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)

Escherichia coli alkaline phosphatase catalyzes the hydrolysis of a wide variety of phosphomonoesters at similar rates, and the reaction proceeds through a phosphoenzyme intermediate. The active site region is highly conserved between the E. coli and mammalian alkaline phosphatases. The three-dimensional structure of the E. coli enzyme indicates that Lys-328, which is replaced by histidine in all mammalian alkaline phosphatases, is bridged to the phosphate through a water molecule. This water molecule is also hydrogen bonded to Asp-327, a bidendate ligand of the one of the two zinc atoms. Here we report the use of site-specific mutagenesis to convert Lys-328 to both histidine and alanine. Steady-state kinetic studies above pH 7.0 indicate that both mutant enzymes have altered pH versus activity profiles compared to the profile for the wild-type enzyme. At pH 10.3, in the presence of Tris, the Lys-328----Ala enzyme is approximately 14-fold more active than the wild-type enzyme. At the same pH in the absence of Tris the Lys-328----Ala enzyme is still 6-fold more active than the wild-type enzyme. Both mutant enzymes have lower phosphate affinities than the wild-type enzyme at all pH values investigated. Pre-steady-state kinetics at pH 5.5 reveal that the Lys-328----Ala enzyme behaves very similar to the phosphate-free wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A water-mediated salt link in the catalytic site of Escherichia coli alkaline phosphatase may influence activity. 190 46

The effects of the histidine modifier, diethyl pyrocarbonate (DEPC), on brush-border membrane transport systems were studied in rat kidney. DEPC caused a strong inhibition of sodium-dependent phosphate and D-glucose uptake. Phosphate uptake remained linear up to 10 s in control and DEPC-treated membrane vesicles. The D-glucose carrier was more sensitive than the phosphate carrier with half-times of inhibition being 4 and 7 min, respectively. Sodium-independent phosphate and D-glucose uptake remained unaffected by DEPC. Intravesicular volume and two enzyme activities endogenous to the luminal membrane (alkaline phosphatase and aminopeptidase M) remained unaffected by DEPC. Increasing the preincubation pH from 5 to 9 increased phosphate transport inhibition caused by DEPC from 73 to 88% in the presence of DEPC. Hydroxylamine was able to completely reverse phosphate uptake inhibition by DEPC (100%), but only partially reversed the D-glucose uptake inhibition (16%). Sodium or substrate (D-glucose or phosphate) in the preincubation media were unable to protect their respective carriers from DEPC. Sodium-dependent transport of L-glutamine, L-phenylalanine, L-leucine, L-alanine, L-glycine, beta-alanine and L-proline were inhibited at different levels ranging from 70 to 90%. Three transport processes were found insensitive to DEPC modification: L-glutamate, L-lysine and D-fructose. None of the amino acid transporters was protected against DEPC by sodium and/or their respective substrates. Sodium influx was inhibited by DEPC (47%) in the absence of any substrate. Our results show a differential sensitivity of sodium-dependent transporters to DEPC and suggest an important role for histidine residues in the molecular mechanisms of these transporters. More experiments are in progress to further characterize the residue(s) involved in these transport inhibitions by DEPC.
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PMID:Kidney brush-border membrane transporters: differential sensitivity to diethyl pyrocarbonate. 191 27

Human placental and germ cell alkaline phosphatases (PLAP and GCAP, respectively), are characterized by their differential sensitivities to inhibition by L-leucine, EDTA, and heat. Yet, they differ by only 7 amino acids at positions 15, 67, 68, 84, 241, 254, and 429 within their respective 484 residues. To determine the structural basis and the amino acid(s) involved in these physicochemical differences, we constructed three GCAP mutants by site-directed mutagenesis and six GCAP/PLAP chimeras and then expressed these alkaline phosphatase mutants in COS-1 cells. We report that the differential reactivity of PLAP and GCAP depends critically on a single amino acid at position 429. GCAP with Gly-429 is strongly inhibited by L-leucine, EDTA, and heat, whereas PLAP with Glu-429 is resistant. By substituting Gly-429 of GCAP with a series of amino acids, we demonstrate that the relative sensitivities of these mutants to L-leucine, EDTA, and heat inhibition are, in general, parallel. Mutants in the order of resistance to these treatments are: Glu (most resistant), Asp/Ile/Leu, Gln/Val/Lys, Ser/His, and Arg/Thr/Met/Cys/Phe/Trp/Tyr/Pro/Asn/Ala/Gly (least resistant). However, the Ser-429 and His-429 mutants were more resistant to EDTA and heat inhibition than the wild-type GCAP, but were equally sensitive to L-leucine inhibition. Structural analysis of mammalian alkaline phosphatase modeled on the refined crystal structure of Escherichia coli alkaline phosphatase indicates that the negative charge of Glu-429 of PLAP, which simultaneously stabilizes the protein as a whole and the metal binding specifically, probably acts through interactions with the metal ligand His-320 (His-331 in E. coli alkaline phosphatase). Replacement of codon 429 with Gly in GCAP leads to destabilization and loosening of the metal binding. The data suggest that the natural binding site for L-leucine may be near position 429, with the amino and carboxyl groups of L-leucine interacting with bound phosphate and His-432 (His-412 in E. coli alkaline phosphatase), respectively.
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PMID:Mutation of a single amino acid converts germ cell alkaline phosphatase to placental alkaline phosphatase. 193 59

