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)

We have recently described a procedure for the purification of microtubule associated protein 1B (MAP1B) from calf brain [Pedrotti, B., & Islam K. (1995) Cell Motil. Cytoskeleton 30, 301-309], and this study further characterizes the purified protein and its interaction with microtubules. We show that purified MAP1B (1) is thermostable; (2) is mainly phosphorylated at the casein kinase II (CKII) sites but only partially phosphorylated at the proline-directed protein kinase (PDPK) sites; (3) both the CKII and PDPK sites can be dephosphorylated by alkaline phosphatase; and (4) dephosphorylation results in an increased mobility on SDS-PAGE gels. The ability of MAP1B to interact with microtubules was also examined and shows that (1) phosphorylated (1B-P), alkaline phosphatase-treated (1B-AP), and heat-treated (1B-P), alkaline phosphatase-treated (1B-AP), and heat-treated (1B-HT) MAP1B bind to taxol-stabilized microtubules; (2) 1 mol of 1B-P, 1B-AP, or 1B-HT each binds about 13-14 tubulin dimers; (3) light chain interaction with MAP1B heavy chain is not affected by AP- or heat-treatment; (4) MAP1B can be displaced from taxol-stabilized microtubules by titration with salt; (5) higher salt concentrations are required to displace 1B-AP compared with 1B-P from taxol-stabilized microtubules; and (6) MAP2 is able to displace both 1B-P and 1B-AP from taxol-stabilized microtubules. The role of phosphorylation in regulating MAP1B interaction with microtubules and light chains is discussed.
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PMID:Characterization of microtubule-associated protein MAP1B: phosphorylation state, light chains, and binding to microtubules. 860 40

To detect Bence-Jones protein (BJP) in serum we precipitated intact immunoglobulins (Ig) using polyethylene glycol (PEG) and subjected the BJP in solution to electrophoresis in agarose gel, followed by transfer to a polyvinylidene difluoride membrane, and immunoenzymatic staining (successively using rabbit anti-human light/heavy chain of Ig, biotinylated swine anti-rabbit Ig, and alkaline phosphatase-conjugated streptavidin). Treatment with PEG effectively reduced background staining of polyclonal Ig in the immunoblots, although intact monoclonal Ig was not always completely removed. To compare the present method with immunoelectrophoresis (IEP), we selected samples from patients demonstrating BJP by IEP in both serum and urine (n = 40), serum only (n = 18), and urine only (n = 32); 21 of these patients had BJP alone and 69 had BJP in addition to intact monoclonal Ig. Efficiency of detection of BJP in serum was increased by the present method: serum BJP was detected in 70 patients by the present method versus 58 by IEP. The present method demonstrated single BJP bands in the samples from 16 patients (kappa, n = 7; lambda, n = 9) and multiple BJP bands (range: 2-9) in the samples from 54 patients (kappa, n = 31; lambda, n = 23). This method could be useful for detecting BJP in serum from patients suspected of having light chain gammopathy (without the need for urine testing) and may complement urine testing in patients excreting polyclonal free light chains of Ig.
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PMID:Detection of Bence-Jones protein in serum by immunoblotting. 872 21

In this report, we describe the expression system that enabled us to produce in Escherichia coli the Fab fragment of a mouse IgM that has previously been shown to inhibit the binding of IgG to autoantigens by interacting with their variable regions. In our system, both light chain and heavy chain fragments were put under the control of the malE promoter. The light chain was fused to the MalE signal sequence, while the heavy chain variable and first constant region were fused to the alkaline phosphatase signal sequence. In this system, after induction of the promoter with maltose, the Fab fragment could be detected in a periplasmic extract of the bacteria by Western blotting and also by ELISA. This Fab fragment was purified on a goat anti-mouse immunoglobulin immunoadsorbent and biotinylated. The Fab fragment produced by E.coli reacted with the trinitrophenyl (TNP) hapten and F(ab')2 fragments of mouse IgG and these reactivities could be specifically inhibited by the corresponding soluble antigens. The dissociation constants of this Fab were 1.65 x 10(-6) M for TNP and 5 x 10(-6) M for IgG F(ab')2 fragments, indicating that the affinity of the Fab fragment compared with that of the whole IgM molecule was similar for TNP but was lower for IgG F(ab')2 fragments.
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PMID:Bacterial secretion of the Fab fragment of a mouse monoclonal IgM that reacts with IgG variable regions. 874 23

