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)

Subcutaneous implantation of demineralized diaphyseal bone matrix in allogeneic rats results in the local induction of endochondral bone differentiation. We have explored the potential of three dissociative extractants, 4 M guanidine hydrochloride (Gdn . HCl), 8 M urea/1 M NaCl, and 1% NaDodSO4 at pH 7.4, containing protease inhibitors to solubilize putative inductive molecules in the bone matrix. Extraction of bone matrix with any one of these extracts resulted in the loss of the bone inductive property. The solubilized extracts were then reconstituted with the residue by dialysis against water. The various reconstituted matrices were bioassayed for bone inductive potential by quantitation of alkaline phosphatase activity and 45Ca incorporation on day 12 after implantation. There was complete recovery of biological activity after reconstitution of the residues with each of the three extracts. Polyacrylamide gel electrophoresis of the extracts revealed similar protein profiles. Gel filtration of the 4 M Gdn. HCl extract on Sepharose CL-4B showed a heterogeneous broad peak. When fractions of that peak containing proteins less than 50,000 daltons were reconstituted with inactive 4 M Gdn . HCl-treated bone matrix and then implanted, new bone was induced. These observations demonstrate the dissociative extraction and successful biological reconstitution of bone inductive macromolecules in demineralized bone matrix.
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PMID:Dissociative extraction and reconstitution of extracellular matrix components involved in local bone differentiation. 695 Apr 1

Alkaline phosphatase was purified from plasma membrane of rat ascites hepatoma AH-130 cells by chromatographies on DEAE-cellulose and Affi-Gel Blue and preparative polyacrylamide gel electrophoresis. The yield of the purified enzyme, finally as the denatured subunits, was about three times better than that obtained previously [J. Biochem. (1978) 83, 1471-1483]. The purified enzyme, a glycoprotein, was analyzed for amino acid and carbohydrate compositions. The composition of the carbohydrate moiety, which amounted to about 18% by weight, indicated that both N- and O-glycosidic sugar chains were contained in the protein. The purified alkaline phosphatase (as the denatured subunits) was used to produce monospecific antibody, which was found to have the same immunological reaction with the native antigen as the antibody raised against the partially purified native enzyme. Immunological analysis by Ouchterlony double gel diffusion and quantitative immunoprecipitation demonstrated that the hepatoma enzyme was identical with that of the liver. In standard polyacrylamide gel electrophoresis at pH 8.9, the hepatoma alkaline phosphatase migrated a little more slowly than the liver enzyme. Both enzymes, however, showed identical mobility after complete removal of sialic acid from them with neuraminidase. These results suggest that the only difference between the two enzymes was in the carbohydrate moiety, especially in the sialic acid content.
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PMID:Chemical and immunological characterization of rat ascites hepatoma alkaline phosphatase: a comparison with the liver enzyme. 706 59

1. Acid phosphatase (AcPase) from potato tubers was purified by tannic acid fractionation, DEAE-cellulose chromatography, filtration on Bio-Gel P-150 and affinity chromatography on Con A-Sepharose. The enzyme was purified 260-fold and was electrophoretically homogeneous; its mol. mass is about 69 000. 2. The carbohydrate component accounts for 16.6% of the total enzyme weight and includes mannose (5.6%), rhamnose (3.4%), glucose (2.5%), galactose (1.5%) and glucosamine (3.6%). In the amino acid composition aspartic acid, glutamic acid, serine and glycine account for 37.7% of total amino acid residues. 3. Optimum pH is at 5.0-5.3. The enzyme activity was reduced by half after 30 min incubation at 60 degrees C, and was fully abolished after 2 h incubation at 70 degrees C. The enzyme is a nonspecific phosphomonoesterase; aromatic phosphomonoesters and inorganic pyrophosphate can serve as substrates. Apparent Km values were 1.25 mM and 40 mM for p-nitrophenylphosphate and inorganic pyrophosphate, respectively. The enzyme is inhibited by MoO42-, Zn2+, Hg2+ and urea. Inhibition caused by urea was reversible at urea concentration below 9 M.
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PMID:Acid phosphatase of potato tubers (Solanum tuberosum L). Purification, properties, sugar and amino acid composition. 715 77

