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)

Extracellular, membrane-bound vesicles are widely regarded to be the initial site of calcification in a variety of tissues under normal and pathological conditions. Alkaline phosphatase is believed to play a vital role in this process by hydrolysing ester phosphates or mineral inhibitors, e.g. inorganic phosphates. In the present study, matrix vesicles from normal and rachitic rat growth plates were compared with regard to specific activity of alkaline phosphatase, total vesicle protein and ultrastructural distribution of alkaline phosphatase activity. Matrix vesicles were released from normal or rachitic growth plates by collagenase digestion and isolated by differential centrifugation. Enzyme cytochemical localization involving a cerium capture method was performed on vesicles collected by vacuum filtration on Millipore filters. SDS gels and Western blots on fractions of both normal and rachitic matrix vesicles showed major proteins to be almost identical and confirmed the presence of alkaline phosphatase in both. Total matrix vesicle protein ((mg total matrix vesicle protein/rat) x 10(2)) per rat was significantly greater for the rachitic animals (9.0 +/- 2.0 vs. 4.0 +/- 1.0), P less than 0.0001. Alkaline phosphatase specific activity (units alkaline phosphatase/mg vesicle protein) in the rachitic and normal matrix vesicles was 25.29 +/- 9.36 and 18.78 +/- 3.37, respectively (0.05 less than P less than 0.1). Electron dense cerium phosphate deposits were localized to the outer membrane surface of matrix vesicles derived from both types of rats. This data, the first to quantify the relationship between rickets, matrix vesicle protein and alkaline phosphatase specific activity, suggests that matrix vesicles from rachitic and normal rats have biochemical and morphological similarity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Increased matrix vesicle protein in rachitic rat epiphyseal growth plates. 165 31

Sodium butyrate (butyrate), 5-azacytidine (5Aza-C), dimethyl sulfoxide (DMSO), and dimethyl formamide (DMF) were applied to a human melanoma cell line for the purpose of inducing pigmentation and terminal differentiation. The results are summarized as follows: 1) butyrate, DMSO, and DMF had a strong cytostatic effect, arresting cells in the G1 phase of the cycle; 2) butyrate caused a morphological change to spindle shape whereas DMSO and DMF produced rounded cells, without affecting the levels of vimentin and intermediate filaments; 3) tyrosinase activity and melanization were stimulated by DMSO and DMF but not by butyrate; 4) butyrate induced several membrane-bound enzyme activities (alkaline phosphatase and gamma-glutamyl transpeptidase); 5) changes in the expression of antigens related to tyrosinase activity (2B7 and 5C12) only partly corresponded to the changes in enzyme activity; 6) expression of the melanosomal B8G3 antigen was decreased by butyrate, DMSO, and DMF; and 7) the action of DMF resembled that of DMSO whereas 5Aza-C had little effect. The results indicate that these differentiating agents activate different sets of genes, the melanogenic pathway being activated independently of gamma-glutamyltranspeptidase. The down regulation of B8G3 antigen by these agents may provide a common focus for understanding the essential action of differentiation inducers in melanoma cells.
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PMID:In vitro phenotypic alteration of human melanoma cells induced by differentiating agents: heterogeneous effects on cellular growth and morphology, enzymatic activity, and antigenic expression. 171 Mar 61

We describe a rapid non-radioactive DNA typing of the serological types DR1-DRw10 using polymerase chain reaction (PCR)-amplified DNA and 15 sequence-specific oligonucleotides (SSO) which are labelled enzymatically at their 3' end with one digoxigenin (DIG). The hybridized SSOs were detected using anti-DIG alkaline phosphatase and Fab fragments and visualization was obtained with the chemiluminescent substate 3-(2'-spiroadamantan)-4-(3''-phosphoryloxy)-phenyl-1,2-di o xetan (AMPPD). The results were identical with those of the previously used 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/4-nitrobluetetrazolium chloride (NBT) system. The use of AMPPD is more rapid and allows the repeated rehybridization of the membrane-bound DNA.
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PMID:Non-radioactive oligotyping for HLA-DR1-DRw10 using polymerase chain reaction, digoxigenin-labelled oligonucleotides and chemiluminescence detection. 177 77

