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Journal of Bone and Mineral Research :... Feb 2018Alkaline phosphatase can be considered "our favorite enzyme" for reasons apparent to those who diagnose and treat metabolic bone diseases or who study skeletal biology....
Alkaline phosphatase can be considered "our favorite enzyme" for reasons apparent to those who diagnose and treat metabolic bone diseases or who study skeletal biology. Few might know, however, that alkaline phosphatase likely represents the most frequently assayed enzyme in all of medicine. Elevated activity in the circulation is universally recognized as a marker for skeletal or hepatobiliary disease. Nevertheless, the assay conditions in many ways are nonphysiological. The term alkaline phosphatase emerged when it became necessary to distinguish "bone phosphatase" from the phosphatase in the prostate that features an acidic pH optimum. Beginning in 1948, studies of the inborn-error-of-metabolism hypophosphatasia would identify the natural substrates and establish the physiological role of alkaline phosphatase, including in biomineralization. Here, we recount the discovery in 1923 and then eventual naming of this enzyme that remains paramount in our field. © 2017 American Society for Bone and Mineral Research.
Topics: Alkaline Phosphatase; Enzyme Assays; History, 20th Century; Humans; Terminology as Topic
PubMed: 28727174
DOI: 10.1002/jbmr.3225 -
Calcified Tissue International Apr 2016Hypophosphatasia (HPP) results from ALPL mutations leading to deficient activity of the tissue-non-specific alkaline phosphatase isozyme (TNAP) and thereby extracellular... (Review)
Review
Hypophosphatasia (HPP) results from ALPL mutations leading to deficient activity of the tissue-non-specific alkaline phosphatase isozyme (TNAP) and thereby extracellular accumulation of inorganic pyrophosphate (PPi), a natural substrate of TNAP and potent inhibitor of mineralization. Thus, HPP features rickets or osteomalacia and hypomineralization of teeth. Enzyme replacement using mineral-targeted TNAP from birth prevented severe HPP in TNAP-knockout mice and was then shown to rescue and substantially treat infants and young children with life-threatening HPP. Clinical trials are revealing aspects of HPP pathophysiology not yet fully understood, such as craniosynostosis and muscle weakness when HPP is severe. New treatment approaches are under development to improve patient care.
Topics: Alkaline Phosphatase; Animals; Humans; Hypophosphatasia; Mice
PubMed: 26590809
DOI: 10.1007/s00223-015-0079-1 -
BMC Pediatrics Jan 2019Necrotizing enterocolitis (NEC) is a devastating disease of intestinal inflammation that primarily affects premature infants. A potential risk factor for necrotizing...
BACKGROUND
Necrotizing enterocolitis (NEC) is a devastating disease of intestinal inflammation that primarily affects premature infants. A potential risk factor for necrotizing enterocolitis is exposure of the premature neonatal intestine to environmental bacteria and their proinflammatory products such as lipopolysaccharide. The metalloenzyme alkaline phosphatase (ALP) has been shown to reduce lipopolysaccharide-mediated inflammation. Additionally, premature rat pups have reduced alkaline phosphatase activity and expression as compared to full term pups. To explore the possibility that the human premature neonatal intestine has a paucity of alkaline phosphatase activity, we measured endogenously produced intestinal alkaline phosphatase activity in meconium as a function of gestational age. To test whether breast milk could serve as a source of exogenous alkaline phosphatase to the neonatal intestine through ingestion, we measured alkaline phosphatase activity in breast milk across a range of time points post-birth.
METHODS
Alkaline phosphatase activity was quantified in 122 meconium samples from infants of gestational ages ranging from 24 to 40 weeks and in 289 breast milk samples collected from 78 individual mothers between days 2-49 post-birth.
RESULTS
We observed a strong positive correlation between the meconium alkaline phosphatase activity and gestational age, with preterm infants having lower meconium alkaline phosphatase activities than early term or term infants. Breast milk alkaline phosphatase activity was highest in the first week post-birth, with peak alkaline phosphatase activity at day 2 post-birth, followed by relatively low alkaline phosphatase activity in weeks 2-7.
CONCLUSIONS
Our results are consistent with the two major risk factors for necrotizing enterocolitis development, preterm birth and lack of breast milk feeding, both contributing to a paucity of alkaline phosphatase activity and impaired capacity to detoxify proinflammatory bacterial products such as lipopolysaccharide.