Microvillous membrane fractions from human term placentae were prepared by differential centrifugation. Extration of membranes with PBS-EDTA or KCI removed soluble cytoplasmic components and serum proteins excepting trace amounts of albumin and transferrin. PAGE-SDS revealed 11 components in the Triton solubilized crude fraction after PBS-EDTA extraction. Membrane components solubilized with Triton were not fractionated by gel filtration on Bio-Gel A-50 m but DEAE-cellulose chromatography partially resolved these components. Three fractions were obtained by stepwise elution of absorbed materials using increasing concentrations of NaCl in the equilibrating buffer. These fractions were characterized using SDS-PAGE. The material unabsorbed to the DEAE contained two components of small molecular weight and one of them showed a positive PAS stain. The first eluted protein peak showed nine components, seven of which stained with PAS. The bulk of glycoproteins with molecular weights greater than 130 000 daltons were found in this fraction. The second eluted peak from DEAE was rich in components with molecular weights less than 42 000 daltons. Four components in this fraction were not identified in the other two ion-exchange fractions. Bands representing mobilities of albumin, transferrin and alkaline phosphatase were observed in DEAE-cellulose fractions; however, 12 components of unknown structure were revealed.
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PMID:Characterization of solubilized microvillous membrane proteins and glycoproteins from human placental syncytiotrophoblast. 723 34

Alkaline phosphatase has been purified from microsomes of chicken epiphyseal cartilage by first selectively extracting certain adventitious proteins with 0.25 M trichloroacetate. The membrane-bound enzyme was then solubilized by 1% cholate in buffered 33% saturated ammonium sulfate and purified by column chromatography on Bio-Gel A-5m, extraction with 1-butanol, and ion exchange chromatography on DEAE-Bio-Gel A. The purified alkaline phosphatase from the cartilage membrane had a subunit molecular weight of 53,000 and a holoenzyme weight of 207,000-220,000, indicating a tetramer. The pH optima for p-nitrophenylphosphate, ATP, and pyrophosphate hydrolysis were 10.3, 9.0, and 8.5, respectively. Values of Vmax (in micromoles/min/mg) were 220, 3.1, and 0.8, respectively. Substrate inhibition was pronounced at values of pH below 8.5. Inhibition of p-nitrophenylphosphate hydrolysis at pH 10.3 showed that phosphate and arsenate were competitive inhibitors (KI = 1.88 and 0.15 mM, respectively) and levamisole was an uncompetitive inhibitor (KI = 0.32 mM), while L-phenylalanine and ZnCl2 were mixed inhibitors (KI = 15.8 and 0.02 mM, respectively). Inhibition by preincubation in 1 mM EDTA was reversible by readdition of 0.25 mM MgCl2 nd 20 microM ZnCl2. The data indicate that this membrane-bound alkaline phosphatase from chicken epiphyseal cartilage is a Zn2+ and possibly Mg2+-containing enzyme. While the subunit molecular weight and kinetic properties of the enzyme are quite typical of vertebrate alkaline phosphatases, the tightness of binding to the membrane lipids, the extreme sensitivity to substrate inhibition, and the tetrameric conformation of the holoenzyme are unusual.
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PMID:Purification and initial characterization of intrinsic membrane-bound alkaline phosphatase from chicken epiphyseal cartilage. 725 97

beta-Hexosaminidase (Hex) activity was determined in bile from 18 patients with cholestasis, six patients without cholestasis and in ten normal liver biopsies. The difference in the mean activities in bile from patients with and without cholestasis was not significant. Only about 0.5 promille of total liver Hex activity was lost per day via the bile flow. Gel chromatography showed that enzyme forms present in bile had higher molecular weights than the forms present in liver tissue, indicating that the biliary enzyme was not routed through the lysosomes before release into the bile. In 32 patients with cholestasis, plasma Hex was increased compared to controls, and correlated to bilirubin. The activity was significantly higher in patients with severe cholestasis than in patients with less severe forms of cholestasis, but no significant difference in Hex activity was observed between patients with benign or malignant biliary obstruction. No significant difference was noted between patients with cholestasis for less than 1 week compared to those whose illness had lasted more than 1.5 weeks. The impact of biliary obstruction on plasma Hex is further illustrated by the observation that decompression lowered plasma Hex as well as bilirubin and alkaline phosphatase.
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PMID:Beta-hexosaminidase in bile and plasma from patients with cholestasis. 767 43

As alternatives to radiolabeled DNA sequencing, chemiluminescent and chromogenic sequencing methods can be comparable in both sensitivity and resolution. Chemiluminescent/chromogenic detection procedures are safer because they completely eliminate the handling and use of radioisotopes. One method involves standard dideoxy DNA sequencing reactions that are initiated with biotinylated primers, separated by gel electrophoresis, transferred onto nylon membrane and detected utilizing chemiluminescent 1,2-dioxetane substrates for alkaline-phosphatase. Alkaline phosphatase is linked to the biotinylated sequencing products by a streptavidin/alkaline phosphatase conjugate (SAAP). In this paper we describe an optimized procedure for transferring sequencing gels. The procedure is based on a semidry method developed at Hoefer Laboratories using the GeneSweep Sequencing Gel Transfer Unit. Transfer is rapid, uniform and reliable from gel to gel. We also describe automation of the development process using a fully programmable Gel/Membrane Processor that automates delivery, incubation and disposal of reagents. All crucial points for electrotransfer of sequencing gels and the detection of biotinylated DNA sequencing reaction products are discussed.
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PMID:Innovations in non-isotopic DNA sequencing: using an electrotransfer unit to blot sequencing gels and an automated membrane processor for detecting DNA sequences. 772 38

