-
Analytical Sciences : the International... 2012Here, we present a label-free, simple and signal-on architecture for fluorescent alkaline phosphatase (ALP) biosensor utilizing an SYBR Green I (SG) assisted...
Here, we present a label-free, simple and signal-on architecture for fluorescent alkaline phosphatase (ALP) biosensor utilizing an SYBR Green I (SG) assisted fluorescence amplification method. The strategy relies on the fact that ALP provides a significant barrier to lambda exonuclease (λ exo) activity by a dephosphorylating DNA hairpin probe (HP), and SG shows a considerable fluorescence intensity enhancement upon binding to double-stranded DNA (dsDNA) than single-stranded DNA (ssDNA). Our method is simple, sensitive and selective, which can also successfully detect the activity of ALP in complex biological fluids. The results have revealed that the method allows a specific and quantitative assay of the target ALP with a wide linear response range from 0.4 to 200 U/mL and a detection limit of 0.05 U/mL.
Topics: Alkaline Phosphatase; Animals; Benzothiazoles; Biosensing Techniques; Cattle; DNA; DNA Probes; Diamines; Fluorescent Dyes; Humans; Organic Chemicals; Quinolines
PubMed: 22975916
DOI: 10.2116/analsci.28.881 -
Mediators of Inflammation 2017Over the past few years, the role of intestinal alkaline phosphatase (IAP) as a crucial mucosal defence factor essential for maintaining gut homeostasis has been... (Review)
Review
Over the past few years, the role of intestinal alkaline phosphatase (IAP) as a crucial mucosal defence factor essential for maintaining gut homeostasis has been established. IAP is an important apical brush border enzyme expressed throughout the gastrointestinal tract and secreted both into the intestinal lumen and into the bloodstream. IAP exerts its effects through dephosphorylation of proinflammatory molecules including lipopolysaccharide (LPS), flagellin, and adenosine triphosphate (ATP) released from cells during stressful events. Diminished activity of IAP could increase the risk of disease through changes in the microbiome, intestinal inflammation, and intestinal permeability. Exogenous IAP exerts a protective effect against intestinal and systemic inflammation in a variety of diseases and represents a potential therapeutic agent in diseases driven by gut barrier dysfunction such as IBD. The intestinal protective mechanisms are impaired in IBD patients due to lower synthesis and activity of endogenous IAP, but the pathomechanism of this enzyme deficiency remains unclear. IAP has been safely administered to humans and the human recombinant form of IAP has been developed. This review was designed to provide an update in recent research on the involvement of IAP in intestinal inflammatory processes with focus on IBD in experimental animal models and human patients.
Topics: Alkaline Phosphatase; Animals; Gastrointestinal Tract; Humans; Inflammation; Intestines
PubMed: 28316376
DOI: 10.1155/2017/9074601 -
Science (New York, N.Y.) Jul 2021Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We...
Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a microfluidic platform for high-throughput expression, purification, and characterization of more than 1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), we performed more than 670,000 reactions and determined more than 5000 kinetic and physical constants for multiple substrates and inhibitors. We uncovered extensive kinetic partitioning to a misfolded state and isolated catalytic effects, revealing spatially contiguous regions of residues linked to particular aspects of function. Regions included active-site proximal residues but extended to the enzyme surface, providing a map of underlying architecture not possible to derive from existing approaches. HT-MEK has applications that range from understanding molecular mechanisms to medicine, engineering, and design.
Topics: Alkaline Phosphatase; Biocatalysis; Catalytic Domain; Flavobacterium; Hydrolysis; Kinetics; Microfluidics; Models, Molecular; Mutation; Oxygen; Phosphates; Protein Conformation; Protein Folding; Thermodynamics
PubMed: 34437092
DOI: 10.1126/science.abf8761 -
Journal of Bacteriology Apr 1978In Bacillus subtilis Marburg strain, single-point mutations in the phoP locus brought about simultaneous losses of the major activities of alkaline phosphatase (APase)...
