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 subcellular localization of aminopeptidase N (previously called aminoendopeptidase) has been investigated. This enzyme was found to be partially released (30-40%) by osmotic shock or by converting Escherichia coli K10 cells to spheroplasts. However, in all other E. coli strains (K12, B/r, MRE 600, ML 308) tested, this enzyme is not released at all by these procedures and thus behaves like a cytoplasmic enzyme. The crypticity of aminopeptidase N is surprisingly low, 75-85% of the enzyme activity is directly assayable in intact cells of any E. coli strain. Various inhibitors of transport systems do not interfer with this assay. Aminopeptidase activity could also be assayed in spheroplasts, even when an insolubilized substrate was used, which suggests a surface location of this enzyme. As well, N-ethylmaleimide (0.4 mM), under conditions which do not allow penetration in the cytoplasm, caused 70% inhibition of aminopeptidase N. Binding of 125I-labeled antiaminopeptidase N antibody to spheroplasts (from K12 strain) was used to assay the orientation of aminopeptidase N in the membrane. This enzyme is exposed on the outer surface of the cytoplasmic membrane. Confirmation of this orientation was obtained by comparing the accessibility of aminopeptidase, alkaline phosphatase and beta-galactosidase to fluorescamine in intact cells. Only 16% of the total beta-galactosidase was labeled with this fluorescent reagent whereas 44-45% of the aminopeptidase N and 59% of the alkaline phosphatase were labeled. Electron microscopic visualization of insolubilized reaction products of aminopeptidase N within the cells showed that these products are located at the poles of the cells. Neither mutant cells which were devoid of aminopeptidase N activity nor parental strains with the enzyme activity inhibited with phenylmercuric chloride contained the characteristic black caps. Thus, it appears that the periplasm is enlarged at the poles of the cells and that the reaction product is mainly located in these places. Investigation of the type of interactions of aminopeptidase N with the plasma membrane only revealed that aminopeptidase N has mainly an electrostatic interaction with the outer surface, probably mediated by magnesium ion bridges. Additional interactions are involved since disruption of the integrity of the cytoplasmic membrane is required to totally release this enzyme.
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PMID:Aminopeptidase N from Escherichia coli. Unusual interactions with the cell surface. 32 10

Escherichia coli KG980, a vitamin B6 auxotroph derived from E. coli K12, utilized the three unphosphorylated forms of vitamin B6, more or less effectively, for growth, but did not utilize the three phosphate forms at concentrations ranging from 10(-7) to 10(-5) M in the minimum medium of Davis and Mingioli. The bacterium, however, utilized the phosphate forms, although less effectively than the unphosphorylated forms, in the phosphate starving medium of Garen and Levinthal which is known to derepress alkaline phosphatase. Correspondingly, the phosphate forms of 3H-labeled vitamin B6 in the minimum medium were not taken up by the bacterial cells grown in the same medium, but were taken up when the phosphate starving medium was used for growth and uptake experiments; the unphosphorylated forms were taken up with either of the media used. After 30-min incubation of the cells grown in the phosphate starving medium with 3H-pyridoxine phosphate added to the same medium, the main extracellular 3H-vitamin B6 was found to be pyridoxine, evidently indicating an involvement of alkaline phosphatase action. It is concluded from these results that the phosphate forms of vitamin B6 can be taken up and utilized only after dephosphorylation but not taken up in their intact form. The initial rate of 3H-pyridoxal and 3H-pyridoxamine uptake in the minimum medium showed saturation kinetics. The Km and Vmax values were 1.2 X 10(-6) M and 62 pmoles/min/mg (dry cell weight) for pyridoxal; 11 X 10(-6) M and 65 pmoles/min/mg for pyridoxamine. 3H-Pyridoxine uptake apparently consisted of a high-affinity saturable component, whose Km value was tentatively estimated to be 2.2 X 10(-6) M, and an unsaturable component. The uptake rates of these three unphosphorylated vitamin B6 compounds compared at limiting concentrations for the growth of E. coli KG980 appear to be essentially in good agreement with the response pattern of this bacterium to the three compounds.
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PMID:Uptake and utilization of vitamin B6 and its phosphate esters by Escherichia coli. 32 31

