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 phosphorylation state of human and bovine spinal cord neurofilaments (NF) was studied by direct phosphate analysis and carbocyanine dye ("Stains-all") binding to NF polypeptides resolved on SDS-polyacrylamide gels. Electrophoretically purified NF-H (200 kDa), NF-M (160 kDa), and NF-L (68 kDa) of human origin contained 24, 18, and 4 mol phosphate/mol protein, whereas bovine NF contained 53, 23, and 5 mol phosphate/mol protein, respectively. Incubation of NF preparations with E. coli alkaline phosphatase removed about 55% of the phosphate from NF-H, about 30% of the phosphate from both human and bovine NF-M, but did not change the phosphate content of NF-L. This treatment also inhibited or substantially reduced the binding of electroblotted NF-H and NF-M to 2 anti-NF monoclonal antibodies known to recognize phosphorylated sites on projection side arms. "Stains-all" was found to be a very sensitive probe for detection of phosphorylated cytoskeletal proteins. Without the phosphatase treatment, NF and other phosphoproteins, MAP1, MAP2, tubulin, and tau, all bound the carbocyanine dye on SDS gels, forming blue dye-protein complexes. Measured densitometrically at 615 nm, the staining intensity (relative units/mol protein) was 9, 9, and 3 for human and 10, 13, and 6 for bovine NF-H, NF-M, and NF-L, respectively. NF-H bound the dye less efficiently than was expected from its phosphate content. After phosphatase treatment, NF-H, with half of its phosphate residues remaining, no longer formed blue complex with "Stains-all," the staining intensity of NF-M decreased by 20-40%, and the staining of NF-L was not changed.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphatase and carbocyanine dye binding define different types of phosphate groups in mammalian neurofilaments. 244 39

1. Liver and bone alkaline phosphatase isoenzymes were solubilized with the zwitterionic detergent sulphobetaine 14, and purified to homogeneity by using a monoclonal antibody previously raised against a partially-purified preparation of the liver isoenzyme. Both purified isoenzymes had a specific activity in the range 1100-1400 mumol/min per mg of protein with a subunit Mr of 80,000 determined by SDS/polyacrylamide gel electrophoresis. Butanol extraction instead of detergent solubilization, before immunoaffinity purification of the liver enzyme, resulted in the same specific activity and subunit Mr. The native Mr of the sulphobetaine 14-solubilized enzyme was consistent with the enzyme being a dimer of two identical subunits and was higher than that of the butanol-extracted enzyme, presumably due to the binding of the detergent micelle. 2. Pure bone and liver alkaline phosphatase were used to raise further antibodies to the two isoenzymes. Altogether, 27 antibody-producing cell lines were cloned from 12 mice. Several of these antibodies showed a greater than 2-fold preference for bone alkaline phosphatase in the binding assay used for screening. No antibodies showing a preference for liver alkaline phosphatase were successfully cloned. None of the antibodies showed significant cross-reaction with placental or intestinal alkaline phosphatase. Epitope analysis of the 27 antibodies using liver alkaline phosphatase as antigen gave rise to six groupings, with four antibodies unclassified. The six major epitope groups were also observed using bone alkaline phosphatase as antigen. 3. Serum from patients with cholestasis contains soluble and particulate forms of alkaline phosphatase. The soluble serum enzyme had the same size and charge as butanol-extracted liver enzyme on native polyacrylamide-gel electrophoresis. Cellulose acetate electrophoresis separated the soluble and particulate serum alkaline phosphatases as slow- and fast-moving forms respectively. In the presence of sulphobetaine 14 all the serum enzyme migrated as the slow-moving form on cellulose acetate electrophoresis. Monoclonal anti-(alkaline phosphatase) immunoadsorbents did not bind the particulate form of alkaline phosphatase in cholestatic serum but bound the soluble form. In the presence of sulphobetaine 14 all the cholestatic serum alkaline phosphatase bound to the immunoadsorbents. 4. The electrophoretic and immunological data are consistent with both particulate and soluble forms of alkaline phosphatase in cholestatic serum being derived from the hepatocyte membrane.
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PMID:The preparation of monoclonal antibodies to human bone and liver alkaline phosphatase and their use in immunoaffinity purification and in studying these enzymes when present in serum. 245 2