Osteoblasts, the bone-forming cells, synthesize the macromolecules of the bone matrix including: type I collagen; osteocalcin; osteonectin; osteopontin; proteoglycan I and II; bone sialoprotein; matrix gla-protein; bone glycoprotein 75; several other proteins, which have not been extensively characterized; growth factors, including transforming growth factor beta and fibroblast growth factor. Osteoblasts also have high levels of the membrane-bound enzyme, alkaline phosphatase, which plays a role in matrix mineralization, and receptors for tissue-specific hormones, such as parathyroid hormone, as well as many other hormones, cytokines and growth factors, which regulate bone growth, differentiation and metabolism. The expression of these various proteins, most of which are not unique to bone but which together characterize the bone phenotype, is induced during osteoblastic differentiation in a stepwise fashion, suggestive of multiple regulatory factors. The detailed sequence of the expression of osteoblastic genes in situ has not been fully characterized. It appears that type I collagen and alkaline phosphatase are expressed early during the commitment to the osteoblastic phenotype, whereas osteopontin and osteocalcin appear late during osteoblastic differentiation. Diversity among "osteoblastic" cells is also apparent, probably not all osteoblastic cells express all the features. A large number of osteoblastic models are currently available to study the expression of osteoblast-related genes in vitro. These include primary cultures from calvaria or trabecular bone from several species, including humans, osteosarcoma-derived cell lines, and experimentally immortalized cells. Some of these in vitro models, especially the calvaria-derived cultures, undergo changes which mimic osteoblastic differentiation in vivo. The study of these and other cell models started providing insights into the regulation of gene expression in osteoblastic cells. In addition to a vast body of information on the conditions required for the expression of various proteins in culture and their regulation by hormones and growth factors, more detailed information on specific genes has recently been obtained. For example, regulation of type I collagen gene expression has been studied in osteosarcoma cell lines where 1,25(OH)2 vitamin D3 was shown to act via specific DNA segment(s) in the 5' flanking region of the gene, while parathyroid hormone affected gene expression by altering the stability of the transcripts. TGF beta 1, which stimulates osteogenesis, was shown to promote the transcription of osteopontin and type I collagen, the latter effect requiring the binding site for the transactivating protein, nuclear factor I.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Gene expression in osteoblastic cells. 180 5

Guinea pig neutrophils contain membrane-bound and soluble phosphatases that catalyze the dephosphorylation of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]. The activities were 5.1 +/- 0.2 and 1.3 +/- 0.2 (SD; n = 5) nmoles phosphate (Pi) released/min/10(7) cell equivalents, respectively. The membrane-bound enzyme dephosphorylated many substrates (e.g., beta-glycerophosphate), exhibited alkaline pH optima, and was inhibited by levamisole. In contrast, the soluble phosphatase was specific for Ins(1,4,5)P3, exhibited a neutral pH optimum, and was insensitive to levamisole. A cerium-based ultrastructural cytochemical procedure was employed to identify the subcellular sites of the membrane-bound activity. Staining was observed on the exterior of the plasmalemma and in a population of granules. Staining in the granules was observed only in permeabilized cells. Treatment of neutrophils with p-diazobenzenesulfonate (DBSA) (4.0 mM) for 20 min at 37 degrees C blocked the cytochemical reaction on the cell surface using beta-glycerophosphate as the substrate, but did not affect the staining of the granules on subsequent permeabilization. In biochemical studies, this treatment with DBSA inhibited the membrane-bound activity by c. 50% but did not affect the soluble phosphatase. Therefore, the membrane-bound phosphatase is, in fact, an alkaline phosphatase that resides in locales not accessible to Ins(1,4,5)P3 generated during cell stimulation. Breakdown of Ins(1,4,5)P3 generated during cell stimulation, therefore, would be catalyzed by the soluble enzyme.
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PMID:Biochemical and cytochemical studies on enzymes that dephosphorylate inositol (1,4,5)-trisphosphate in neutrophils. 184 59

The Escherichia coli mannitol permease catalyzes the concomitant transport and phosphorylation of D-mannitol. This 68-kDa protein consists of a membrane-bound, N-terminal domain involved in mannitol binding and translocation and a C-terminal, cytoplasmic domain responsible for mannitol phosphorylation. Secondary-structure prediction methods suggest that the N-terminal half of the permease spans the membrane approximately seven times in alpha-helical segments, but these data cannot conclusively predict the structure. We have used gene fusions between mtlA (encoding the permease) and 'phoA (encoding alkaline phosphatase lacking its signal sequence) to further investigate the topology of the mannitol permease. Initially, fusions were constructed by using a lambda TnphoA vector and in vitro cloning of 'phoA into naturally occurring restriction sites in mtlA. However, the former method gave severe problems with insertion "hot-spots" in our vector systems, and the latter method was limited by the number of useful restriction sites. Therefore, we developed a nested-deletion method for creating mtlA-phoA fusions. 'phoA was first cloned downstream from the part of mtlA encoding the membrane-bound half of the permease. This construct was then treated with the appropriate restriction enzymes and with exonuclease III to create random fusions. An analysis of greater than 40 different fusion clones constructed by these methods provides strong evidence for six membrane-spanning regions in the mannitol permease with three relatively short periplasmic loops and two large cytoplasmic loops in the membrane-bound half of the protein.
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PMID:Membrane topology analysis of Escherichia coli mannitol permease by using a nested-deletion method to create mtlA-phoA fusions. 194 74

