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

---

Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

tolA mutants of Escherichia coli K-12 release periplasmic proteins into the extracellular medium; they are sensitive to growth inhibitors such as cholic acid and tolerant to group A colicins and filamentous bacteriophage. Suppressor mutants of the tolA-876 allele were isolated by selecting for cholic acid resistant clones that did not release periplasmic ribonuclease I. One class of tolA suppressor strains carried mutations in the staA gene (for suppressor of tolA) located a 41 min. tolA-876 staA strains partially recovered a wild-type phenotype: they exported alkaline phosphatase and beta-lactamase into the periplasm and only released very low amounts of periplasmic proteins; moreover, they were sensitive to E1 and A colicins and more resistant than tolA-876 staA+ strains to various growth inhibitors. Furthermore, tolA-876 staA-2 and tolA+staA-2 mutants were 10- to 2700-times more resistant than staA+ strains to bacteriophages TuIa, TuIb and T4, and TuII whose receptors are major outer membrane proteins OmpF, OmpC and OmpA, respectively. SDS-PAGE analysis suggested that cell envelopes of staA or staA+ strains contained similar amounts of these proteins but characterization of strains carrying ompF (or C or A)-phoA gene fusions showed that mutation stA-2 reduced ompF gene expression by a factor of two. Analysis of double mutants strains carrying mutation staA-2 and a tolA, tolB, excC or excD periplasmic-leaky mutation showed that staA suppression was allele specific which suggested that proteins TolA and StaA might directly interact.
...
PMID:Isolation and characterization of extragenic suppressor mutants of the tolA-876 periplasmic-leaky allele in Escherichia coli K-12. 204 Apr 38

RNP, Sm and SS-B nuclear antigens from calf thymus were studied with respect to the size distribution on sucrose gradients as well as to the molecular integrity and related structural changes when they were subjected to enzymatic digestions under different conditions. Making a difference with RNP particles, the Sm size distribution is concentration dependent, a property in accordance with the complexity of the Sm particles in comparison with the RNPs. The use of combined effects of temperature, endogenous proteases and RNase A, allowed us to gain insight into the limits of stability of the three antigenic particles. Following treatments in the absence of RNAse A, the degradation products (32-38 Kd molecular weight) of the 70 Kd RNP polypeptide remain stable and associated with other molecules within the RNP particle. It was also found that the phosphate groups of the SS-B protein moiety are only accessible to alkaline phosphatase if the RNA of the SS-B particle is degraded by the action of RNAse A.
...
PMID:RNP, Sm and SS-B antigens from calf thymus: molecular stability upon enzymatic digestion. 209 30

Data from differential scanning calorimetry (DSC) may be used to estimate very large binding constants that cannot be conveniently measured by more conventional equilibrium techniques. Thermodynamic models have been formulated to describe interacting systems that involve either one thermal transition (protein-ligand) or two thermal transitions (protein-protein) and either 1:1 or higher binding stoichiometry. Methods are described for obtaining binding constants and heats of binding by two different methods: calculation or simulation fitting of data. Extensive DSC data on 2'CMP binding to RNase are presented and analyzed by the two methods. It is found that the methods agree when binding sites are completely saturated, but substantial errors arise in the calculation method when site saturation is incomplete and the transition of liganded molecules overlaps that of unliganded molecules. This arises primarily from an inability to determine TM (i.e., the temperature where concentrations of folded and unfolded protein are equal) under weak-binding conditions. Results from simulation show that the binding constants and heats of binding from the DSC method agree quantitatively with corresponding estimates obtained from equilibrium methods when extrapolated to the same temperature. It was also found from the DSC data that the binding constant decreases with increasing concentration of ligand, which might arise from nonideality effects associated with dimerization of 2'CMP. Simulations show that the DSC method is capable of estimating binding constants for ultratight interactions up to perhaps 10(40) M-1 or higher, while most equilibrium methods fail well below 10(10) M-1. DSC data from the literature on a number of interacting systems (trypsin-soybean trypsin inhibitor, trypsin-ovomucoid, trypsin-pancreatic trypsin inhibitor, chymotrypsin-subtilisin inhibitor, subtilisin BPN-subtilisin inhibitor, RNase S protein-RNase S peptide, avidin-biotin, ovotransferrin-Fe3+, superoxide dismutase-Zn2+, alkaline phosphatase-Zn2+, and assembly of regulatory and catalytic subunits of aspartate transcarbamoylase) were analyzed by simulation fitting or by calculation. Apparent single-site binding constants ranged from ca. 10(5) to 10(20) M-1, while the interaction constant for assembly of aspartate transcarbamoylase was estimated as 10(37) in molarity units. For most of these systems, the DSC interaction constants compared favorably with other literature estimates, for some it did not for reasons unknown, while for still others this represented the first estimate. Simulations show that for proteins having two binding sites for the same ligand within a single cooperative unit, ligand rearrangement will occur spontaneously during a DSC scan as the transition temperature of the unliganded protein is approached.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:Study of strong to ultratight protein interactions using differential scanning calorimetry. 220 24

