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 F1-20 protein is a novel neuronal-specific, synapse-associated protein that is expressed nonuniformly in mouse brain. Expression of the F1-20 protein is developmentally regulated in a pattern coincident with active synaptogenesis and synaptic maturation. Here we report the cloning of the cDNA sequence for the F1-20 protein. We found two distinct isoforms of F1-20 cDNA that differed by the presence of 15 additional nucleotides, which does not interrupt the open reading frame. RNase protection analysis and PCR amplification of mouse brain RNA revealed that both isoforms are present in cellular RNA. It is likely that the two F1-20 mRNA isoforms are derived from RNA splicing events utilizing alternative 3' acceptor sites. Analysis of the deduced amino acid sequence for the complete open reading frame revealed that the predominant F1-20 mRNA encodes an 896 amino acid polypeptide with a molecular weight of 91,319 Da. The deduced amino acid sequence does not contain a signal sequence, or any extensive hydrophobic regions. The deduced amino acid sequence does contain a number of consensus sequences for protein kinases. Searches of the protein and nucleic acid sequence data bases revealed that the F1-20 protein has not been previously characterized at the primary structure level, although a weak similarity was found between rabbit calpastatin and the C-terminal portion of the F1-20 protein. We then determined biochemically that the F1-20 protein is a substrate for Ca(2+)-dependent proteolysis, which is specifically inhibited by calpain inhibitors in vitro. This indicates that the F1-20 protein is a substrate for neuronal calpain. We observed that treatment of a synaptosomal lysate with alkaline phosphatase led to an increase in the electrophoretic mobility of the F1-20 protein, as well as to an increase in the sharpness of the electrophoretic band. This indicates that the F1-20 protein is phosphorylated in vivo.
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PMID:Characterization of a novel synapse-specific protein. II. cDNA cloning and sequence analysis of the F1-20 protein. 160 33

This study compares the synthesis of mutant type I collagen in cultured dermal fibroblasts and trabecular osteoblasts that were isolated from a patient with moderately severe osteogenesis imperfecta (type IV). Previous study of this patient's dermal fibroblasts revealed a 2000 dalton deletion located in cyanogen bromide peptide 4 of alpha 2(I)-collagen. The phenotype of the bone cell cultures was defined by a 3-4 day logarithmic phase doubling time, predominantly type I collagen production over type III and alkaline phosphatase activity 13.5 times dermal fibroblast levels. The current study revealed that both fibroblasts and osteoblasts synthesized a normal and a shortened alpha 2(I) chain, each as the product of separate alleles. Following pepsin treatment of the procollagens, a shortened alpha 1(I) chain was also seen in both cell types. Cyanogen bromide peptide mapping of osteoblast alpha-chains demonstrated the same deletions in the cyanogen bromide peptide 4 as observed in the fibroblast cyanogen bromide maps. PAGE analysis of oligonucleotide-specific cDNA that was reverse transcribed from RNA isolated from fibroblasts and osteoblasts also demonstrated the presence of two bands, one the normal size of alpha 2(I) cDNA and a second species that was smaller by 54 base pairs. Sequencing of polymerase chain reaction-amplified cDNA fragments revealed an in-frame deletion of exon 12. This finding was confirmed by the RNase protection method. Genomic DNA sequencing detected a T----G point mutation in the second position of the 5' splice donor site of intron 12. Therefore, in this patient with osteogenesis imperfecta there was no qualitative alteration in the osteoblast-specific expression of this mutant alpha 2(I)-collagen allele compared to dermal fibroblasts.
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PMID:Expression of mutant alpha (I)-procollagen in osteoblast and fibroblast cultures from a proband with osteogenesis imperfecta type IV. 164 48

The steroid-binding capacity of the adrenocortical pregnenolone-binding protein (PBP) is effectively destroyed by extreme temperature (boiling water for 2-5 min); however, the boiled preparation contains a factor that potentiates ligand binding when readded to native PBP. Treatment of the boiled fraction with calf intestinal alkaline phosphatase at pH 9 reverses the stimulatory effect on PBP activity. Additionally, if native PBP is first incubated with alkaline phosphatase, which converts it to a nonbinding form, activity can be fully restored in a dose-dependent manner by the addition of the boiled preparation. The factor (itself devoid of binding capacity) can also be generated by exposing native PBP to acidic conditions (pH 4). The molecule is small (mol wt, less than 2000), as judged by Sephadex G-25 gel filtration and equilibrium dialysis. It is not retained on Concanavalin-A-Sepharose and is not extractable with a variety of organic solvents. The factor remains active after lyophilization and has a net negative charge at pH 7.4 (determined by DEAE-cellulose chromatography). While the binding capacity of native PBP is destroyed by a variety of proteases, the heat-stable factor is unaffected by similar treatment. Additionally, factor activity is not susceptible to RNase, DNase, or lipase digestion. Thus, the protein moiety of the PBP has an absolute requirement for a distinct phosphorylated heat-stable factor for expression of ligand-binding activity, and it may be through this factor that binding activity is regulated. It is not yet known whether the factor is acting allosterically or actually functions as part of the steroid-binding site.
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PMID:Adrenocortical pregnenolone-binding protein activity requires a small heat-stable factor: evidence that regulation by phosphorylation/dephosphorylation occurs at the level of the factor, not the protein. 177 Sep 49

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)
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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.
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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.
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PMID:Reconstitution of influenza virus RNA-nucleoprotein complexes structurally resembling native viral ribonucleoprotein cores. 235 55

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.
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PMID:Detection of c-fms and CSF-1 RNA by in situ hybridization. 244 33

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.
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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.
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PMID:A single, phosphate-repressible deoxyribonuclease, DNase A, secreted in Aspergillus nidulans. 267 10

The oncogene product p53, isolated from SV3T3 cells where it forms a complex with simian virus 40 large tumor antigen (T antigen) in the nucleus, has been found to be phosphorylated at at least four distinct sites on the 390 amino acid protein. Separation of tryptic phosphopeptides has permitted identification of two sites as Ser-312 and Ser-389, and permitted analysis of the types of phosphate bonds. The peptide containing Ser-312 separates electrophoretically into three charged forms; two are resistant to dephosphorylation by both alkaline phosphatase and alkaline hydrolysis, suggesting a phosphodiester. The carboxyl-terminal phosphopeptide containing Ser-389 was alkaline phosphatase-resistant and liberated four ribonucleoside monophosphates upon base or RNase hydrolysis, suggesting that Ser-389 may be covalently linked to RNA. Phosphorylation of Ser-389 decreased markedly at the nonpermissive temperature in simian virus 40 tsA58-transformed cells, indicating a dependence on native T antigen function and a possible role in transformation by T antigen. Two additional phosphorylation sites, one involving serine and one involving threonine, probably reside in the amino-terminal segment of p53 and appear to be peptide-phosphate monoesters.
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PMID:Mapping of phosphomonoester and apparent phosphodiester bonds of the oncogene product p53 from simian virus 40-transformed 3T3 cells. 300 31


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