Malpha2-3 is a monoclonal antibody that partially mimics the nicotinic acetylcholine receptor (AChR). Its three-dimensional structure has been previously predicted by molecular modeling, suggesting that 29 complementarity determining region (CDR) residues and 2 framework residues are exposed to solvent. To identify the antibody residues that bind to the antigen, i.e. snake toxin that binds specifically to AChR, we (i) produced the scFv form of Malpha2-3 fused to alkaline phosphatase, in the periplasmic space of Escherichia coli; (ii) submitted approximately 75% of exposed residues of the fused scFv to individual or combined mutations, and (iii) identified the residues whose mutations affect scFv binding to the toxin, using a sensitive enzyme-linked immunosorbent assay. 11 critical residues were identified, including 8 heavy chain residues, 2 framework residues, and 1 light chain residue. They cover a surface of approximately 800 A2, with a subset of most critical residues (VHD31, VHY32, and VHG101) and several aromatic residues. This functional architecture not only constitutes a plausible complementary binding surface for the snake toxin but also offers a structural basis to ultimately understand the capacity of the antibody to partially mimic AChR.
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PMID:The functional architecture of an acetylcholine receptor-mimicking antibody. 929 23

Comprehensive preparations of antibodies against various kinds of proteins in cells would be useful in proteome research and antibody-based research. Here we report the panning of a human antibody heavy chain variable domain (VH) phage library against a cytosolic fraction of rat liver to obtain antibodies specific for certain cytoplasmic proteins. Rat liver specimens were homogenized and subjected to differential centrifugation. A 125000 x g supernatant (rat liver cytosol, RLC) was immobilized onto a nitrocellulose membrane and subjected to phage VH library panning. For efficient assessment of binding phages, we established a system that was a combination of monoclonal phage ELISA and quantitative dot blotting of phages. The VH genes of the binding phages were selected and expressed as VH--bacterial alkaline phosphatase (PhoA) conjugates (VH/RLC--PhoAs) in Escherichia coli. One of the VH/RLC--PhoAs stained one major band on Western blotting of RLC and also stained the cytoplasm of hepatocytes histochemically. This is the first report of phage library panning against the cytosolic fraction of cells to obtain human VH fragments, and the application of those human VH fragments to histochemical study.
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PMID:Phage library panning against cytosolic fraction of cells using quantitative dot blotting assay: application of selected VH to histochemistry. 1186 Oct 66

The crystal structure of a mutant form of the single-chain fragment (scFv), derived from the monoclonal anti-His tag antibody 3D5, in complex with a hexahistidine peptide has been determined at 2.7 A resolution. The peptide binds to a deep pocket formed at the interface of the variable domains of the light and the heavy chain, mainly through hydrophobic interaction to aromatic residues and hydrogen bonds to acidic residues. The antibody recognizes the C-terminal carboxylate group of the peptide as well as the main chain of the last four residues and the last three imidazole side-chains. The crystals have a solvent content of 77% (v/v) and form 70 A-wide channels that would allow the diffusion of peptides or even small proteins. The anti-His scFv crystals could thus act as a framework for the crystallization of His-tagged target proteins. Designed mutations in framework regions of the scFv lead to high-level expression of soluble protein in the periplasm of Escherichia coli. The recombinant anti-His scFv is a convenient detection tool when fused to alkaline phosphatase. When immobilized on a matrix, the antibody can be used for affinity purification of recombinant proteins carrying a very short tag of just three histidine residues, suitable for crystallization. The experimental structure is now the basis for the design of antibodies with even higher stability and affinity.
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PMID:Crystal structure of the anti-His tag antibody 3D5 single-chain fragment complexed to its antigen. 1205 74

We demonstrate the importance of optimizing the balance of light chain (LC) and heavy chain (HC) expression to achieve high level production of Fab' fragments in the Escherichia coli periplasm. The LC:HC balance has been controlled by varying the codon usage of the signal peptide (SP) and 5' mature domain coding regions. Different SP coding regions have been identified from a codon wobble-based library using alkaline phosphatase (AP) as a reporter gene. A plasmid system that enables random combination of these variant SP coding regions is used to construct optimized Fab' expression plasmids. These small plasmid libraries facilitated selection of optimal Fab' expression plasmids and resulted in increases of periplasmic yield, up to 580 mgL(-1) from E. coli fermentations and will enable rapid variable region subcloning and selection of future Fab(') expression plasmids.
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PMID:A plasmid system for optimization of Fab' production in Escherichia coli: importance of balance of heavy chain and light chain synthesis. 1240 86

