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)

Alterations of the renal function in the isolated perfused rat kidney system after application of two bacterial RNases, Bacillus intermedius RNase (binase) and ribonuclease produced by Bacillus amyloliquefaciens (barnase), were investigated with two different treatment regimens in comparison with catalytically inactive derivates of the enzymes, photooxidated at the active site His101 binase and inactive mutant His102Gln barnase. For the in vitro approach the test enzymes were dissolved in the perfusion media and applied to the kidney after removal from the animal. Alternatively, the test ribonucleases were administered to rats in vivo and the renal effects were assessed in the isolated perfused rat kidney 1 and 6 h after treatment. In the in vitro regimen both active enzymes induced time- and concentration-dependent nephrotoxicity reflected in enhancement of urinary protein excretion, decline of glucose reabsorption, increase of gamma-glutamyltranspeptidase and alkaline phosphatase activities in urine. In vivo administration of active binase induced functional impairment of the isolated perfused organ in a similar way. None of the inactive RNases in both regimens and at all concentrations tested altered any renal parameter. The results suggest that RNA degradation may be involved in the nephrotoxic effects of bacillar RNases.
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PMID:Nephrotoxic effects of bacterial ribonucleases in the isolated perfused rat kidney. 916 Jan 9

Cytokine-driven activation of hepatic stellate cells (HSC) in tissue injury and inflammation is a key pathogenetic event in liver fibrogenesis leading to an expanded pool of matrix producing myofibroblasts (MFB) which represent the transformed counterpart of HSC. We hypothesize that expansion of the pool of MFB might also be accomplished by modulation of apoptosis, which plays an opposite and complementary role to mitosis in the cellular homeostasis. We characterized the susceptibility of HSC in primary culture and of MFB in secondary culture to apoptosis induced by the soluble Fas ligand (sFasL) and related the effects to the expression levels of Fas (APO-1/CD95) and some major proapoptotic and contra-apoptotic protooncogenes. MFB showed a dose-dependent apoptotic reaction upon exposure to sFasL as evidenced by a strong increase of nucleosomal DNA fragments, loss of cellular DNA, positive TUNEL reaction, and annexin staining. The effect was found only if protein synthesis (cycloheximide) or RNA synthesis (actinomycin D) were arrested. HSC maintained for various times in primary culture were completely resistant to sFasL in combination with cycloheximide, but in late primary cultures (day 7 onward) an increasing susceptibility to sFasL-mediated apoptosis was developed. By semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis and alkaline phosphatase-anti-alkaline phosphatase staining Fas receptor was identified both in HSC and MFB at comparable expression levels. The expression of the contra-apoptotic protooncogenes bcl-2 and bcl-xl was found to be much stronger in early HSC than in late HSC and MFB as shown by ribonuclease protection assay. The expression of bcl-2 was additionally confirmed by semiquantitative RT-PCR and immunoblotting. Proapoptotic bax was found in comparable quantities at the RNA level in HSC and MFB but at the protein level MFB showed increased bax expression. It is concluded that transformation of HSC to MFB is paralleled by an increasing sensitivity to sFasL-mediated apoptosis, which might be related to a strong decrease of bcl-2 and bcl-xl expression, leading to a preponderance of proapoptotic gene expression in MFB. Modulation of apoptotic susceptibility of transforming HSC could be an important complementary pathway in the pathogenesis of fibrosis.
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PMID:Transformation-dependent susceptibility of rat hepatic stellate cells to apoptosis induced by soluble Fas ligand. 969 16