The ultrastructure of crystal formation in vitro associated with extracellular membrane-bound matrix vesicles (MV) isolated from rat incisor pulp was studied in Dulbecco's modified Eagle's medium (DMEM) supplemented with an organic phosphate, Na-beta-glycerophosphate (BGP). Matrix vesicles were isolated from basal regions of the pulps using a collagenase digestion and ultra-centrifugation method. Isolated MV contained alkaline phosphatase (ALP) activity and had diameters of 30-200 nm. Membrane structures of the isolated MV were well preserved. Incubation of MV in DMEM in the presence of BGP caused the development of bilaminar electron densities associated with the vesicle membrane. These preceded crystal deposition which was observed in the culture medium after 3 days. Both heat-inactivated MV incubated with BGP, and fresh MV incubated in the absence of BGP failed to show crystal formation, even after 3 days. Staining of demineralized sections of mineralized MV with uranyl acetate and lead citrate, revealed numerous needle-like structures similar in shape to the untreated crystals. Electron diffraction patterns of the newly formed crystals revealed a pattern consistent with hydroxyapatite. The requirement of BGP for mineralization of these MV and the long lag time before crystal formation is probably due to the low calcium (Ca) x inorganic phosphate (Pi) ion product in the original medium. The requirement of ALP activity which would cause hydrolysis of BGP and a rise in Pi would favor the precipitation of biologic apatite from the culture medium.
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PMID:Matrix vesicles isolated from apical pulp of rat incisors: crystal formation in low Ca x Pi ion-product medium containing beta-glycerophosphate. 196 82

A novel highly sensitive non-radioactive DNA labeling and detection system based on the ELISA principle has been developed. DNA is modified with the cardenolide-hapten digoxigenin by enzymatic incorporation of digoxigenin-labeled deoxyuridine-triphosphate with Klenow enzyme. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Following hybridization of membrane-bound target-DNA with a digoxigenin-labeled probe, the hybrids are detected by an ELISA reaction using digoxigenin-specific antibodies covalently coupled to the marker enzyme alkaline phosphatase [(Dig):CIAP]. This binding of antibody: marker enzyme-conjugate is followed by an enzyme-catalysed coupled redox reaction with the colour substrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium salt (NBT) giving rise to a deep-blue coloured, water-insoluble precipitate directly adhering to the membrane. The digoxigenin system allows the detection of 0.1 pg homologous DNA within 16 h in dot- and Southern-blots on nitrocellulose or nylon membranes avoiding any significant background even after a prolonged period of color development. Due to its high sensitivity and specificity, the new system is appropriate for detection of single-copy genes in genomic blots as well as for Northern, slot, colony, plaque and in situ hybridizations.
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PMID:Non-radioactive labeling and detection of nucleic acids. I. A novel DNA labeling and detection system based on digoxigenin: anti-digoxigenin ELISA principle (digoxigenin system). 207 99

The enzyme TEM beta-lactamase constitutes a versatile gene-fusion marker for studies on membrane proteins and protein export in bacteria. The mature form of this normally periplasmic enzyme displays readily detectable and distinctly different phenotypes when localized to the bacterial cytoplasm versus the periplasm, and thus provides a useful alternative to alkaline phosphatase for probing the topology of cytoplasmic membrane proteins. Cells producing translocated forms of beta-lactamase can be directly selected as ampicillin-resistant colonies, and consequently a beta-lactamase fusion approach can be used for positive selection for export signals, and for rapid assessment of whether any protein expressed in Escherichia coli inserts into the bacterial cytoplasmic membrane. The level of ampicillin resistance conferred on a cell by an extracytoplasmic beta-lactamase derivative depends on its level of expression, and therefore a beta-lactamase fusion approach can be used to directly select for increased yields of any periplasmic or membrane-bound gene products expressed in E. coli.
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PMID:Beta-lactamase as a probe of membrane protein assembly and protein export. 207 55

Four alkaline phosphatase forms from adult rat femur were distinguished on polyacrylamide gel electrophoresis: two soluble forms of Mr 165,000 and 110,000 in the water extract, and three membrane-bound forms of Mr 130,000, 110,000 and 100,000 extractable with deoxycholate. Alkaline phosphatase after SDS-treatment disintegrated into three kinds of monomers: of Mr 80,000, 65,000 and 50,000. The soluble fraction (extract I) contained subunits of Mr 80,000 and 55,000--whereas the pellet fraction (extract II), subunits of Mr 65,000 and 50,000. Since for native forms only three types of subunits were found it seems that, apart from homodimers, there are also some heterodimers composed of the Mr 65,000 and 50,000 subunits forming the native enzyme of Mr 110,000-115,000. Two denatured monomers: of Mr 80,000 and 50,000 may form two native homodimeric forms of Mr 165,000 and 100,000 while in the pellet two monomers: of Mr 65,000 and 50,000 may correspond to three native alkaline phosphatase forms: of Mr 130,000, 110,000-115,000 and 100,000. Probably the Mr 110,000-115,000 form is a heterodimer composed of subunits of Mr 65,000 and 50,000.
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PMID:Alkaline phosphatase from adult rat femur. 210 Aug 96


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