Molecular-dynamic calculations predict that, if Tyr24 and Asn84 are each replaced by a Cys residue, it should be possible to form a third disulfide bond in ribonuclease T1 (RNase T1) between these residues, with only minimal conformational changes at the catalytic site. The gene encoding such a mutant variant of RNase T1 (Tyr24----Cys24, Asn84----Cys84) was constructed by the cassette mutagenesis method using a chemically synthesized gene. In order to reduce the toxic effect of the mutant enzyme (RNase T1S) on an Escherichia coli host, we arranged for the protein to be secreted into the periplasmic space by using a vector that harbors a gene for an alkaline phosphatase signal peptide under the control of the trp promoter. The nucleolytic activity of RNase T1S toward pGpC was approximately the same as that of RNase T1 at 37 degrees C (pH 7.5). Moreover, at 55 degrees C, RNase T1S retained nearly 70% of its activity while the activity of the wild-type enzyme was reduced to less than 10%. RNase T1S was also more resistant to denaturation by urea than the wild-type enzyme. However, unlike RNase T1, RNase T1S was irreversibly and almost totally inactivated by boiling at 100 degrees C for 15 min.
...
PMID:A thermoresistant mutant of ribonuclease T1 having three disulfide bonds. 234 14

Reconstitution of influenza virus nucleoprotein (NP)-RNA complexes was performed with segment 8 RNA, which was synthesized in vitro from cDNA, and NP purified from virions. Under optimum conditions established using a filter binding assay and a gel retardation assay, NP was found to bind any RNA longer than 15 nucleotides. NP-RNA complexes formed at 30 degrees C are more resistant to high concentrations of NaCl than those formed at 0 degrees C. Treatment of NP with N-ethylmaleimide gave no effect on its RNA binding activity, whereas treatment with alkaline phosphatase enhanced its RNA binding activity. The newly developed "reverse-printing" method of RNase V1-treated complexes revealed that reconstituted NP-RNA complexes carry RNase V1-sensitive sites as do native ribonucleoprotein (RNP) cores (RNA polymerase-NP-RNA complexes), implying that RNA-NP complexes structurally similar to native RNP cores are reconstituted from isolated components.
...
PMID:Reconstitution of influenza virus RNA-nucleoprotein complexes structurally resembling native viral ribonucleoprotein cores. 235 55

Exposure for 20 min of stationary phase cells of Salmonella typhimurium to a combined triple stress system (TSS) treatment comprising hypochlorite derived 5 ppm free available chlorine in solution acidified with 1% succinate (pH 2.5) and at a chill shock temperature of 5 degrees C resulted in symptoms of injury. Cells became sensitive to 40 micrograms/ml lysozyme, 50 micrograms/ml actinomycin D and 100 micrograms/ml ribonuclease B, to which control cells were resistant. Metabolic injury was indicated by reduction in colony forming ability of stressed cells on minimal salts glucose agar M9 medium. There was no detectable leakage loss of 260-280 nm-absorbing materials. This was also confirmed by assay of the cellular RNA material components. Loss of alkaline phosphatase activity was observed in the stressed cells. The intensity of induced cellular damage as measured by lysozyme sensitivity was greatest in the cells exposed to the complete TSS, followed by those stressed in 1% succinate at 5 degrees C, then 5 ppm chlorine at 5 degrees C and the singular chill shock stress at 5 degrees C, respectively. The magnitudes of cellular damage, however, were suggestive of synergistic interactions among the component stress factors of the TSS. The findings obtained indicated impairment of the structural integrity and functional capabilities of the permeability barriers and the inactivation of certain periplasmic enzymes. The resultant cumulative cellular damage from the TSS exposure may therefore enhance greater sensitivity of treated cells to subsequent stress factors.
...
PMID:Mechanisms of triple stress-mediated damage in stationary phase cells of Salmonella typhimurium exposed to succinate-acidified hypochlorite system at 5 degrees C. 242

Recent studies have demonstrated by Northern blot analysis that both the c-fms proto-oncogene and the CSF-1 gene are expressed during human monocytic differentiation. In order to examine c-fms and CSF-1 expression at the cellular level, we have applied alkaline phosphatase detection of biotinylated v-fms and CSF-1 cDNA probes in situ. Using this approach, we demonstrate that c-fms and CSF-1 transcripts are detectable in HL 60 cells induced along the monocytic lineage but not in uninduced cells. The specific detection of these transcripts is further supported by the absence of histochemical staining in RNase-treated cells and when using pBR322 plasmid without insert as the biotinylated probe. Finally, the results indicate that most of the induced HL-60 cells have detectable levels of both c-fms and CSF-1 RNA. This approach should be useful for studying expression of these genes in populations of leukemic blasts and normal hematopoietic cells.
...
PMID:Detection of c-fms and CSF-1 RNA by in situ hybridization. 244 33

