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 nucleotide sequence of the alkaline phosphatase (APase) gene (phoA) of Escherichia coli strain 294 has been determined. Pre-APase has a total of 471 amino acids (aa) including a signal sequence of 21 aa. The derived aa sequence differs from that obtained by protein sequencing by the presence of aspartic acid instead of asparagine at positions 16 and 36, and glutamic acid instead of glutamine at position 197. Two open reading frames (ORF1 and ORF2) located downstream from phoA or upstream from proC have been found. ORF1 encodes a putative presecretory protein of 106 aa with a signal sequence of 21 or 22 aa. If this protein is actually produced, it may be one of the smallest periplasmic proteins in E. coli.
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PMID:Nucleotide sequence of the alkaline phosphatase gene of Escherichia coli. 353 24

The effect of varying the amino acid concentrations of the culture medium on matrix vesicle formation was studied in primary cultures of chicken epiphyseal growth plate chondrocytes grown in Dulbecco's modified Eagle's medium (DME) supplemented with 10% fetal bovine serum (FBS). Decreasing the levels of free amino acids in the culture medium to levels of one-half, one quarter, and one eighth of the values normally present in DME caused a progressive decline in matrix vesicle (MV) formation. Increasing the level in the culture medium of those amino acids that are enriched in extracellular fluid (ECF) of growth plate cartilage significantly increased formation of matrix vesicles (MV), as assayed by the alkaline phosphatase (AP) activities present in high-speed sediments from spent culture media. However, adjusting the levels of all amino acids to match those of the ECF produced the greatest stimulation of MV formation. Of the amino acids that are notably enriched in ECF, glutamate (GLU), alanine (ALA), serine (SER), asparagine (ASN), and taurine (TAU) individually enhanced MV production, whereas proline (PRO), glycine (GLY), and aspartate (ASP) had essentially no effect. The simple combination of ECF levels of ALA and GLU resulted in a stimulation of MV formation equal to that observed when the eight aforementioned amino acids were elevated to ECF levels. Other combinations of ASP and GLY, or of TAU, SER, and ASN showed some stimulation, but at a lower level. Increasing the amino acid concentrations, alone or in combination, also increased the levels of cellular AP, and to a lesser extent cellular protein. While increases in cellular AP were generally correlated with increased formation of AP-rich MV, this was not uniformly true. These results indicate that in addition to hormones and growth factors, nutritional factors such as the levels of amino acids are also critical for normal phenotypic expression, growth, and matrix formation by epiphyseal chondrocytes.
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PMID:Effect of amino acid levels on matrix vesicle formation by epiphyseal growth plate chondrocytes in primary culture. 394 89

The combined enzymological investigation including determination of the total activity of asparagine transaminase and alanine transaminase, two serum enzymes, alkaline phosphatase, gamma-glutamyl transpeptidase, acetyl cholinesterase, and butyryl cholinesterase was applied to two groups of pregnant women with pyelonephritis treated with ampicillin (12 patients) and roscillin (14 patients). The investigation was performed at the following stages: before the treatment, on the 7th and on the 12th day of the treatment. No statistically significant differences in the average values of the activity of the above enzymes at these stages were observed in patients of the both groups which indicated the absence of the hepatotoxic effect of the preparations on the patients of a group as a whole. An increase in the levels of transaminases recorded in some patients after discontinuation of the treatment course was evident of a possible cytotoxic effect of the drugs without the signs of cholestasis. The effect was connected with the initial functional renal insufficiency.
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PMID:[Enzymological evaluation of the hepatotoxicity of ampicillin and its therapeutic form, roscillin, in the treatment of pyelonephritis in pregnancy]. 399 43

The differentiation of aggregates of certain teratocarcinoma stem cell lines begins with the formation of an outer layer of primary endoderm cells characterized by the production of plasminogen activator and the absence of histochemically detectable alkaline phosphatase activity. After several days of culture these outer cells develop into a mixture of two types of terminally differentiated endoderm: parietal endoderm which produces a thick layer of underlying basement membrane and visceral endoderm which produces alpha-fetoprotein (AFP). We report here that in the presence of tunicamycin, a drug that inhibits glycosylation of N-asparagine linked glycoproteins, a primary endoderm-like cell is formed which is alkaline phosphatase negative and plasminogen activator positive. However, terminal differentiation of these cells is inhibited as manifested by the lack of accumulation of a thick basement membrane and the absence of immunologically detected AFP. Such inhibition is reversible following removal of the tunicamycin. Terminal differentiation of endoderm depends, therefore, upon N-asparagine linked glycoproteins.
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PMID:Tunicamycin reversibly inhibits the terminal differentiation of teratocarcinoma stem cells to endoderm. 618 42