Two out of three rabbit anti-helodermin antisera previously shown to be useful for radioimmunoassay failed to detect up 100 ng of the peptide helodermin spotted directly on a nitrocellulose membrane. A lesser shortcoming of dot immunoassay was encountered with porcine PHI (peptide histidine-isoleucinamide), a member of the same peptide family, and an anti-PHI antiserum. To improve antigen-antibody interactions in the solid phase, we compared five methods of prior peptide immobilization. The best result was obtained when the nitrocellulose membrane was pretreated for 1 min in 4% ovalbumin, followed by 10 min activation with 2.5% glutaraldehyde, before peptide spotting. After cross-linking, the peptide was immunodetected with a F(ab')2 fragment of an anti-rabbit IgG coupled to alkaline phosphatase. The peptide cross-linking method was capable of increasing the sensitivity of ensuing immunodetection by more than 1000-fold, i.e., made feasible the detection of 0.1 ng peptide/dot in cases when the direct spotting method was inefficient. The sensitivity of this new, reliable and simple dot immunoassay for peptides was comparable to the conventional dot assay of directly immobilized large proteins.
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PMID:The sensitivity of dot immunoassay for the peptides helodermin, histidine-isoleucinamide (PHI) and histidine-methioninamide (PHM) increases after peptide cross-linking to proteins prefixed on nitrocellulose. 223 Jan 34

Most of the essential cellular components, like nucleic acids, lipids and sugars, are phosphorylated. The phosphate equilibrium in Escherichia coli is regulated by the phosphate (Pi) input from the surrounding medium. Some 90 proteins are synthesized at an increased rate during Pi starvation and the global control of the cellular metabolism requires cross-talk with other regulatory mechanisms. Since the Pi concentration is normally low in E. coli's natural habitat, these cells have devised a mechanism for synthesis of about 15 proteins to accomplish two specific functions: transport of Pi and its intracellular regulation. The synthesis of these proteins is controlled by two genes (the phoB-phoR operon), involving both negative and positive functions. PhoR protein is a histidine protein kinase, induced in Pi starvation and is a transmembrane protein. It phosphorylates the regulator protein PhoB which is also Pi starvation-induced. The PhoB phosphorylated form binds specifically to a DNA sequence of 18 nucleotides (the pho Box), which is part of the promoters of the Pho genes. The genes controlled by phoB constitute the Pho regulon. The repression of phoA (the gene encoding alkaline phosphatase) by high Pi concentrations in the medium requires the presence of an intact Pst operon (pstS, pstC, pstA, pstB and phoU) and phoR. The products of pstA and pstC are membrane bound, whereas the product of pstS is periplasmic and PstB and PhoU proteins are cytoplasmic. The function of the PhoU protein may be regulated by cofactor nucleotides and may be involved in signaling the activation of the regulon via PhoR.
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PMID:From cell membrane to nucleotides: the phosphate regulon in Escherichia coli. 224 34

Rabbit skeletal muscle and liver fructose 1,6-diphosphate aldolases autophosphorylate in the presence of inorganic phosphate at physiological and alkaline pH. ATP as well as nonhydrolyzable ATP analogues inhibits autophosphorylation. Autophosphorylation of aldolases abolishes catalytic activity, which is restored upon treatment with alkaline phosphatase. Limited proteolysis of aldolase preferentially hydrolyzes the COOH terminus and liberates a phosphorylated peptide. Treatment of rabbit aldolases with carboxypeptidase, which liberates the COOH terminal residue Tyr 363, although modifying catalytic activity does not affect autophosphorylation. Amino acid analyses are consistent with results of autophosphorylation of the COOH terminus showing residue His 361 in muscle aldolase and Tyr 361 in liver aldolase. Phosphate lability in acid pH by phosphorylated muscle aldolase but not by phosphorylated liver aldolase corroborates the amino acid assignment. Autophosphorylation of the aldolases in the crystalline state is consistent with an intramolecular mechanism. The pH dependence of autophosphorylation being dependent on the enzyme's physical state (soluble or crystalline) is not inconsistent with crystallization stabilizing a conformer having different amino acid pka values and/or reactivities than those of the soluble state.
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PMID:Inactivation of mammalian fructose diphosphate aldolases by COOH terminus autophosphorylation. 227 41