To examine the relationship between folding and export competence by the twin-arginine translocation (Tat) pathway we analyzed the subcellular localization of fusions between a set of eight putative Tat leader peptides and alkaline phosphatase in isogenic Escherichia coli strains that either allow or disfavor the formation of protein disulfide bonds in the cytoplasm. We show that export by the Tat translocator is observed only in strains that enable oxidative protein folding in the cytoplasm. Further, we show that other disulfide-containing proteins, namely single-chain Fv and heterodimeric F(AB) antibody fragments, are export-competent only in strains having an oxidizing cytoplasm. Functional, heterodimeric F(AB) protein was exported from the cytoplasm by means of a Tat leader peptide fused to the heavy chain alone, indicating that the formation of a disulfide-bonded dimer preceeds export. These results demonstrate that in vivo only proteins that have attained the native conformation are exported by the Tat translocator, indicating that a folding quality-control mechanism is intrinsic to the export process. The ability to export proteins with disulfide bonds and the folding proofing feature of the Tat pathway are of interest for biotechnology applications.
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PMID:Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway. 1272 69

Bone marrow stroma fibroblastoid cells (BMSFC) develop from a single clone of cells within each of the in vitro fibroblastoid colonies (CFU-F) derived from either murine or human bone marrow. All of the clones represented by these colonies displayed antigenic and product markers for osteoblast, smooth muscle, and adipocyte lineages when tested separately for each marker. Separate sets of fibroblastoid colonies derived from the same individual donor's culture tested positive with antibodies specific for smooth muscle-specific heavy chain myosin (SMMHC), smooth muscle alpha actin-1, bone sialoprotein, osteocalcin, or alkaline phosphatase, and developed von Kossa-positive deposits shown by X-ray microanalysis and electron diffraction to be hydroxyapatite. Individual cells were positive for both SMMHC and osteocalcin. All cells in the multiple clones tested were capable of metabolizing a fatty acid to form intracellular lipid droplets. PCR transcripts obtained from the human cell cultures that provided these BMSFC clones were consistent with the immunocytochemical findings. Transcripts for PPAR (gamma)-2 and Cbfa-1 were dependent upon the culture medium content, suggesting an osteoblast/adipocyte differentiation switch point. Cell lineage specificity for markers and RNA transcripts was determined by comparison to skin fibroblast controls. These findings demonstrate a high degree of interlineage plasticity in vitro for BMSFC.
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PMID:Intraclonal plasticity for bone, smooth muscle, and adipocyte lineages in bone marrow stroma fibroblastoid cells. 1456 92

Malignant lymphomas in the female genital tract are rare, and those arising from this tissue system are extremely uncommon. Most pertinent reports lack clear references to the accepted classifications or failed to apply immunomarkers and molecular techniques for a reliable diagnosis. We analyzed a large group of patients with primary and secondary lymphomas of the female genital tract classified on the basis of the recent WHO consensus. A total of 186 patients with malignant lymphoma detected in the female genital tract were selected from the files of the Kiel Lymphoma Registry covering the period of 1974 to 2004. Stringent criteria were applied to separate systemic versus secondary lymphomas. All cases were reviewed on the basis of conventionally stained sections, relevant immunohistochemistry using the alkaline phosphatase/anti-alkaline phosphatase technique, and clinical information, as far as available. When required, gene rearrangement analysis was performed, including TCR-gamma chain gene and the three FR fragments of the IgG heavy chain gene. In addition, typical chromosomal translocations were detected by means of the FISH technique to verify the diagnosis, where needed. Thirty-seven percent of the cases were systemic lymphomas and 63% were mostly extranodal lymphomas primary to the female genital tract. The adnexa were involved in 87 cases, followed by uterine corpus in 23 cases, uterine cervix in 17 cases, portio in 9 cases, vagina in 11 cases, and vulva including clitoris in 8 cases. In 31 cases, two or more adjacent sites were involved. In both (primary and secondary) groups, the adnexa were the prevailing site of involvement. As expected, the overwhelming majority of cases were of B phenotype. The most frequent type of lymphoma proved to be diffuse large B-cell lymphoma, closely followed by follicular lymphoma, including all 3 grades of malignancy. Burkitt lymphoma showed a rather similar frequency. Marginal zone lymphoma occurred exclusively as primary lesions in the uterine mucosa. Lymphoplasmacytic lymphoma was restricted to the vulvo-vaginal area and occurred in women over 60 years of age. In conclusion, our study provides a thorough overview of various types of lymphoma affecting the female genital tract primarily or secondarily, which were classified on the basis of a widely accepted WHO classification. Although quite rare, our report should remind the pathologist of considering malignant lymphomas while reading biopsies taken from female genital organ.
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PMID:Lymphomas of the female genital tract: a study of 186 cases and review of the literature. 1693 68


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