Gaseous CO2 was used as an antisolvent to induce the fractional precipitation of alkaline phosphatase, insulin, lysozyme, ribonuclease, trypsin, and their mixtures from dimethylsulfoxide (DMSO). Compressed CO2 was added continuously and isothermally to stationary DMSO solutions (gaseous antisolvent, GAS). Dissolution of CO2 was accompanied by a pronounced, pressure-dependent volumetric expansion of DMSO and a consequent reduction in solvent strength of DMSO towards dissolved proteins. View cell experiments were conducted to determine the pressures at which various proteins precipitate from DMSO. The solubility of each protein in CO2-expanded DMSO was different, illustrating the potential to separate and purify proteins using gaseous antisolvents. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) was used to quantify the separation of lysozyme from ribonuclease, alkaline phosphatase from insulin, and trypsin from catalase. Lysozyme biological activity assays were also performed to determine the composition of precipitates from DMSO initially containing lysozyme and ribonuclease. SDS-PAGE characterizations suggest that the composition and purity of solid-phase precipitated from a solution containing multiple proteins may be accurately controlled through the antisolvent's pressure. Insulin, lysozyme, ribonuclease, and trypsin precipitates recovered substantial amounts of biological activity upon redissolution in aqueous media. Alkaline phosphatase, however, was irreversibly denaturated. Vapor-phase antisolvents, which are easily separated and recovered from proteins and liquid solvents upon depressurization, appear to be a reliable and effective means of selectively precipitating proteins.
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PMID:Protein purification with vapor-phase carbon dioxide. 1009 36

We describe a method for obtaining radioactive fingerprints from nonradioactive ribonucleic acid. Fragments derived by T1 ribonuclease digestion of RNA are dephosphorylated with bacterial alkaline phosphatase. When these fragments are used as primers for the reaction of primer dependent polynucleotide phosphorylase with [alpha-(32)P]GDP in the presence of T1 ribonuclease the 3'-hydroxyl group of each fragment becomes phosphorylated. The degree of phosphorylation is reasonably uniform. The method has been applied to T1 ribonuclease digests of Escherichia coli tRNA(Met) (f); the oligonucleotides were further analyzed by spleen phosphodiesterase digestion. In a similar manner fingerprints of pancreatic ribonuclease digests of RNA can be obtained, when [alpha-(32)P]UDP, polynucleotide phosphorylase and pancreatic ribonuclease are used.
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PMID:Fingerprinting nonradioactive ribonucleic acid with the aid of polynucleotide phosphorylase. 1079 69

RNase U2 is an endoribonuclease secreted by the fungus Ustilago sphaerogena. Its genomic DNA (rnu2), containing an intron of 116 bp, has been isolated and cloned. The corresponding cDNA has also been synthesized. The recombinant RNase U2 was successfully produced in Pichia pastoris, fused to the yeast alkaline phosphatase signal peptide. The recombinant RNase U2, purified by affinity chromatography, contains three extra amino acids at its amino-terminal end and retains the enzymatic and spectroscopic properties of the natural fungal protein.
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PMID:Ribonuclease U2: cloning, production in Pichia pastoris and affinity chromatography purification of the active recombinant protein. 1093 Jul 32

Snake envenomation employs three well integrated strategies: prey immobilization via hypotension, prey immobilization via paralysis, and prey digestion. Purines (adenosine, guanosine and inosine) evidently play a central role in the envenomation strategies of most advanced snakes. Purines constitute the perfect multifunctional toxins, participating simultaneously in all three envenomation strategies. Because they are endogenous regulatory compounds in all vertebrates, it is impossible for any prey organism to develop resistance to them. Purine generation from endogenous precursors in the prey explains the presence of many hitherto unexplained enzyme activities in snake venoms: 5'-nucleotidase, endonucleases (including ribonuclease), phosphodiesterase, ATPase, ADPase, phosphomonoesterase, and NADase. Phospholipases A(2), cytotoxins, myotoxins, and heparinase also participate in purine liberation, in addition to their better known functions. Adenosine contributes to prey immobilization by activation of neuronal adenosine A(1) receptors, suppressing acetylcholine release from motor neurons and excitatory neurotransmitters from central sites. It also exacerbates venom-induced hypotension by activating A(2) receptors in the vasculature. Adenosine and inosine both activate mast cell A(3) receptors, liberating vasoactive substances and increasing vascular permeability. Guanosine probably contributes to hypotension, by augmenting vascular endothelial cGMP levels via an unknown mechanism. Novel functions are suggested for toxins that act upon blood coagulation factors, including nitric oxide production, using the prey's carboxypeptidases. Leucine aminopeptidase may link venom hemorrhagic metalloproteases and endogenous chymotrypsin-like proteases with venom L-amino acid oxidase (LAO), accelerating the latter. The primary function of LAO is probably to promote prey hypotension by activating soluble guanylate cyclase in the presence of superoxide dismutase. LAO's apoptotic activity, too slow to be relevant to prey capture, is undoubtedly secondary and probably serves principally a digestive function. It is concluded that the principal function of L-type Ca(2+) channel antagonists and muscarinic toxins, in Dendroaspis venoms, and acetylcholinesterase in other elapid venoms, is to promote hypotension. Venom dipeptidyl peptidase IV-like enzymes probably also contribute to hypotension by destroying vasoconstrictive peptides such as Peptide YY, neuropeptide Y and substance P. Purines apparently bind to other toxins which then serve as molecular chaperones to deposit the bound purines at specific subsets of purine receptors. The assignment of pharmacological activities such as transient neurotransmitter suppression, histamine release and antinociception, to a variety of proteinaceous toxins, is probably erroneous. Such effects are probably due instead to purines bound to these toxins, and/or to free venom purines.
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PMID:Ophidian envenomation strategies and the role of purines. 1173 31