Morphological mon mutants and a chain-forming envC mutant of Escherichia coli K12 are hypersensitive to detergents and to various other antibacterial agents. Electron microscopy shows that the cytoplasmic membrane of growing mon and envC cells is dissolved by detergents, and that the cytoplasm dissociates into two parts of unequal density. The mutant envC is sensitive to actinomycin D and to rifampicin, is lysed by lysozyme in the absence of EDTA, and releases alkaline phosphatase in the absence of osomotic shock, indicating a gross perturbation of its outer membrane.
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PMID:Morphological mutants of Escherichia coli: nature of the permeability barrier in mon and envC cells. 35 22

Protein content of membranes in wild type strains of E. coli K12 and K10 and in mutants defective in alkaline phosphatase regulator genes: E. coli C85 (R1-R2+p+) and E. coli C4(R1+R2-P+) under the conditions of repression and derepression of this enzyme was studied. Correlation between the content in membranes of minor component with the molecular weight 30,700 and the state of the regulatory system of alkaline phosphatase biosynthesis was shown. This protein was absent in the membranes of the repressed cells of wild type strains and in the membranes of nonrepressible (constitutive) mutant E. coli C4. Probably the protein with molecular weight 30,700 is a product of the regulatory gene R2 and its binding with the membrane determines its regulatory function.
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PMID:[Protein content of E. coli membrane under conditions of repressed and derepressed biosynthesis of alkaline phosphatase]. 80 86

ODC was purified to homogeneity from E. coli K12 MG1655 strain transformed with a pBR322 plasmid carrying the ODC gene. This preparation was homogeneous as it was analyzed by SDS-polyacrylamide gel electrophoresis. From this preparation the amino-terminal sequence analysis was obtained. The native ODC of E. coli is activated by ATP, GTP, CTP and UTP at 10(-3) M concentration to around 170-300%. Our results indicate that the recombinant ODC is activated only by GTP and UTP at 10(-3) M 370% and 300%, respectively. When the recombinant ODC was incubated with calf intestine alkaline phosphatase, this inactive ODC can be reversibly activated allosterically only by GTP or UTP at a concentration of 10(-6) or 10(-5) M. That GTP or UTP can allosterically convert the inactive form of ODC to an active form suggests that these analogues may be the in vivo physiological regulators of ODC.
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PMID:Allosteric activation by nucleotides of the inactive by phosphatase ornithine decarboxylase of Escherichia coli. 144 81

The FhuA protein (formerly TonA) is located in the outer membrane of Escherichia coli K12. Fusions between fhuA and phoA genes were constructed. They determined proteins containing a truncated but still active alkaline phosphatase of constant size and a variable FhuA portion which ranged from 11%-90% of the mature FhuA protein. The fusion sites were nearly randomly distributed along the FhuA protein. The FhuA segments directed the secretion of the truncated alkaline phosphatase across the cytoplasmic membrane. The fusion proteins were proteolytically degraded up to the size of alkaline phosphatase and no longer reacted with anti-FhuA antibodies. The fusion proteins were more stable in lon and pep mutants lacking cytoplasmic protease and peptidases, respectively. The larger fusion proteins above a molecular weight of 64,000 dalton were predominantly found in the outer membrane fraction. They were degraded by trypsin when cells were converted to spheroplasts so that trypsin gained access to the periplasm. In contrast, FhuA protein in the outer membrane was largely resistant to trypsin. It is concluded that the larger FhuA'-'PhoA fusion proteins were associated with, but not properly integrated into, the outer membrane.
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PMID:Probing FhuA'-'PhoA fusion proteins for the study of FhuA export into the cell envelope of Escherichia coli K12. 285 32

