Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It is believed that one or more basic residues at the extreme amino terminus of precursor proteins and the lack of a net positive charge immediately following the signal peptide act as topological determinants that promote the insertion of the signal peptide hydrophobic core into the cytoplasmic membrane of Escherichia coli cells with the correct orientation required to initiate the protein export process. The export efficiency of precursor maltose-binding protein (pre-MBP) was found to decrease progressively as the net charge in the early mature region was increased systematically from 0 to +4. This inhibitory effect could be further exacerbated by reducing the net charge in the signal peptide to below 0. One such MBP species, designated MBP-3/+3 and having a net charge of -3 in the signal peptide and +3 in the early mature region, was totally export defective. Revertants in which MBP-3/+3 export was restored were found to harbor mutations in the prlA (secY) gene, encoding a key component of the E. coli protein export machinery. One such mutation, prlA666, was extensively characterized and shown to be a particularly strong suppressor of a variety of MBP export defects. Export of MBP-3/+3 and other MBP species with charge alterations in the early mature region also was substantially improved in E. coli cells harboring certain other prlA mutations originally selected as extragenic suppressors of signal sequence mutations altering the hydrophobic core of the LamB or MBP signal peptide. In addition, the enzymatic activity of alkaline phosphatase (PhoA) fused to a predicted cytoplasmic domain of an integral membrane protein (UhpT) increased significantly in cells harboring prlA666. These results suggest a role for PrlA/SecY in determining the orientation of signal peptides and possibly other membrane-spanning protein domains in the cytoplasmic membrane.
 ...
PMID:Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations. 172 28

A simple method for the comparison and identification of protein epitopes recognized by monoclonal antibodies directly on thin-layer plates and 3MM paper chromatograms is described. Enzyme digests of myelin basic protein were separated on thin-layer plates and 3MM paper, fixed with glutaraldehyde and probed directly with affinity-purified mouse monoclonal antibodies. Detection of the immunoreactive peptides was enhanced using a second rabbit anti-mouse immunoglobulin and finally located using an alkaline phosphatase-conjugated anti-rabbit immunoglobulin. By probing the same enzyme digests of MBP with various monoclonal antibodies raised against MBP, a different binding 'pattern' of reactive peptides is rapidly obtained for monoclonal antibodies of differing specificities. This procedure was extended to the identification of the antigenic determinant using synthetic peptides. The major advantages of this procedure are its simplicity, non-radioactive nature and speed. Furthermore, there is the possibility of sequencing immunoreactive peptides eluted from the 3MM paper.
 ...
PMID:Rapid characterization of protein epitopes recognized by monoclonal antibodies using direct probing on thin-layer and paper chromatograms. 243 71

Dichloromethylenebisphosphonate (Cl2-MBP), a compound structurally related to inorganic pyrophosphate but resistant to hydrolysis of endogenous phosphatase to yield inorganic phosphate, inhibits bone resorption and soft tissue mineralization in vivo. Previously, we have shown that bone cells isolated from rat calvaria respond profoundly to the exposure of Cl2MBP. To determine whether the cellular effects evoked by Cl2MBP are confined to a particular bone cell type, calvaria from 1 day postnatal rats were subjected to a sequential time-dependent enzyme digestion, yielding five bone cell populations marked by differences in PTH response, alkaline phosphatase activity and collagen, as well as hyaluronic acid synthesis. Culturing these bone cell populations with Cl2MBP revealed that previously observed results found with mixed bone cells (inhibition of cell proliferation, diminution of hyaluronic acid synthesis, and increase in alkaline phosphatase) were limited to cell populations which, according to the isolation scheme, stem from the outer tissue layer(s) of the calvaria. Collagen synthesis, however, was found to be equally increased regardless of cell type. These present results indicate that the action of Cl2MBP on bone may be cell specific.
 ...
PMID:Differential response of bone cells isolated by sequential digestion to dichloromethylenebisphonate in culture. 609 52

An ELISA method for the quantitation in vitro of HCV serine proteinase activity was developed. A peptide substrate, Ac-Gly-Glu-Ala-Gly-Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Ser-Tyr-Thr-Trp-Thr-L ys (biotin) -OH (Sub-1), was hydrolyzed by a recombinant NS3 proteinase fused with maltose binding protein (MBP-NS3) into a product with a free amino moiety at the N-terminus. The product was immobilized, and the amino moiety was analyzed by digoxigenin labeling followed by immunological reaction with anti-digoxigenin-alkaline phosphatase conjugate and then the colorimeteric reaction. This method is suited for the high throughput screening of inhibitors, and the screening can be accelerated by automatic operation.
 ...
PMID:An enzyme-linked immunosorbent assay for detecting proteolytic activity of hepatitis C virus proteinase. 917 84