Topics: Alkaline Phosphatase; Enterocolitis, Necrotizing; Gestational Age; Humans; Infant; Infant, Newborn; Intestines; Milk, Human
PubMed: 30606146
DOI: 10.1186/s12887-018-1379-1 -
Tissue Non-Specific Alkaline Phosphatase and Vascular Calcification: A Potential Therapeutic Target.Current Cardiology Reviews 2019Vascular calcification is a pathologic phenomenon consisting of calcium phosphate crystal deposition in the vascular walls. Vascular calcification has been found to be a... (Review)
Review
Vascular calcification is a pathologic phenomenon consisting of calcium phosphate crystal deposition in the vascular walls. Vascular calcification has been found to be a risk factor for cardiovascular diseases, due to its correlation with cardiovascular events and mortality, and it has been associated with aging, diabetes, and chronic kidney disease. Studies of vascular calcification have focused on phosphate homeostasis, primarily on the important role of hyperphosphatemia. Moreover, vascular calcification has been associated with loss of plasma pyrophosphate, one of the main inhibitors of calcification, thus indicating the importance of the phosphate/pyrophosphate ratio. Extracellular pyrophosphate can be synthesized from extracellular ATP by ecto-nucleotide pyrophosphatase/ phosphodiesterase, whereas pyrophosphate is hydrolyzed to phosphate by tissuenonspecific alkaline phosphatase, contributing to the formation of hydroxyapatite crystals. Over the last decade, vascular calcification has been the subject of numerous reviews and studies, which have revealed new agents and activities that may aid in explaining the complex physiology of this condition. This review summarizes current knowledge about alkaline phosphatase and its role in the process of vascular calcification as a key regulator of the phosphate/pyrophosphate ratio.
Topics: Alkaline Phosphatase; Humans; Vascular Calcification
PubMed: 30381085
DOI: 10.2174/1573403X14666181031141226 -
Journal of the American Chemical Society Jan 2017Chemiluminescence (CL) has a higher signal-to-noise ratio than fluorescence, but the use of CL to track an enzyme-instructed self-assembly (EISA) process has not been...
Chemiluminescence (CL) has a higher signal-to-noise ratio than fluorescence, but the use of CL to track an enzyme-instructed self-assembly (EISA) process has not been reported. In this work, by coincubation of the hydrogelator precursor Fmoc-Phe-Phe-Tyr(HPO)-OH (1P) and the CL agent AMPPD (2) with alkaline phosphatase (ALP), we employed CL to directly characterize and image the simultaneous EISA process of 1P. Hydrogelation processes of 1P with and without 2 and the CL properties of 2 with and without 1P under ALP catalysis were systematically studied. The results indicated that 2 is an ideal CL indicator for ALP-triggered hydrogelation of 1P. Using an IVIS optical imaging system, we obtained time-course CL images of 2 to track the simultaneous hydrogelation process of 1P in the same solution. We envision that our CL method could be employed to track more biological EISA events in the near future.
Topics: Alkaline Phosphatase; Biocatalysis; Gels; Kinetics; Luminescence; Molecular Conformation; Optical Imaging; Particle Size
PubMed: 28064496
DOI: 10.1021/jacs.6b11041 -
Biomolecules Nov 2021Intestinal alkaline phosphatase (IAP) is a multi-functional protein that has been demonstrated to primarily protect the gut. The role of IAP in maintaining intestinal... (Review)
Review
Intestinal alkaline phosphatase (IAP) is a multi-functional protein that has been demonstrated to primarily protect the gut. The role of IAP in maintaining intestinal homeostasis is underscored by the observation that IAP expression is defective in many gastrointestinal-related disorders such as inflammatory bowel disease IBD, necrotizing enterocolitis, and metabolic syndrome and that exogenous IAP supplementation improves the outcomes associated with these disorders. Additionally, studies using transgenic IAP-knock out (IAP-KO) mouse models further support the importance of the defensive role of IAP in the intestine. Supplementation of exogenous IAP and cellular overexpression of IAP have also been used in vitro to dissect out the downstream mechanisms of this protein in mammalian cell lines. Some of the innate immune functions of IAP include lipopolysaccharide (LPS) detoxification, protection of gut barrier integrity, regulation of gut microbial communities and its anti-inflammatory roles. A novel function of IAP recently identified is the induction of autophagy. Due to its critical role in the gut physiology and its excellent safety profile, IAP has been used in phase 2a clinical trials for treating conditions such as sepsis-associated acute kidney injury. Many excellent reviews discuss the role of IAP in physiology and pathophysiology and here we extend these to include recent updates on this important host defense protein and discuss its role in innate immunity via its effects on bacteria as well as on host cells. We will also discuss the relationship between IAP and autophagy and how these two pathways may act in concert to protect the gut.