The "SOS Chromotest" has recently been introduced by P. Quillardet et al. (1982; Quillardet and Hofnung, 1985), who use strain PQ37 of Escherichia coli K12 to test for genotoxicity. We have modified the procedure in order to optimize the determination of beta-galactosidase and alkaline phosphatase activities, and, where possible, to allow measurements to be made automatically. Kinetic determination is quicker, more sensitive and avoids interference by coloured compounds. Modification of the metabolic activation system increases the sensitivity of the test for progenotoxicity.
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PMID:Kinetic determination of enzymatic activity and modification of the metabolic activation system in the SOS chromotest. 309 30

An 11.6-kilobase (kb) region of a 34-kb fragment of Escherichia coli DNA that encodes the K1 capsular polysaccharide genes is necessary for translocation of the K1 polysaccharide to the bacterial cell surface. This 11.6-kb region contains a gene, kpsD, encoding a 60-kilodalton protein. The kpsD gene was localized to a 2.4-kb PstI-BamHI fragment. Cells harboring a Tn1000 insertion in kpsD did not synthesize the 60-kilodalton protein and did not express polysaccharide on the cell surface. Immunodiffusion and rocket immunoelectrophoresis of cell extracts, however, demonstrated that K1 polysaccharide was synthesized by these cells. We present evidence that the kpsD gene product is synthesized as a precursor and that the processed form is located in the periplasmic space. Analysis of alkaline phosphatase activity of a kpsD-phoA fusion demonstrated that kpsD expression was under positive regulation. A 260-base-pair AluI fragment located within the kpsD coding sequence was used as a probe and was found to hybridize to chromosomal DNA from E. coli that synthesizes the K2, K5, K7, K12, and K13 capsular polysaccharides but not K3 and K100. These results suggest that the kpsD gene product may be required for export not only of K1 but for other K antigens as well.
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PMID:Translocation of capsular polysaccharides in pathogenic strains of Escherichia coli requires a 60-kilodalton periplasmic protein. 311 65

Mutants of Escherichia coli K12 carrying exc mutations inducing the release of the plasmid pBR322-encoded beta-lactamase (EC 3.5.2.6) into the extracellular medium were analysed and compared with previously described excretory mutants carrying lky mutations associated with the release of alkaline phosphatase and to tolA and tolB mutants, originally described as tolerant towards various colicins. The exc, lky and tol mutations mapped near the gal operon at min 16.5 of the E. coli linkage map. A genetic analysis presented in this paper showed that some exc and lky mutations belonged to the tolA and tolB complementation groups. Furthermore, we identified a third cistron, excC, involved in the excretion of periplasmic enzymes but distinct from the two others.
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PMID:tolA, tolB and excC, three cistrons involved in the control of pleiotropic release of periplasmic proteins by Escherichia coli K12. 331 62

The SOS-function-inducing activities of 36 furylethylenes were characterized in Escherichia coli K12. The induction of the SOS function was assayed by monitoring the beta-galactosidase activity in the sulA::lacZ fusion strain PQ 37. To correct for the inhibitory effects of test compounds on mRNA or protein synthesis, the level of the constitutive alkaline phosphatase was assayed in parallel. Tested furylethylenes included nine alkylesters and eleven N-alkylamides of 5-nitro-2-furylacrylic acid (NFAA) and fourteen derivatives differing not only in substituents at exocyclic double bond, but also in the position 5 of the furan ring. The induction of the SOS-function by the derivatives depends on the presence of 5-nitrofuran centre in their molecule; side chains in the position 2 modify the degree of SOS response. SOS-inducing potency of n-alkyl congeners decreases with increasing lipophilicity. Effect of derivatives with branched alkyl substituents is lower than expected from the behavior of the n-alkyl homologues. All derivatives with positive effect on SOS-function in E. coli show mutagenic activity on Salmonella typhimurium TA98 in Ames test.
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PMID:Relationships between structure of 5-nitro-2-furylethylenes and their SOS-function-inducing activities in Escherichia coli. 351 69


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