A method has been described for isolation of the specific cytoplasmic granules of rabbit polymorphonuclear leucocytes. Homogeneous suspensions of leucocytes were disrupted by lysis in 0.34 M sucrose. This procedure liberated the cytoplasmic contents of the cell and dissolved a considerable proportion of the nuclei. Following disruption, the sucrose lysate was separated into three fractions by differential centrifugation, i.e. 400 g or nuclear pellet, 8,200 g or granule pellet and the postgranule supernate. Microscopic examination revealed that the 8,200 g pellet was composed of intact granules as well as occasional mitochondria. The other two fractions were morphologically heterogeneous. Studies with isolated granules demonstrated their lysis by a variety of weak acids and surface-active agents. When buffered solutions were employed between the ranges of pH 2.0 and 9.0, granule lysis began at pH 5.5 and was complete at pH 4.0. Chemical analysis disclosed that the granule pellet contained protein and phospholipid with only traces of nucleic acids. Approximately 70 to 80 per cent of the total cellular antimicrobial agent phagocytin was present in the granule fraction. This material was liberated from the granules by acid (pH 5.0 or lower). Studies on selected enzymes showed that acid phosphatase, alkaline phosphatase, nucleotidase, ribonuclease, deoxyribonuclease, and beta glucuronidase were predominantly localized in the granule fraction. Approximately 50 per cent of total cellular lysozyme and cathepsin were also present in the 8,200 g pellet. Disruption of the granules was associated with the release of the majority of granule protein and enzymes in a non-sedimentable form. The properties and composition of rabbit polymorphonuclear leucocyte granules seem to be analogous to those of liver lysosomes.
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PMID:The isolation and properties of the specific cytoplasmic granules of rabbit polymorphonuclear leucocytes. 1369 90

Fibroblast growth factors (FGFs) are involved in stimulation of angiogenesis in tumors and other pathological circumstances. Increased activity of normal skeletal muscles resulting from chronic electrical stimulation is a very potent stimulus for capillary growth but a relationship between the initiation of this angiogenesis and the involvement of autocrine growth factors has yet to be established. Although FGF expression has been reported in muscles stimulated for 3 weeks, capillary growth is underway significantly earlier, beginning around 3 days. The present experiments have therefore studied the possible involvement of basic fibroblast growth factor (FGF-2) in stimulated rat fast skeletal muscles prior to, and coincident with, capillary growth. Muscle contractions were induced via electrodes implanted in the vicinity of the peroneal nerve and maintained for 8h/day for 2, 4 or 7 days. Capillary/fiber ratio (C/F), based on staining of capillary endothelium for alkaline phosphatase, was not changed in either extensor digitorum longus (EDL) or tibialis anterior (TA) after 2 days stimulation, but increased in TA stimulated for 4 days and in both muscles after 7 days. The expression of mRNA for FGF-2, detected by ribonuclease protection assay, was decreased in all stimulated muscles compared with control or contralateral muscles; immunohistochemistry showed FGF-2 gene product in nerves and larger blood vessels but not in capillaries. There was no evidence from immunohistochemistry for up-regulation of receptors flg and bek for FGF-2. The presence of FGF-2, flg and bek in arterioles may indicate a possible role for FGF-2 in the regulation of blood flow since we have previously shown it to be a dilator of small arterioles. However, based on the lack of correlation between changes in capillary density and the expression of mRNA and protein for FGF-2 and its receptors, it is unlikely that it is directly linked with the initiation of angiogenesis resulting from chronic activity in skeletal muscles.
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PMID:Lack of involvement of basic fibroblast growth factor (FGF-2) in capillary growth in skeletal muscles exposed to long-term contractile activity. 1451 78

