Partial reactivation of the helicase activity was found as well when NS1(O) was incubated with protein kinase C.
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PMID:Biochemical activities of minute virus of mice nonstructural protein NS1 are modulated In vitro by the phosphorylation state of the polypeptide. 973 39

Hydropathy profile analysis of the amino acid sequence of the Na+/proline transporter of Escherichia coli (PutP) suggests that the protein consists of 12 transmembrane domains (TMs) which are connected by hydrophilic loops (Nakao, T., Yamato, I., and Anraku, Y. (1987) Mol. Gen. Genet. 208, 70-75). We have tested this prediction by applying a gene fusion approach in combination with a Cys accessibility analysis and site-specific proteolysis. Characterization of a series of PutP-alkaline phosphatase (PhoA) and PutP-beta-galactosidase (LacZ) hybrid proteins yields a reciprocal activity pattern of the reporter proteins that is in agreement with the topology of TMs III to XII of the 12-helix model. Placement of the PutP-PhoA and PutP-LacZ junction sites closer to the N terminus does not yield conclusive results. As a prerequisite for further topology studies, a functional PutP molecule devoid of all five native Cys residues (Cys-free PutP) is generated. Subsequently, amino acids in Cys-free PutP are replaced individually with Cys, and the accessibility of the sulfhydryl groups is analyzed. Surprisingly, Cys residues placed close to the N terminus of PutP (Ile-3 --> Cys, Thr-5 --> Cys) or into putative TM II (Ser-71 --> Cys, Glu-75 --> Cys) are highly accessible to membrane permeant and impermeant thiol reagents in intact cells. In contrast, Cys at the C terminus (Ser-502 --> Cys) reacts only with the membrane permeant but not with the impermeant reagent in intact cells. These results contradict the 12-helix motif and indicate a periplasmic location of the N terminus whereas the C terminus faces the cytoplasm. In addition, a transporter with Cys in place of Leu-37 (putative periplasmic loop (pL2) shows the same accessibility pattern as the Cys at the C terminus. Furthermore, PutP which has been purified and reconstituted into proteoliposomes in an inside-out orientation, is readily cleaved by the endoproteinase AspN before Asp-33 (pL2), Asp-112 (putative cytoplasmic loop (cL3), Asp-262 (cL7), and Asp-356 (cL9). These results suggest a cytosolic location of Asp-33 and Leu-37, thereby implying the formation of an additional TM formed by amino acids of pL2. Based on these observations, a new secondary structure model is proposed according to which the protein consists of 13 TMs with the N terminus on the outside and the C terminus facing the cytoplasm. The 13-helix structure is discussed as a common topological motif for all members of the Na+/solute cotransporter family.
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PMID:Topology of the Na+/proline transporter of Escherichia coli. 975 72

Overexpression of bovine growth hormone (bGH) in transgenic (PEPCK-bGH) mice induces resistance to insulin, which is compensated by a major increase in insulin levels. In these animals, hepatic insulin receptors (InsRs) are downregulated while tyrosine kinase activity of wheat germ agglutinin (WGA)-purified InsRs towards exogenous substrates is unexpectedly increased. By normalizing insulinemia, we attempted to determine whether the alterations detected in the early steps of insulin signal transduction are due to exposure to chronically high GH levels or are secondary to hyperinsulinemia. Transgenic PEPCK-bGH animals were treated with a single intraperitoneal administration of streptozotocin (STZ) or were deprived of food for 48 h, to normalize insulin levels. Both fasting and STZ treatment were effective in reducing insulin blood levels to control values or below, while GH levels remained unchanged (STZ treatment) or increased (fasted animals). In the liver of untreated transgenic mice, the number of InsRs as determined by 125I-insulin binding was significantly diminished (65+/-5% and 60+/-6% of normal values in microsomes and solubilized membranes respectively;P<0.01 vs control mice). In treated transgenic mice, the number of InsRs increased to values similar to or slightly higher than those found in normal control mice (STZ-treated: 139+/-26% and 126+/-8%; fasted: 128+/-5% (P<0.05) and 102+/-1.5%, for microsomes and solubilized membranes respectively). Neither treatment altered InsR affinity. InsR concentration in liver as determined by immunoblotting using an antibody against the beta-subunit of the insulin receptor was found to be reduced in transgenic mice (69+/-3% of normal values,P<0.001) and was normalized after both STZ treatment (105+/-4%) and fasting (109+/-4%). Insulin-stimulated autophosphorylation activity of InsRs in transgenic mice was increased (154+/-13%,P<0.01 compared with the control group), essentially normalized by STZ treatment (96+/-14%), and reduced by fasting, to below the values measured in normal control mice (56+/-15%,P<0.05). The potential influence of basal serine/threonine (Ser/Thr) phosphorylation of the InsR beta-subunit on the regulation of the InsRs from transgenic mice was also investigated. The autophosphorylation activity of WGA-purified InsRs from all groups of mice studied was essentially unchanged after dephosphorylation with alkaline phosphatase or mild trypsinization. Consequently, our results suggest that the observed changes in InsR number and autophosphorylation activity in the liver of bGH transgenic mice are directly related to changes in insulin blood levels, and that Ser/Thr phosphorylation is apparently not involved in the regulation of the InsR autophosphorylation activity in this model of insulin resistance.
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PMID:Role of hyperinsulinemia on hepatic insulin receptor concentration and autophosphorylation in the presence of high growth hormone levels in transgenic mice overexpressing growth hormone gene. 979 37