Intestinal-like alkaline phosphatase was found to be expressed in the intestinal 407 cell line. This enzyme was identified by use of monoclonal antibodies specific for human placental (H7 and HPMS-1) and intestinal alkaline phosphatase (2HIMS-1 and 2HIMS-3) separately. Purification of this isozyme by use of two different monoclonal antibody immunoaffinity chromatographies demonstrates a single protein band on SDS-polyacrylamide gel electrophoresis indicating that this enzyme is not formed as a heterodimer. The apparent monomer subunit molecular weight and the dimer molecular weight of this isozyme were determined to 70000 and 160000, respectively. The enzyme is a homodimer according to molecular weight determinations. Furthermore, this isozyme is neuraminidase sensitive and comparatively heat stable, properties also characteristic for the placental enzyme. Our data suggest that the intestinal-like alkaline phosphatase in the intestinal 407 cell line displays properties intermediate of the intestinal and placental isozymes which may reflect the existence and reexpression of a new primitive isozyme.
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PMID:Evidence for the expression of a primitive intestinal-like alkaline phosphatase in the intestinal 407 cell line. 246 Jan 3

The effect of matrix components extracted Bovine periodontal ligament (PDL) on cell proliferation and alkaline phosphatase activity of cultured human periodontal ligament fibroblast (HPLF) were examined in order to understand the cell-tissue interaction of periodontal ligament. Bovine PDL tissue was sequentially extracted with 0.05 M Tris HCl buffer, pH 7.4, containing 1M NaCl or 4M GdmCl. After seeding 24 hours, the cultured HPLF were exposed to the extracts for two through eight days. Nine days after seeding, HPLF indicated four times high activity on ALP and 1.6 times the amount of total protein than those of control (without extract), while DNA synthesis increased only 1.2 times. On the contrary, the NaCl extract depressed the ALP activity of HPLF. The GdmCl extract enhanced both the total protein and ALP activity in dose-dependently. The ALP increasing activity of GdmCl extract on HPLF is stable to heat (78 degrees C, 20 min) and collagenase treatment but partially inactivated by trypsin digestion. Since the GdmCl extract also induced colony formation of NRK-49F cell in soft agarose, it was suggested that the extract contain EGF and TGF-beta like factor. Molecular size of this factor was estimated as 20-50Kd using Sepharose CL-6B gel chromatography. Furthermore, this factor from Sepharose CL-6B were separated into two forms by ion exchange CM-Sepharose column chromatography. Purified preparation from reversed phase column chromatography contained 14Kd, 15Kd, 17Kd, 20Kd, 28Kd40Kd, and 46Kd components on SDS-PAGE. This factor may accumulate in extracellular matrix, and may play a role of cell-tissue interaction and homeostasis in periodontal ligament.
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PMID:[Biochemical study on the TGF-beta like growth factor derived from bovine periodontal ligament]. 248 40