The nonspecific alkaline phosphatase of Saccharomyces sp. strain 1710 has been shown by phosphatase cytochemistry to be exclusively located in the vacuole, para-Nitrophenyl phosphate-specific alkaline phosphatase is not detected by this procedure because the activity of this enzyme is sensitive to the fixative agent, glutaraldehyde. To determine whether the oligosaccharide of nonspecific alkaline phosphatase is necessary to transport the enzyme into the vacuole, protoplasts were derepressed in the absence or in the presence of tunicamycin, an antibiotic which interferes with the glycosylation of asparagine residues in proteins. The location of the enzyme in the tunicamycin-treated protoplasts, as determined by electron microscopy and subcellular fractionation, was identical to its location in control protoplasts. In addition, carbohydrate-free alkaline phosphatase was found in vacuoles from tunicamycin-treated protoplasts. Our findings indicate that the asparagine-linked carbohydrate moiety does not determine the cellular location of the enzyme.
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PMID:Asparagine-linked carbohydrate does not determine the cellular location of yeast vacuolar nonspecific alkaline phosphatase. 681 17

Carboxypeptidase Y, a vacuolar enzyme from Saccharomyces cerevisiae, was digested with endo-beta-N-acetyl-D-glucosaminidase H to release the four oligosaccharide chains that are linked to asparagine in the glycoprotein. The oligosaccharides were fractionated into a neutral and acidic component, and the latter proved to phosphorylated. From its gel filtration pattern, the neutral fraction was shown to be a mixture of at least four homologs, the smallest of which had a proton NMR spectrum almost identical to that given by an IgM oligosaccharide with eight mannoses and one N-acetylglucosamine [Cohen, R. E. & Ballou, C. E. (1980) Biochemistry 19, 4345--4358]. The yeast oligosaccharide has one additional mannose unit in an alpha 1 leads to 3 or alpha 1 leads to 6 linkage, whereas the larger homologs appear to have two, three, and four more mannose units. One phosphorylated oligosaccharides with a mannose/phosphate ratio of 12.5 was reduced with NaB3H4 and then subjected to mild acid hydrolysis. This released mannose and mannobiose that were glycosidically linked to the phosphate group, whereas complete acid hydrolysis yielded D-mannose 6-phosphate. The recovered oligosaccharide phosphomonoester, which contained 11 or 12 mannose units, was digested exhaustively with alpha-mannosidase, and the product of this reaction was treated with alkaline phosphatase, which yielded radioactive Man3GlcNAcH2. These results suggest that the mannosidase-resistant phosphorylated oligosaccharide has the structure Man leads to P leads to 6 alpha Man leads to alpha Man leads to 6 beta Man leads to 4GlcNAcH2, in which some of the phosphate groups are substituted with mannobiose instead of mannose. A second phosphorylated oligosaccharide with a mannose/phosphate ratio of 6.5 probably contains two phosphodiester groups, but its structure has not been investigated in detail.
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PMID:Carbohydrate chains on yeast carboxypeptidase Y are phosphorylated. 701 28

An increase in the number of beta 1,6 branches of the trimannosyl core of asparagine-linked oligosaccharides has been shown to be directly correlated with the metastatic potential of cultured tumour cells. The Phaseolus vulgaris leukoagglutinating lectin (PHA-L) binds to beta 1,6 branches of tri- and tetra-antennary oligosaccharides. We have applied digoxigenin- and biotin-conjugated PHA-L to establish a non-radioactive detection system for beta 1,6 branches, which can be used in lectin blotting as well as light and electron microscopic cytochemistry. For this purpose the HCT116 human colon carcinoma cell line and colon carcinoma tissue were investigated. Digoxigenin-conjugated PHA-L in conjunction with alkaline phosphatase-conjugated anti-digoxigenin antibodies was superior to biotin-conjugated PHA-L in lectin blotting with respect to sensitivity and specificity. Similarly, the digoxigenin conjugated PHA-L in conjunction with gold-labelled anti-digoxigenin antibodies resulted in more intense specific staining and lower background compared to biotin-conjugated PHA-L visualized with a streptavidin immunogold complex. The specificity of lectin binding in blotting and cytochemical studies was demonstrated by the absence of staining when the lectin was omitted or preabsorbed with glycoprotein, and following pretreatment of the cellular homogenates or tissue sections by N-glycosidase F. Our results demonstrate that digoxigenin-conjugated PHA-L provides high sensitivity and specificity for histochemical and blotting techniques and is amenable for quantification. The technique should have applications in tumour research.
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PMID:Use of Phaseolus vulgaris leukoagglutinating lectin in histochemical and blotting techniques: a comparison of digoxigenin- and biotin-labelled lectins. 750 28