We report a case of hyperphosphatasemia in a 35-year-old patient with hepatitis B who underwent an orthotopic xenogeneic liver transplant. Marked increases in total alkaline phosphatase (ALP; EC 3.1.3.1) activity began 5 days posttransplantation (six times human normal) and increased to approximately 17 times normal at day 11. Increased ALP persisted for > 40 days and steadily increased to 75 times normal in the patient's last 30 days. Gel electrophoresis detected both liver (LALP) and biliary (high-molecular-mass, BALP) isoforms. LALP measured with ion-exchange columns revealed an activity time course pattern similar to that of total ALP. Results for BALP activity also obtained with ion-exchange columns exhibited broad variability, ranging from 2 to 428 times normal.
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PMID:Changes in biliary (high-molecular-mass) and liver isoforms of alkaline phosphatase after baboon-to-human liver transplantation. 791 70

The protozoan parasite Leishmania mexicana secretes a heavily glycosylated 100-kDa acid phosphatase (sAP) which is associated with one or more polydisperse proteophosphoglycans. Most of the glycans in this complex were released using mild acid hydrolysis conditions that preferentially cleave phosphodiester linkages. The released saccharides were shown to consist of monomeric mannose and a series of neutral and phosphorylated glycans by Dionex high performance liquid chromatography, methylation analysis, exoglycosidase digestions, and one-dimensional 1H NMR spectroscopy. The neutral species comprised a linear series of oligosaccharides with the structures [Man alpha 1-2]1-5Man. The phosphorylated oligosaccharides were characterized as PO4-6Gal beta 1-4Man and PO4-6[Glc beta 1-3]Gal beta 1-4Man. The attachment of these glycans to the polypeptide backbone via the linkage, Man alpha 1-PO4-Ser, is suggested by: 1) the finding that more than 60% of the serine residues in the polypeptide are phosphorylated and 2) the resistance of the phosphoserine residues to alkaline phosphatase digestion unless the sAP was first treated with either mild acid (to release all glycans) or jack bean alpha-mannosidase (to release neutral mannose glycans). Analysis of the partially resolved components of the complex indicated that the most of the O-linked glycans on the 100-kDa phosphoglycoprotein comprised mannose and the mannose-oligosaccharides. In contrast the major O-linked glycans on the proteophosphoglycan were short phosphoglycan chains, containing on average two repeat units per chain. In addition to the O-linked glycans, both components in the sAP complex contained N-linked glycans. The N-glycanase F-released glycans were characterized by Bio-Gel P4 chromatography and exoglycosidase digestions to be the biantennary oligomannose type with the structures Glc1Man6GlcNAc2 and Man6GlcNAc2. The O-linked glycans of the sAP complex are similar to those found in the phosphoglycan chains of the abundant surface lipophosphoglycan, but differ in having much shorter phosphoglycan chains and a more diverse series of mannose cap oligosaccharides. These data suggest that there are marked differences in the ability of different glycosyltransferases to utilize peptide-linked versus glycolipid-linked acceptors.
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PMID:O- and N-glycosylation of the Leishmania mexicana-secreted acid phosphatase. Characterization of a new class of phosphoserine-linked glycans. 792 59

This study reports the selection and characterization of osteogenic precursors from human bone marrow which were isolated by two "clonings" and successive subculturing. These cell lines express alkaline phosphatase activity. Gel electrophoresis of [3H]-proline labeled cultures showed that the cloned cells produce only type I collagen. They synthetize osteocalcin and osteonectin. They respond to 1,25 dihydroxy vitamin D3 by increasing osteocalcin synthesis and secretion, and to parathyroid hormone by increasing cyclic AMP synthesis. After the third subculture in the absence of beta-glycerophosphate, these cell lines formed lots of clusters which exhibit high alkaline phosphatase activity and positive von Kossa staining. X-ray energy spectrum shows that these cells are surrounded by "budding" structures containing calcium and phosphorus with a ratio Ca:P identical to those of pure hydroxyapatite. This process was associated with 45Ca uptake into the cells. All these data support the selection of osteogenic cells which may be of considerable clinical importance.
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PMID:Human bone marrow stromal cells express an osteoblastic phenotype in culture. 840 13


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