The major acid phosphatase form (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) was purified from the soluble extract of barley roots. The enzyme is homogeneous on polyacrylamide gel electrophoresis and moves as a single band of Mr approximately 38,000 in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme was Mr 77,600 and 79,000 as determined, respectively, by gel filtration on a Sephadex G-100 column and by density gradient ultracentrifugation. The isoelectric point was about 6.28. The enzyme is competitively inhibited by molybdate (Ki = 9 x 10(-7) M). NaF, Ag(+), Hg(2+), Pb(2+) and Zn(2+) are also inhibitors, while other cations showed no effect. The enzyme hydrolyzes a wide variety of natural and synthetic phosphate esters. In particular, the enzyme seems to be active on ATP, o-phosphotyrosine, o-phosphoserine and glucose 1-phosphate. The pH dependence studies between pH 4-8 using p-nitrophenylphosphate as substrate and diethylpyrocarbonate inactivation indicate the presence of essential histidine residue at the active site.
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PMID:Multiple forms of barley root acid phosphatase: purification and some characteristics of the major cytoplasmic isoenzyme. 229 73

A young man receiving high dose cytosine arabinoside (3g/m2 every 12 hours) for promyelocytic leukemia developed rapidly increasing hyperbilirubinemia and hepatorenal syndrome. The patient had been treated previously with courses of standard dose cytosine arabinoside without hepatic or renal complications. His condition rapidly deteriorated, and he required hemodialysis. The total bilirubin increased to 45.4 mg/dL, but alkaline phosphatase remained normal. Twelve days after starting chemotherapy, the patient died of hepatorenal failure. Liver necropsy revealed mild bile stasis and microvesicular steatosis. We suspect high dose cytosine arabinoside played a major role in causing impairment of bilirubin transport within the hepatocyte in this patient.
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PMID:Jaundice and hepatorenal syndrome associated with cytosine arabinoside. 231 16

The effect of lithium and other antipsychotic drugs on the renal function in patients with manic-depressive disorders has been investigated. Thirty-four patients (5 males and 29 females) treated with lithium and 21 patients (6 males and 15 females) on other antipsychotic drugs were studied. A control group of 10 persons consisting of healthy subjects, all of whom were taking no medication was also studied. No significant differences in the treatment duration were present between the patients investigated. Although few patients on lithium had glomerular filtration reduced, no statistically significant difference in creatinine clearance was found between the groups. None of the patients had a disturbance in the reabsorption of glucose, amino acids (histidine, lysine, valine, glutamine, glycine, serine, taurine, threonine, alanine, isoleucine) and beta 2-microglobulin. Patients treated with lithium had a significantly reduced urine concentration and higher daily diuresis than did the other two studied groups. A significantly higher overnight elimination of alkaline phosphatase was found in a group of patients taking other antipsychotic drugs. The attained results suggest tubular lesions in patients with manic-depressive psychosis occurring in the association with the prophylactic use of lithium and, at same time, the possibility of the other in association with the other antipsychotic drugs.
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PMID:[Effect of long-term use of lithium on kidney function]. 236 20

In this study a double immunohistochemical staining procedure is described for the simultaneous demonstration of antigen expressing cells and replicating cells in rat thymus. As markers for cell surface antigen expression a monoclonal antibody against Ia-expressing cells (His 19) and a monoclonal antibody against cells of the monocyte-macrophage lineage (ED2) were used. Replicating cells were demonstrated by the incorporation of 5-bromodeoxyuridine (BrdUrd). Tissue pieces were fixed in a periodate-lysine-paraformaldehyde fixative and embedded in glycol methacrylate. To demonstrate Ia-expressing cells or ED2-positive macrophages in plastic embedded sections a digestion with trypsin is necessary. The staining procedure was applied sequentially and was performed with a peroxidase and an alkaline phosphatase labeled reagent yielding respectively a brown and a blue reaction product. Results with this staining procedure on plastic embedded sections of rat thymus, an organ with a high DNA synthesizing capacity, showed incorporation of BrdUrd predominantly in the cortex. ED2-positive macrophages were only found in the cortex. The Ia-positive epithelial reticular cells demonstrated extremely well their stellate form.
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PMID:Simultaneous immunohistochemical demonstration of antigen expression and 5-bromodeoxyuridine incorporation in plastic embedded sections. 243 84


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