Using site-directed mutagenesis, an aspartate side chain involved in binding metal ions in the active site of Escherichia coli alkaline phosphatase (Asp-369) was replaced, alternately, by asparagine (D369N) and by alanine (D369A). The purified mutant enzymes showed reduced turnover rates (kcat) and increased Michaelis constants (Km). The kcat for the D369A enzyme was 5,000-fold lower than the value for the wild-type enzyme. The D369N enzyme required Zn2+ in millimolar concentrations to become fully active; even under these conditions the kcat measured for hydrolysis of p-nitrophenol phosphate was 2 orders of magnitude lower than for the wild-type enzyme. Thus the kcat/Km ratios showed that catalysis is 50 times less efficient when the carboxylate side chain of Asp-369 is replaced by the corresponding amide; and activity is reduced to near nonenzymic levels when the carboxylate is replaced by a methyl group. The crystal structure of D369N, solved to 2.5 A resolution with an R-factor of 0.189, showed vacancies at 2 of the 3 metal binding sites. On the basis of the kinetic results and the refined X-ray coordinates, a reaction mechanism is proposed for phosphate ester hydrolysis by the D369N enzyme involving only 1 metal with the possible assistance of a histidine side chain.
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PMID:Kinetics and crystal structure of a mutant Escherichia coli alkaline phosphatase (Asp-369-->Asn): a mechanism involving one zinc per active site. 770 48

His-412 in wild-type Escherichia coli alkaline phosphatase is a direct ligand to one of the two zinc atoms critical for the function of the enzyme. To investigate the function of this residue, site-specific mutagenesis was used to substitute His-412 with asparagine and alanine, generating mutant enzymes H412N and H412A, respectively. Both mutant enzymes show a 5-fold decrease in kcat and 30-fold increase in Km when compared to the corresponding kinetic parameters for the wild-type enzyme. In contrast to the wild-type enzyme, Tris and ethanolamine inhibit both the mutant enzymes by inhibiting the hydrolysis reaction and not participating in the transferase reaction; furthermore, both mutants have lower zinc and phosphate content than the wild-type enzyme. The addition of Zn2+ to the H412N and H412A enzymes restores catalytic activity to within 2-fold of the value for the wild-type enzyme, but more importantly the presence of Zn2+ completely restores substrate affinity. The similarity in the kinetic parameters for the H412N and H412A enzymes in the absence and presence of zinc suggests that the asparagine side chain does not play a significant role in coordinating zinc. Furthermore, both the asparagine and alanine substitutions reduce the affinity of the resulting enzymes for zinc. The pH profiles for the two mutant enzymes are different than the pH profile observed for the wild-type enzyme, suggesting that the amino acid substitutions may have altered the pKa of the zinc coordinated water molecule that is critical in the second step of the mechanism. These data suggest that His-412 does not directly participate in the catalytic mechanism but is mainly involved in zinc binding, and therefore is also indirectly involved in substrate binding and product release.
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PMID:Mutations at histidine 412 alter zinc binding and eliminate transferase activity in Escherichia coli alkaline phosphatase. 798 32

Major histocompatibility (MHC) class II antigens are heterodimeric cell surface glycoproteins consisting of an alpha and a beta chain. Although one-dimensional SDS-polyacrylamide gel electrophoresis analysis of purified MHC class II antigens shows a single diffuse band for each chain, multiple spots of identical molecular size were observed for each chain when analyzed by two-dimensional electrophoresis. The basis of this heterogeneity has not been clearly defined and has been predicted partially to be due to glycosylation and/or phosphorylation of the mature protein. To investigate the role of the three N-linked oligosaccharides of the alpha and beta chains in determining the isoelectric point of each chain, affinity-purified MHC class II antigens from human and rat sources were deglycosylated using asparagine amidase. The complete enzymatic removal of all three N-linked oligosaccharides was confirmed by SDS-polyacrylamide gel electrophoresis as well as by four different lectin-linked Western blot analyses. Two-dimensional gel analysis of the deglycosylated molecules shows no significant difference from the fully glycosylated chains. We have expressed truncated forms of the HLA DR2 chains which lack the transmembrane and cytoplasmically exposed regions in Escherichia coli. Two-dimensional electrophoresis of these single chains also reveal multiple banding patterns. The two-dimensional banding patterns described are unaffected by exposure to acidic or basic conditions, increased gel running time in the first dimension, treatment of the proteins with alkaline phosphatase to remove any potential phosphorylation, or preincubation in the presence of iodoacetamide. Multiple forms of recombinant alpha and beta chains were also observed in Tris-glycine-urea gels which merged into a single band in the presence of SDS. In addition, partially fractionated bands from preparative isoelectric focusing gels, when refocused, showed an identical number of multiple spots spanning the same range of isoelectric points. These results together suggest that each polypeptide chain of MHC class II antigens may exist in multiconformational forms, and the observed charge heterogeneity is independent of glycosylation and phosphorylation of the proteins.
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PMID:Intramolecular charge heterogeneity in purified major histocompatibility class II alpha and beta polypeptide chains. 814 5


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