The binding of aldosterone (ALDO) to the mineralocorticoid receptor (MR) induces a conformational change of the protein referred to as 'transformation'. This feature can be evidenced in vivo by the capacity of the MR to interact with chromatin, and in vitro by the ability of the MR to bind to DNA strands or to shift the sedimentation coefficient (S) to lower values. The transformation process allows MR to work as a transcription factor after interacting with specific sequences of DNA. The signal transduction pathway for the MR transformation remains unknown. As a first step towards elucidating the mechanism of steroid-dependent MR transformation, we asked if the MR-signaling pathway is affected by the phosphorylation status of the MR-heterocomplex, and how that pathway may be regulated. Incubation of preformed [3H]ALDO-MR complex with bovine intestinal alkaline phosphatase led to an increase in the rate of MR-transformation (measured as 9.4-5.4S shift). This alkaline phosphatase-dependent MR transformation was inhibited by the specific alkaline phosphatase-type inhibitor levamisole, and was not evident in incubations performed with acid phosphatases. A direct correlation between the DNA-cellulose binding capacity of the [3H]ALDO-MR complex and the percentage of transformed 5.4S MR form was also observed. When rat kidney cytosol was incubated in the absence of both exogenous phosphatase and stabilizing agents (such as molybdate or vanadate), MR transformation also took place, in a time- and temperature-dependent process. In contrast with the inhibitory effect observed upon alkaline phosphatase-promoted transformation, levamisole was unable to inhibit the endogenous transforming activity of MR, suggesting that an endogenous phosphatase other than those which belong to the alkaline-type may be responsible for that transformation. Tautomycin, a polyketide produced by the soil bacteria Streptomyces which inhibits serine/threonine phosphatases of the PP1/PP2A subgroup, was able to inhibit the endogenous phosphatase activity in a concentration-dependent form (Ki(app)=7.35 nM). These results support the idea that the endogenous renal activity involved in the regulation of rat kidney MR transformation may be a protein phosphatase which belongs to the PP1/PP2A subgroup.
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PMID:Tautomycin inhibits phosphatase-dependent transformation of the rat kidney mineralocorticoid receptor. 986 32

Chronic occupational exposure to organophosphorus and carbamate-type pesticides significantly inhibits acetylcholinesterase activity and causes morbidity. This study on mice was designed to evaluate their amino profile and to identify signs of hepatic dysfunction following their chronic exposure to mixtures of organophosphorus pesticides. Laboratory mice were exposed to a formulated mixture of the six organophosphorus pesticides (Dimethoate, Chlorpyrifos, Profenofos, Pirimiphos methyl, Triazophos and Dimethoate) most commonly used in agriculture in this region of the Middle East. Doses (10% of LD50 of the mixture) were given once a week by gavage in corn oil for 7 weeks; the control group was given only corn oil. At the end of the exposure period, mice were culled and blood samples were collected to determine erythrocyte acetylcholinesterase activity, biochemical markers of liver function and concentrations of serum amino acids. Erythrocyte acetylcholinesterase activity and total serum proteins decreased significantly in the exposed group. Serum concentrations of alanine aminotransferase and aspartate aminotransferase, alanine, glutamic acid, glycine, isoleucine, leucine, methionine, ornithine, proline, serine, threonine and valine were significantly increased in the exposed mice, while serum levels of cystine were decreased significantly. There were also non-significant increases in serum alkaline phosphatase, gama-glutamyl transpeptidase and some of the other amino acids. Chronic exposure to mixtures of organophosphorus pesticides is associated with decreased acetylcholinesterase activity, hepatic dysfunction and disturbance of amino acids profile. Biochemical indices of hepatocellular injury and disturbed amino acid metabolism may be of value as markers of chronic exposure to such pesticides.
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PMID:Hepatic injury and disturbed amino acid metabolism in mice following prolonged exposure to organophosphorus pesticides. 1002 66