In Bacillus subtilis Marburg strain, single-point mutations in the phoP locus brought about simultaneous losses of the major activities of alkaline phosphatase (APase) and alkaline phosphodiesterase (APDase). Revertants recovered the two activities. APases with APDase activity were purified from the membrane fraction of B. subtilis 6160-BC6 and from the culture fluid of an APase-secreting B. subtilis mutant strain, RAN 1. In addition to these major APases with APDase activity, at least two kinds of phosphodiesterase (PDase) without phosphatase activity were found in the cytoplasmic supernatants of RAN 1 and an APase-less B. subtilis mutant strain, SP25. Another minor APase with a molecular weight of about 80,000, which had almost no PDase activity, was isolated from the membrane fraction of strain 6160-BC6. Enzyme distribution in subcellular fractions from various strains cultured in high- and low-phosphate media was analyzed. The PDases did not cross-react with rabbit antiserum against the RAN 1 APase with APDase activity. The main component of the PDases had a molecular weight of about 80,000 and was most active at pH 8.0. These results suggest that APase with APDase activity is different from PDases detected in cytoplasmic supernatants and that phoP is the structural gene for the phosphate-repressible APase with APDase activity.
Topics: Alkaline Phosphatase; Bacillus subtilis; Epitopes; Genes; Hydrogen-Ion Concentration; Molecular Weight; Phosphoric Diester Hydrolases; Subcellular Fractions
PubMed: 77271
DOI: 10.1128/jb.134.1.108-114.1978 -
BMC Oral Health Apr 2020Bisphosphonate coating of dental implants is a promising tool for surface modification aiming to improve the osseointegration process and clinical outcome. The... (Meta-Analysis)
Meta-Analysis
BACKGROUND
Bisphosphonate coating of dental implants is a promising tool for surface modification aiming to improve the osseointegration process and clinical outcome. The biological effects of bisphosphonates are thought to be mainly associated with osteoclasts inhibition, whereas their effects on osteoblast function are unclear. A potential of bisphosphonate coated surfaces to stimulate osteoblast differentiation was investigated by several in vitro studies with contradictory results. The purpose of this systematic review and meta-analysis was to evaluate the effect of bisphosphonate coated implant surfaces on alkaline phosphatase activity in osteoblasts.
METHODS
In vitro studies that assessed alkaline phosphatase activity in osteoblasts following cell culture on bisphosphonate coated titanium surfaces were searched in electronic databases PubMed/MEDLINE, Scopus and ISI Web of Science. Animal studies and clinical trials were excluded. The literature search was restricted to articles written in English and published up to August 2019. Publication bias was assessed by the construction of funnel plots.
RESULTS
Eleven studies met the inclusion criteria. Meta-analysis showed that coating of titanium surfaces with bisphosphonates increases alkaline phosphatase activity in osteoblasts after 3 days (n = 1), 7 (n = 7), 14 (n = 6) and 21 (n = 3) days. (7 days beta coefficient = 1.363, p-value = 0.001; 14 days beta coefficient = 1.325, p-value < 0.001; 21 days beta coefficient = 1.152, p-value = 0.159).
CONCLUSIONS
The meta-analysis suggests that bisphosphonate coatings of titanium implant surfaces may have beneficial effects on osteogenic behaviour of osteoblasts grown on titanium surfaces in vitro. Further studies are required to assess to which extent bisphosphonates coating might improve osseointegration in clinical situations.
Topics: Alkaline Phosphatase; Animals; Cell Differentiation; Cells, Cultured; Dental Implants; Diphosphonates; Osseointegration; Osteoblasts; Surface Properties; Titanium
PubMed: 32334598
DOI: 10.1186/s12903-020-01089-4 -
The Biochemical Journal Aug 19791. Halobacterium cutirubrum alkaline phosphatase is associated in crude extracts with a phosphodiesterase. 2. The enzymes were stabilized in buffers containing both...