Immobilization of proteins on microplate wells by simple adsorption (e.g., for ELISA) is convenient, but it can be inefficient, especially if proteins are hydrophilic or small in size. This problem was alleviated by the use of polyvinylbenzyl lactonoylamide (PVLA). PVLA is strongly adsorbed to the hydrophobic well surface, and its lactonamide part can be oxidized with periodate to generate aldehydo groups. Proteins are then immobilized covalently to the aldehydo groups by reductive amination under mild conditions. Using this method, henceforth termed the PVLA method, alkaline phosphatase (AP) was immobilized to microplates six- to sevenfold greater than by simple adsorption (as measured by activity). Similarly, the activity of immobilized mannose-binding protein A (MBP-A) was 4- to 8-fold higher by the PVLA method than by simple adsorption. The PVLA-coated plates needed as little as 200 ng of MBP-A per well to have a sufficient amount of MBP-A immobilized for the measurement of binding of 125I-labeled mannosylated bovine serum albumin (125I-Man-BSA), but unmodified plates required as much as 20 micrograms/well MBP-A to obtain the same response. Recommended conditions for the PVLA method are 40 microliters of 2 mg/ml of PVLA for coating, 1 mM NaIO4 for the generation of the aldehydo groups, and a 2-h reductive amination at 37 degrees C between pH 8 and 9 for the protein ligation.
 ...
PMID:Efficient immobilization of proteins by modification of plate surface with polystyrene derivatives. 917 6

We analysed the properties of mature MBP (maltose-binding protein or MalE protein) fused to an integral cytoplasmic membrane protein of Escherichia coli. Fusion of MalE to the first MalG periplasmic loop enabled a strain defective in the malE gene to utilize maltose. In contrast, fusion of MalE to a cytoplasmic loop did not complement the malE delta 444 deletion. We obtained results highly correlated with those obtained by using alkaline phosphatase as a reporter for the topology of MalG. We discuss the possibility of genetically determining the topology of cytoplasmic membrane proteins by a method based on engineered fusions to MBP.
 ...
PMID:Activity of protein MalE (maltose-binding protein) fused to cytoplasmic and periplasmic regions of an Escherichia coli inner membrane protein. 976 17

Tristetraprolin (TTP) is a hyperphosphorylated protein that destabilizes mRNA by binding to an AU-rich element (ARE). Mice deficient in TTP develop a severe inflammatory syndrome. The biochemical properties of TTP have not been adequately characterized, due to the difficulties in protein purification and lack of a high-titer antiserum. Full-length human TTP was expressed in human HEK293 cells and purified to at least 70% homogeneity. The purified protein was free of endogenous ARE binding activity, and was used for investigating its size, zinc dependency, and binding kinetics for tumor necrosis factor alpha mRNA ARE. A high-titer rabbit antiserum was raised against the MBP-hTTP fusion protein expressed in Escherichia coli. Cellular localization studies of the transfected cells indicated that approximately 80% of the expressed TTP was in the cytosol, with 20% in the nuclei. TTP from both locations bound to the ARE and formed similar complexes. The purified TTP was shown to be intact by N-terminal His-tag purification, C-terminal peptide sequencing, and mass spectrometry analysis. Results from size exclusion chromatography are consistent with the predominant form of active TTP being a tetramer. TTP's ARE binding activity was increased by 10 microM Zn(2+). The half-maximal binding of TTP from HEK293 cells was approximately 30 nM in assays containing 10 nM ARE. This value was about twice that of TTP from E. coli. TTP from HEK293 cells was highly phosphorylated, and its electrophoretic mobility was increased by alkaline phosphatase treatment and somewhat by T271A mutation, but not by PNGase F or S186A mutation. The gel mobility of TTP from E. coli was decreased by in vitro phosphorylation with p42/ERK2 and p38 mitogen-activated protein kinases. These results suggest that TTP's zinc-dependent ARE binding affinity is reduced by half by posttranslational modifications, mainly by phosphorylation but not by glycosylation, in mammalian cells. The results support a model in which each subunit of the TTP tetramer binds to one of the five overlapping UUAUUUAUU sequences of the ARE, resulting in a stable TTP-ARE complex.
 ...
PMID:Expression, purification, and biochemical characterization of the antiinflammatory tristetraprolin: a zinc-dependent mRNA binding protein affected by posttranslational modifications. 1550 35