1. The degree of cell-to-cell coupling between ventricular myocytes of neonatal rats appeared well preserved when studied in the perforated version of the patch clamp technique or, in double whole-cell conditions, when ATP was present in the patch pipette solution. In contrast, when ATP was omitted, the amplitude of junctional current rapidly declined (rundown). 2. To examine the mechanism(s) of ATP action, an 'internal perfusion technique' was adapted to dual patch clamp conditions, and reintroduction of ATP partially reversed the rundown of junctional channels. 3. Cell-to-cell communication was not preserved by a non-hydrolysable ATP analogue (5'-adenylimidodiphosphate, AMP-PNP), indicating that the effect most probably did not involve direct interaction of ATP with the channel-forming proteins. 4. An ATP analogue supporting protein phosphorylation but not active transport processes (adenosine 5'-O-(3-thiotriphosphate), ATPgammaS) maintained normal intercellular communication, suggesting that the effect was due to kinase activity rather than to altered intracellular Ca2+. 5. A broad spectrum inhibitor of endogenous serine/threonine protein kinases (H7) reversibly reduced the intercellular coupling. A non-specific exogenous protein phosphatase (alkaline phosphatase) mimicked the effects of ATP deprivation. The non-specific inhibition of endogenous protein phosphatases resulted in the preservation of substantial cell-to-cell communication in ATP-free conditions. 6. The activity of gap junctional channels appears to require both the presence of ATP and protein kinase activity to counteract the tonic activity of endogenous phosphatase(s).
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PMID:ATP counteracts the rundown of gap junctional channels of rat ventricular myocytes by promoting protein phosphorylation. 1008 44

In OKP cells expressing ETB endothelin receptors, activation of Na+/H+ antiporter activity by endothelin-1 (ET-1) was resistant to low concentrations of ethylisopropyl amiloride, indicating regulation of Na+/H+ exchanger isoform 3 (NHE3). ET-1 increased NHE3 phosphorylation in cells expressing ETB receptors but not in cells expressing ETA receptors. Receptor specificity was not due to demonstrable differences in receptor-specific activation of tyrosine phosphorylation pathways or inhibition of adenylyl cyclase. Phosphorylation was associated with a decrease in mobility on SDS-PAGE, which was reversed by treating immunoprecipitated NHE3 with alkaline phosphatase. Phosphorylation was first seen at 5 min and was maximal at 15-30 min. Phosphorylation was maximal with 10(-9) M ET-1. Phosphorylation occurred on threonine and serine residues at multiple sites. In summary, ET-1 induces NHE3 phosphorylation in OKP cells on multiple threonine and serine residues. ETB receptor specificity, time course, and concentration dependence are all similar between ET-1-induced increases in NHE3 activity and phosphorylation, suggesting that phosphorylation plays a key role in activation.
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PMID:ETB receptor activation leads to activation and phosphorylation of NHE3. 1019 26

The NH2-terminal domain of sonic hedgehog (residue 25-198) was expressed in both yeast and animal cells. The yeast-derived NH2-terminal domain of sonic hedgehog was less active by far than the animal cell-derived counterpart. The yeast-derived NH2-terminal domain of sonic hedgehog lacked 10 amino acids from the NH2-terminus. This cleavage of the yeast-derived NH2-terminal domain of sonic hedgehog might due to Kex 2. In contrast, a mutant yeast-derived NH2-terminal domain of sonic hedgehog (Lys-33 to Thr) retained its NH2-terminus and its activity was comparable to that of the animal cell-derived NH2-terminal domain of sonic hedgehog. The NH2-terminal deleted NH2-terminal domain of sonic hedgehog completely lost its activity, nevertheless it inhibited the alkaline phosphatase activity induced by the animal cell-derived NH2-terminal domain of sonic hedgehog in a dose-dependent manner. These data suggest that the NH2-terminal deleted NH2-terminal domain of sonic hedgehog retains a receptor-binding ability and that the NH2-terminal peptide of the NH2-terminal domain of sonic hedgehog is necessary for its signal transduction.
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PMID:The NH2-terminal region of the active domain of sonic hedgehog is necessary for its signal transduction. 1021 71


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