1. Halobacterium cutirubrum alkaline phosphatase is associated in crude extracts with a phosphodiesterase. 2. The enzymes were stabilized in buffers containing both (NH4)2SO4 and 10 mM-Mn2+. 3. Adsorption chromatography on Sepharose 6B/agarose-gel columns in the presence of 1.4M-(NH4)2SO4 gave a phosphatase-free phosphodiesterase and the alkaline phosphatase associated with some phosphodiesterase activity. 4. Further chromatography of the separated enzymes gave a good recovery of greater than 600-fold purified phosphodiesterase and greater than 3000-fold purified alkaline phosphatase. 5. The requirements of these enzymes and their relationship to each other was examined. 6. A detailed study showed that the alkaline phosphatase was adsorbed at least partially to agarose and dextran columns at all (NH4)2SO4 concentrations from 0.25 to 2M. 7. In contrast, no adsorption of the enzyme or protein standards was evident in 2.5M-KCl/l M-NaCl or 0.25 M-KCl/0.1 M-NaCl, in agreement with previous studies by Louis, Peterkin & Fitt [(1971) Biochem. J. 121, 635-641], thus confirming the validity of gel filtration in 2.5 M-KCl/1 M-NaCl as a method for determining the approximate molecular weights of extremehalophile proteins.
Topics: Alkaline Phosphatase; Ammonium Sulfate; Chlorides; Chromatography, Agarose; Halobacterium; Phosphoric Diester Hydrolases; Substrate Specificity
PubMed: 227360
DOI: 10.1042/bj1810347 -
Biomedicine & Pharmacotherapy =... Jul 2023In normal condition human hair growth occurs through three phases, anagen (growth phase included about 85 % of hairs, last from 2 to 6 years), catagen (transitional...
In normal condition human hair growth occurs through three phases, anagen (growth phase included about 85 % of hairs, last from 2 to 6 years), catagen (transitional phase lasting up to 2 weeks) and telogen (resting phase which last from 1 to 4 months). Natural dynamics of the hair growth process can be impaired by several factors, such as genetic predisposition, hormonal disorders, aging, poor nutrition or stress, which can lead to the slowdown in the growth of hair or even hair loss. The aim of the study was to assess the hair growth promotion effect of marine-derived ingredients, hair supplement Viviscal® and its raw components (marine protein complex AminoMarC®, shark and oyster extract). Cytotoxicity, production of alkaline phosphatase and glycosaminoglycans, as well as expression of genes involved in hair cycle-related pathways were investigated using dermal papilla cells, both immortalized and primary cell lines. Tested marine compounds showed no evidence of cytotoxicity under in vitro conditions. Viviscal® significantly increased proliferation of dermal papilla cells. Moreover, tested samples stimulated cells to produce alkaline phosphatase and glycosaminoglycans. Increased expression of hair cell cycle-related genes was also observed. The obtained results indicate that marine-derived ingredients stimulate hair growth through anagen activation.
Topics: Humans; Hair Follicle; Alkaline Phosphatase; Hair; Cell Division; Cell Cycle
PubMed: 37156114
DOI: 10.1016/j.biopha.2023.114838 -
Journal of the American Chemical Society Feb 2023Enzymes inherently exhibit molecule-to-molecule heterogeneity in their conformational and functional states, which is considered to be a key to the evolution of new...
Enzymes inherently exhibit molecule-to-molecule heterogeneity in their conformational and functional states, which is considered to be a key to the evolution of new functions. Single-molecule enzyme assays enable us to directly observe such multiple functional states or functional substates. Here, we quantitatively analyzed functional substates in the wild-type and 69 single-point mutants of alkaline phosphatase by employing a high-throughput single-molecule assay with a femtoliter reactor array device. Interestingly, many mutant enzymes exhibited significantly heterogeneous functional substates with various types, while the wild-type enzyme showed a highly homogeneous substate. We identified a correlation between the degree of functional substates and the level of improvement in promiscuous activities. Our work provides much comprehensive evidence that the functional substates can be easily altered by mutations, and the evolution toward a new catalytic activity may involve the modulation of the functional substates.
Topics: Alkaline Phosphatase; Escherichia coli; Escherichia coli Proteins; Mutation; Protein Conformation
PubMed: 36706363
DOI: 10.1021/jacs.2c06693 -
The Journal of Biological Chemistry Oct 1988When membrane-bound human liver alkaline phosphatase was treated with a phosphatidylinositol (PI) phospholipase C obtained from Bacillus cereus, or with the proteases...