Polyadenylated [poly(A)+] RNA molecules have been isolated from Methanococcus vannielii by oligodeoxythymidylate-cellulose affinity chromatography at 4 degrees C. Approximately 16% of the label in RNA isolated from cultures allowed to incorporate [3H]uridine for 3 min at 37 degrees C was poly(A)+ RNA. In contrast, less than 1% of the radioactivity in RNA labeled over a period of several generations was contained in poly(A)+ RNA molecules. Electrophoretic separation of poly(A)+ RNA molecules showed a heterogeneous population with mobilities indicative of sizes ranging from 900 to 3,000 bases in length. The population of poly(A)+ RNA molecules was found to have a half-life in vivo of approximately 12 min. Polyadenylate [poly(A)] tracts were isolated by digestion with RNase A and RNase T1 after 3' end labeling of the poly(A)+ RNA with RNA ligase. These radioactively labeled poly(A) oligonucleotides were shown by electrophoresis through DNA sequencing gels to average 10 bases in length, with major components of 5, 9, 10, 11, and 12 bases. The lengths of these poly(A) sequences are in agreement with estimates obtained from RNase A and RNase T1 digestions of [3H]adenine-labeled poly(A)+ RNA molecules. Poly(A)+ RNA molecules from M. vannielii were labeled at their 5' termini with T4 polynucleotide kinase after dephosphorylation with calf intestine alkaline phosphatase. Pretreatment of the RNA molecules with tobacco acid pyrophosphatase did not increase the amount of phosphate incorporated into poly(A)+ RNA molecules by polynucleotide kinase, indicating that the poly(A)+ RNA molecules did not have modified bases (caps) at their 5' termini. The relatively short poly(A) tracts, the lack of 5' cap structures, and the instability of the poly(A)+ RNA molecules isolated from M. vannielii indicate that these archaebacterial poly(A)+ RNAs more closely resemble eubacterial mRNAs than eucaryotic mRNAs.
...
PMID:Polyadenylated, noncapped RNA from the archaebacterium Methanococcus vannielii. 258 34

Tumor necrosis factor (TNF) is a Mr 17,000 cytokine produced by macrophages. We have recently demonstrated that TNF is also produced by transformed human epithelial cells. The present studies have examined TNF expression in human myeloid leukemic cells. We have monitored TNF expression at a cellular level using alkaline phosphatase detection of a biotinylated TNF cDNA probe in situ. Using this approach, TNF transcripts were detectable in HL-60 cells induced along the monocytic lineage by phorbol ester but not in uninduced cells. The specific detection of TNF RNA at a cellular level was supported by the absence of histochemical staining in RNase-treated cells and when using biotinylated pBR322 plasmid without insert. These studies were extended to preparations of purified acute myeloblastic leukemia cells. The results demonstrate that TNF is expressed in myeloblasts in eight of nine patients with AML. In each preparation of myeloblasts with detectable TNF RNA, transcripts were present at 89-98% of the cells. The identification of TNF RNA in situ was also associated with the detection of TNF protein in leukemic blasts by indirect immunofluorescence. Moreover, the detection of TNF protein in these preparations of myeloblasts was confirmed by immunoblotting. However, using this approach to examine AML cells before and after purification indicated that TNF expression is induced as a result of the enrichment procedures. Thus, certain populations of purified myeloid leukemic cells are capable of expressing TNF at both the RNA and protein levels.
...
PMID:Detection of tumor necrosis factor gene expression at a cellular level in human acute myeloid leukemias. 264 77

High levels of nuclease activities were identified in filtrates of Aspergillus cultures after growth in low-but not in high-phosphate media. Deoxyribonuclease activities, characterized extensively by column chromatography, showed a coincident single peak for ss- and ds-DNase which was distinct from the peak for RNase. Both ss-DNase and ds-DNase are endonucleolytic and showed the highest activity in the presence of Ca2+ and Mn2+ (at pH 8.0). They also showed identical heat sensitivities suggesting that a single, phosphate-repressible DNase was secreted. This enzyme, therefore, corresponds to the well-characterized extracellular DNase A of Neurospora. However, the Aspergillus DNase A did not cross-react with antisera to secreted Neurospora nucleases and showed different chromatographic properties, and active peptides of different sizes were visualized on DNA activity gels. The increasing derepression of Aspergillus DNase A by decreasing phosphate levels was similar to that of secreted alkaline phosphatase and these increases were both abolished by the regulatory mutant palcA.
...
PMID:A single, phosphate-repressible deoxyribonuclease, DNase A, secreted in Aspergillus nidulans. 267 10


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---