DNA ligase D (LigD) catalyzes end-healing and end-sealing steps during nonhomologous end joining in bacteria. Pseudomonas aeruginosa LigD consists of a central ATP-dependent ligase domain fused to a C-terminal polymerase domain and an N-terminal 3'-phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at a duplex primer-template with a short 3'-ribonucleotide tract. The phosphodiesterase, which cleaves a 3'-terminal diribonucleotide to yield a primer strand with a ribonucleoside 3'-PO4 terminus, requires the vicinal 2'-OH of the penultimate ribose. The phosphomonoesterase converts the terminal ribonucleoside 3'-PO4 to a 3'-OH. Here we show that the PE domain has a 3'-phosphatase activity on an all-DNA primer-template, signifying that the phosphomonoesterase reaction does not depend on a 2'-OH. The distinctions between the phosphodiesterase and phosphomonoesterase activities are underscored by the results of alanine-scanning, limited proteolysis, and deletion analysis, which show that the two reactions depend on overlapping but nonidentical ensembles of protein functional groups, including: (i) side chains essential for both ribonuclease and phosphatase activity (His-42, His-48, Asp-50, Arg-52, His-84, and Tyr-88); (ii) side chains important for 3'-phosphatase activity but not for 3' ribonucleoside removal (Arg-14, Asp-15, Glu-21, Gln-40, and Glu-82); and (iii) side chains required selectively for the 3'-ribonuclease (Lys-66 and Arg-76). These constellations of critical residues are unique to LigD-like proteins, which we propose comprise a new bifunctional phosphoesterase family.
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PMID:Essential constituents of the 3'-phosphoesterase domain of bacterial DNA ligase D, a nonhomologous end-joining enzyme. 1604 7

DNA ligase D (LigD) performs end remodeling and end sealing reactions during nonhomologous end joining in bacteria. Pseudomonas aeruginosa LigD consists of a central ATP-dependent ligase domain fused to a C-terminal polymerase domain and an N-terminal phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at the 3' end of the primer strand of a primer-template. The phosphodiesterase cleaves a 3'-terminal diribonucleotide to yield a primer strand with a ribonucleoside 3'-PO4 terminus. The phosphomonoesterase converts a terminal ribonucleoside 3'-PO4 or deoxyribonucleoside 3'-PO4 of a primer-template to a 3'-OH. Here we report that the phosphodiesterase and phosphomonoesterase activities are both dependent on the presence and length of the 5' single-strand tail of the primer-template substrate. Although the phosphodiesterase activity is strictly dependent on the 2'-OH of the penultimate ribose, it is indifferent to a 2'-OH versus a2'-H on the terminal nucleoside. Incision at the ribonucleotide linkage is suppressed when the 2'-OH is moved by 1 nucleotide in the 5' direction, suggesting that LigD is an exoribonuclease that cleaves the 3'-terminal phosphodiester. We report the effects of conservative amino acid substitutions at residues: (i) His42, His48, Asp50, Arg52, His84, and Tyr88, which are essential for both the ribonuclease and 3'-phosphatase activities; (ii) Arg14, Asp15, Glu21, and Glu82, which are critical for 3'-phosphatase activity but not 3'-ribonucleoside removal; and (iii) at Lys66 and Arg76, which participate selectively in the 3'-ribonuclease reaction. The results suggest roles for individual functional groups in metal binding and/or phosphoesterase chemistry.
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PMID:Substrate specificity and structure-function analysis of the 3'-phosphoesterase component of the bacterial NHEJ protein, DNA ligase D. 1654 Apr 77


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