Saito et al recently reported that the alkaline phosphatase (ALPase) of human periodontal ligament fibroblasts (HPLF) showed remarkably high activity which was similar, but not identical phenotype, to that present in osteoblasts, and suggested that HPLF could be termed as "osteoblastic fibroblast." The present study attempts to explore the ALPase synthesized on HPLF in relation to 1,25(OH)2D3. These HPLF were obtained by the explantation method and then subcultured in D-MEM containing 2 mg FCSP/ml, 50 micrograms ascorbic acid/ml and penicillin/streptomycin after trypsinization. The HPLF were inoculated at a cell density of 1.25 x 10(4) cells/cm2 in culture wells. After 24hr, the HPLF were treated every two days for 7 days with 0.5-10nM 1,25 (OH)2D3. Then, ALPase activity, DNA and protein contents were assayed by the methods using p-nitrophenylphosphate, diaminobenzoic acid and Coomassie Brilliant Blue, respectively. Also, ALPase was prepared from the confluent HPLF incubated with 5 nM 1,25 (OH)2D3 for 12 days, and digested with and without trypsin. The crude ALPase which was solubilized with 10mM Tris-HCl, pH 7.4 containing 0.2 mM MgCl2 and 0.1% NP-40 was applied to 5-15% gradient SDS-PAGE and stained with beta-naphththylacid phosphate and First Blue BB salt in 60 mM borate buffer pH 9.7. The cell growth which was assayed by DNA contents and the incorporation of 3H-thymidine was decreased by 1,25(OH)2D3. On the other hand, ALPase activity was increased approximately 3.6 fold at 6 day by the addition of 5 nM 1,25(OH)2D3. From the separation of ALPase activity on SDS-PAGE, 110 K and 120-130 K ALPase were identified. The 110 K ALPase, which was not changed by 1,25(OH)2D3, was converted to 100K, releasing 10K peptide after trypsin treatment. This 110K ALPase might be tightly associated with cell membrane structure. The 120-130K ALPase was remarkably increased by 1,25(OH)2D3 on SDS-PAGE and completely digested with trypsin. The ALPase in the cultured HPLF might be located not only on the plasma membrane but also in the extracellular matrix. Therefore, 1,25(OH)2D3 may regulate the cell cycle and also the gene expression of ALPase of HPLF.
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PMID:[Biochemical study of human periodontal ligament fibroblasts--1,25 (OH)2D3 dependent alkaline phosphatase]. 248 52

The pattern of serum proteins separated by SDS-PAGE is typified by the microprotein apolipoprotein A I which is split from high density lipoprotein by SDS. High density lipoprotein is generally retained by the glomerulus and does not appear in the urine even in glomerulopathies. Thus, apolipoprotein A I is generally absent in the SDS-PAGE protein pattern of renal proteinurias. However, in postrenal hematurias and proteinurias apolipoprotein A I can be found by SDS-PAGE, immunoblotting and Ouchterlony test. Using rabbit anti-human-apolipoprotein A I as primary antibody and alkaline phosphatase-conjugated anti-rabbit immunoglobulins as secondary antibody antibody apolipoprotein A I could be detected even at a 1:128,000 dilution of blood. This means a microhematuria of only 8 microliters blood/l urine theoretically can be identified as postrenal. Unfortunately, apolipoprotein A I is not only visible on the immunoblots of postrenal hematurias and proteinurias but could also be seen in renal proteinurias. Thus, only with reservation can apolipoprotein A I be called a marker of postrenal hematuria and proteinuria. On the other hand, most renal proteinurias can be identified reliably by SDS-PAGE and analysis of apolipoprotein A I is superfluous. Apolipoprotein A I, however, could become useful in the differentiation of microhematurias without proteinuria.
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PMID:[Differentiation of renal and postrenal hematuria and proteinuria with SDS polyacrylamide gel electrophoresis and immunoblotting]. 250 May 60

We have purified to homogeneity the regions derived by chymotryptic digestion of the ox neurofilament polypeptides NFH and NFM; the regions, called M1 and M2, are thought to form part of the projecting sidearms of mammalian neurofilaments [Chin, Eagles & Maggs (1983) Biochem. J. 215, 239-252]. They were isolated and purified under non-denaturing conditions and showed no tendency to interact with each other in solution. The Mr values obtained by sedimentation are approx. 61,000 for M1 and 42,000 for M2, considerably lower than the values obtained by SDS/polyacrylamide-gel electrophoresis. These Mr values were unchanged in the presence of 6 M-guanidine hydrochloride, suggesting that the regions exist as monomers in solution. Both M1 and M2 are highly phosphorylated, and there is only a slight change in the sedimentation value upon dephosphorylation. Dephosphorylation of M1 with alkaline phosphatase was more than 90% efficient but was never absolute. Dephosphorylation of M2 was complete. Both M1 and M2 bind Ca2+; in the case of M1, this binding is phosphorylation-dependent. M1 also binds cytochrome c, and dephosphorylation affects binding. In similar conditions, neurofilaments bind at least twice their own mass of cytochrome c, owing to their opposite net charges. No interactions were observed between native or dephosphorylated M1 and M2, and intact neurofilaments under a wide variety of conditions. These results are discussed in terms of the possible roles that neurofilament sidearms might play and throw doubt upon their supposed function of rigidly cross-linking neurofilaments together within the axoplasm of neurons.
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PMID:Characterization of two proteolytically derived soluble polypeptides from the neurofilament triplet components NFM and NFH. 255 34