When membrane-bound human liver alkaline phosphatase was treated with a phosphatidylinositol (PI) phospholipase C obtained from Bacillus cereus, or with the proteases ficin and bromelain, the enzyme released was dimeric. Butanol extraction of the plasma membranes at pH 7.6 yielded a water-soluble, aggregated form that PI phospholipase C could also convert to dimers. When the membrane-bound enzyme was solubilized with a non-ionic detergent (Nonidet P-40), it had the Mr of a tetramer; this, too, was convertible to dimers with PI phospholipase C or a protease. Butanol extraction of whole liver tissue at pH 6.6 and subsequent purification yielded a dimeric enzyme on electrophoresis under nondenaturing conditions, whereas butanol extraction at pH values of 7.6 or above and subsequent purification by immunoaffinity chromatography yielded an enzyme with a native Mr twice that of the dimeric form. This high molecular weight form showed a single Coomassie-stained band (Mr = 83,000) on electrophoresis under denaturing conditions in sodium dodecyl sulfate, as did its PI phospholipase C cleaved product; this Mr was the same as that obtained with the enzyme purified from whole liver using butanol extraction at pH 6.6. These results are highly suggestive of the presence of a butanol-activated endogenous enzyme activity (possibly a phospholipase) that is optimally active at an acidic pH. Inhibition of this activity by maintaining an alkaline pH during extraction and purification results in a tetrameric enzyme. Alkaline phosphatase, whether released by phosphatidylinositol (PI) phospholipase C or protease treatment of intact plasma membranes, or purified in a dimeric form, would not adsorb to a hydrophobic medium. PI phospholipase C treatment of alkaline phosphatase solubilized from plasma membranes by either detergent or butanol at pH 7.6 yielded a dimeric enzyme that did not absorb to the hydrophobic medium, whereas the untreated preparations did. This adsorbed activity was readily released by detergent. Likewise, alkaline phosphatase solubilized from plasma membranes by butanol extraction at pH 7.6 would incorporate into phosphatidylcholine liposomes, whereas the enzyme released from the membranes by PI phospholipase C would not incorporate. The dimeric enzyme purified from a butanol extract of whole liver tissue carried out at pH 6.6 did not incorporate. We conclude that PI phospholipase C converts a hydrophobic tetramer of alkaline phosphatase into hydrophilic dimers through removal of the 1,2-diacylglycerol moiety of phosphatidylinositol. Based on these and others' findings, we devised a model of alkaline phosphatase's conversion into its various forms.
Topics: Alkaline Phosphatase; Bacillus cereus; Cell Membrane; Humans; Kinetics; Liposomes; Liver; Macromolecular Substances; Molecular Weight; Peptide Hydrolases; Phosphatidylcholines; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoric Diester Hydrolases; Solubility
PubMed: 2844768
DOI: No ID Found -
The Biochemical Journal Mar 1993A complete genomic clone and a full-length cDNA coding for bovine intestinal alkaline phosphatase have been isolated and sequenced. The gene (5.4 kb) contains 11 exons...
A complete genomic clone and a full-length cDNA coding for bovine intestinal alkaline phosphatase have been isolated and sequenced. The gene (5.4 kb) contains 11 exons separated by ten small introns at positions identical to those other members of the eukaryotic tissue-specific alkaline phosphatase family. In addition, 1.5 kb of upstream sequences contain putative regulatory elements showing sequence similarity to human and mouse intestinal alkaline phosphatase promoter sequences. To achieve recombinant bovine intestinal alkaline phosphatase expression, the coding region of the gene was subcloned into the pcDNA I eukaryotic expression vector and transfected into Chinese hamster ovary cells. Recombinant bovine intestinal alkaline phosphatase displays enzymatic properties comparable with those of purified native bovine intestinal alkaline phosphatase, a slightly increased thermal stability and, upon desialylation, it shows a homogeneous behaviour in agarose gel electrophoresis and isoelectric focusing. The availability of the recombinant bovine intestinal alkaline phosphatase and the elucidation of its primary sequence will help to accelerate our efforts to obtain the first crystallographic model of a eukaryotic alkaline phosphatase molecule.
Topics: Alkaline Phosphatase; Amino Acid Sequence; Animals; Base Sequence; CHO Cells; Cattle; Cloning, Molecular; Cricetinae; DNA; Intestines; Molecular Sequence Data; Recombinant Proteins; Sequence Homology, Amino Acid
PubMed: 8452539
DOI: 10.1042/bj2900503