Zearalenone (ZEA) is an estrogenic mycotoxin produced by Fusarium sp., and its production on corn and small grains during storage has been of considerable concern. For sensitive ZEA detection, we applied an open sandwich (OS) immunoassay that can noncompetitively detect monovalent antigens utilizing an antigen-induced enhancement of the V(H)/V(L) interaction. We cloned the V(H) and V(L) cDNAs of anti-ZEA mAb to a split-Fv phagemid pKST2, and firstly both V(H) and V(L) fragments were displayed on M13 phage p9 and p7, respectively, using an amber suppressor, TG-1, as a host. The split-Fv phage showed specific binding to immobilized ZEA, which was well inhibited by free ZEA. Then, the V(H)/V(L) interaction and its antigen-dependency were analyzed using a non-suppressor HB2151 as a host to produce V(H)-displaying phage and his/myc-tagged soluble V(L) in the culture supernatant. By capturing V(L) with an anti-myc or -his antibody and probing bound V(H)-phage, ZEA was successfully detected with a superior detection limit as well as a wider working range than those of a competitive assay. Also, essentially the same results were reproduced with purified V(H)-alkaline phosphatase and MBP-V(L) fusion proteins.
 ...
PMID:Sensitive detection of estrogenic mycotoxin zearalenone by open sandwich immunoassay. 1721 26

Small peptides with less than 1000 in molecular weight are not considered amenable to sandwich immunoassays due to their difficulty of simultaneous recognition by two antibodies. As an alternative, we attempted noncompetitive detection of small peptides by open sandwich enzyme-linked immunosorbent assay (OS-ELISA) utilizing the antigen-induced enhancement of antibody VH/VL interaction. Taking fragments of human osteocalcin (BGP), a major non-collagen peptide produced in bone, as model peptides, OS immunoassay was performed using the cloned VH and VL cDNAs from two anti-BGP monoclonal antibodies either recognizing the N- or C-terminal fragment, respectively. When the clones were used for OS-ELISA with immobilized VL fragment and phage-displayed VH fragment, enhanced VH/VL interaction upon BGP addition was observed. Especially the clone for the C-terminal fragment showed a superior detection limit as well as a wider working range than those of competitive assay. The result was reproduced with purified VH-alkaline phosphatase and MBP-VL fusion proteins, where the latter was directly immobilized onto the microplate wells. The minimum detectable fragment was the hexamer including the C-terminus. This simple approach with a single monoclonal antibody with a short measurement time may prove a useful tool in immunodiagnostics as well as in proteomics research.
 ...
PMID:Noncompetitive detection of low molecular weight peptides by open sandwich immunoassay. 1763 82

There is concern that transgenic Bt-crops carry genes that could have undesirable effects on natural and agro-ecosystem functions. We investigated the effect of Bt-cotton (expressing the Cry 1Ac protein) on several microbial and biochemical indicators in a sandy loam soil. Bt-cotton (MRC-6301Bt) and its non-transgenic near-isoline (MRC-6301) were grown in a net-house on a sandy clay loam soil. Soil and root samples were collected 60, 90, and 120 days after sowing. Soil from a control (no-crop) treatment was also included. Samples were analysed for microbial biomass C, N and P (MBC, MBN, MBP), total organic carbon (TOC), and several soil enzyme activities. The microbial quotient (MQ) was calculated as the ratio of MBC-to-TOC. The average of the three sampling events revealed a significant increase in MBC, MBN, MBP and MQ in the soil under Bt-cotton over the non-Bt isoline. The TOC was similar in Bt and non-Bt systems. Potential N mineralization, nitrification, nitrate reductase, and acid and alkaline phosphatase activities were all higher in the soil under Bt-cotton. Root dry weights were not different (P > 0.05), but root volume of Bt-cotton was higher on 90 and 120 days than that of non-Bt cotton. The time of sampling strongly affected the above parameters, with most being highest on 90 days after sowing. We concluded from the data that there were some positive or no negative effects of Bt-cotton on the studied indicators, and therefore cultivation of Bt-cotton appears to be no risk to soil ecosystem functions.
 ...
PMID:Assessment of biological and biochemical indicators in soil under transgenic Bt and non-Bt cotton crop in a sub-tropical environment. 1872 17


1 2 Next >>