Membrane glycoproteins and glycolipids play an important role in epithelial organization, transport and function. To study the effects of exogenous carbohydrates on the expression of glycoproteins, cells of the renal epithelial line LLC-PK1 were cultured on different nutritive carbohydrate sources and on uridine, which is, despite striking differences, known to substitute all essential nutritive functions of glucose. LLC-PK1 cultures were long-term adapted to growth in culture medium containing 0.5, 5, 10 and 25 mM glucose, and 5 mM fructose, galactose and uridine, respectively, as the sole carbohydrate source. These growth conditions elicited adaptive changes in the expression of enzyme activities of alkaline phosphatase and gamma-glutamyltranspeptidase, integral membrane glycoproteins exclusively localized in the apical membrane of LLC-PK1 cells. SDS-PAGE of membrane preparations of adapted LLC-PK1 cells revealed a strong induction of several protein bands between 13.5 and 47 kD in fructose-grown cells, while in plasma membranes of cells grown in galactose several protein bands between 62 and 70 kD decreased. Changes in the secretion pattern of proteins into the culture medium were most prominent in uridine-grown cells compared to controls grown on 25 mM glucose.
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PMID:Modification of membrane protein expression and protein secretion in LLC-PK1 cultures grown on different carbohydrates. 257 49

The additional penicillin-binding protein (PBP) 2' that is important in determining intrinsic resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA) has been purified by affinity chromatography using monoclonal antibodies. Monoclonal antibody 1/423.10.351 reacted in ELISA with detergent extracts of membranes from resistant organisms, but not in immunoblots with PBP 2' separated by SDS-PAGE. Immunoprecipitation experiments showed that antibody 1/423.10.351 reacted with PBP 2' in detergent extracts. The latter antibody, covalently coupled to protein A-Sepharose through the Fc region, served as an affinity matrix to purify PBP 2'. The PBP was detected in immunoblots using a second monoclonal antibody, 2/401.43. Conjugation of this antibody with alkaline phosphatase afforded more rapid detection of PBP 2' for the immunological detection of PBP 2' both in affinity-purified fractions and in resistant strains.
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PMID:Characterisation of a monoclonal antibody and its use in the immunoaffinity purification of penicillin-binding protein 2' of methicillin-resistant Staphylococcus aureus. 261 82

A synthetic gene containing the coding sequence for the human cysteine proteinase inhibitor, cystatin A, was obtained by enzymatic assembly of 20 oligodeoxyribonucleotides which had been chemically synthesized by the solid phase phosphoramidite method. It was cloned into an Escherichia coli plasmid. The expression plasmid for cystatin A was constructed by introducing the synthetic gene downstream of the tac promoter of an E. coli plasmid which is a derivative of pKK223-3 with high copy number. The gene was expressed in E. coli JM109 without IPTG-induction. The expression of cystatin A was detected by SDS-polyacrylamide gel electrophoresis of the E. coli JM109 lysate, followed by immunoblotting using rabbit antiserum raised with human epidermal cystatin A and alkaline phosphatase-conjugated goat anti-rabbit IgG. The result showed that the molecular weight of the expression product is identical with that of the authentic protein and the antigenic properties are also the same. Furthermore, the expression product purified with a CM-papain Sepharose affinity column and FPLC system with a Mono-Q column showed the same inhibitory activity for various cysteine proteinases. Also, purified recombinant cystatin A was found to have identical amino acid composition, NH2-terminal amino acid sequence, and peptide-map on reverse phase HPLC with those of the authentic inhibitor.
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PMID:Studies on chemical synthesis of human cystatin A gene and its expression in Escherichia coli. 266 52


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