Topics: Alkaline Phosphatase; Animals; Autophagy; Disease Models, Animal; GPI-Linked Proteins; Gastrointestinal Diseases; Gene Expression Regulation; Gene Knockout Techniques; Humans; Immunity, Innate; Mice; Mice, Transgenic
PubMed: 34944428
DOI: 10.3390/biom11121784 -
International Journal of Molecular... Oct 2022To develop new alkaline phosphatase inhibitors (ALP), a series of pyrazolo-oxothiazolidine derivatives were synthesized and biologically assessed, and the results showed...
To develop new alkaline phosphatase inhibitors (ALP), a series of pyrazolo-oxothiazolidine derivatives were synthesized and biologically assessed, and the results showed that all of the synthesized compounds significantly inhibited ALP. Specifically, compound displayed the strongest inhibitory activity (IC = 0.045 ± 0.004 μM), which is 116-fold more active than monopotassium phosphate (IC = 5.242 ± 0.472 μM) as a standard reference. The most potent compound among the series () was checked for its mode of binding with the enzyme and shown as non-competitively binding with the target enzyme. The antioxidant activity of these compounds was examined to investigate the radical scavenging effect. Moreover, the MTT assay method was performed to evaluate their toxic effects on the viability of MG-63 human osteosarcoma cells, and all compounds have no toxic effect on the cells at 4 μM. Computational research was also conducted to examine the binding affinity of the ligands with alkaline phosphatase, and the results revealed that all compounds showed good binding energy values within the active site of the target. Therefore, these novel pyrazolo-oxothiazolidine derivatives might be employed as promising pharmacophores for potent and selective alkaline phosphatase inhibitors.
Topics: Humans; Alkaline Phosphatase; Enzyme Inhibitors; Kinetics; Molecular Docking Simulation; Molecular Structure; Structure-Activity Relationship; Pyrazoles; Thiazoles
PubMed: 36362051
DOI: 10.3390/ijms232113262 -
Cells Nov 2021Human umbilical cord perivascular cells (HUCPVCs), harvested from human umbilical cord perivascular tissue, show potential for future use as an alternative to...
Human umbilical cord perivascular cells (HUCPVCs), harvested from human umbilical cord perivascular tissue, show potential for future use as an alternative to mesenchymal stromal cells. Here, we present the results for the characterization of the properties alkaline phosphatase-positive HUCPVCs (ALP(+)-HUCPVCs). These ALP(+)-HUCPVCs were created from HUCPVCs in this study by culturing in the presence of activated vitamin D3, an inhibitor of bone morphogenetic protein signaling and transforming growth factor-beta1 (TGF-β1). The morphological characteristics, cell proliferation, gene expression, and mineralization-inducing ability of ALP(+)-HUCPVCs were investigated at the morphological, biological, and genetic levels. ALP(+)-HUCPVCs possess high ALP gene expression and activity in cells and a slow rate of cell growth. The morphology of ALP(+)-HUCPVCs is fibroblast-like, with an increase in actin filaments containing alpha-smooth muscle actin. In addition to ALP expression, the gene expression levels of type I collagen, osteopontin, elastin, fibrillin-1, and cluster of differentiation 90 are increased in ALP(+)-HUCPVCs. ALP(+)-HUCPVCs do not have the ability to induce mineralization nodules, which may be due to the restriction of phosphate uptake into matrix vesicles. Moreover, ALP(+)-HUCPVCs may produce anti-mineralization substances. We conclude that ALP(+)-HUCPVCs induced from HUCPVCs by a TGF-β1 stimulation possess myofibroblast-like properties that have little mineralization-inducing ability.
Topics: Actin Cytoskeleton; Alkaline Phosphatase; Biomarkers; Calcification, Physiologic; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Cell Shape; Extracellular Matrix; Gene Expression Regulation, Enzymologic; Humans; Umbilical Cord
PubMed: 34831233
DOI: 10.3390/cells10113011 -
Protein Science : a Publication of the... Aug 2021Alkaline phosphatase (ALP), a homo-dimeric enzyme has been widely used in various bioassays as disease markers and enzyme probes. Recent advancements of digital bioassay...
Alkaline phosphatase (ALP), a homo-dimeric enzyme has been widely used in various bioassays as disease markers and enzyme probes. Recent advancements of digital bioassay revolutionized ALP-based diagnostic assays as seen in rapid growth of digital ELISA and the emerging multiplex profiling of single-molecule ALP isomers. However, the intrinsic heterogeneity found among ALP molecules hampers the ALP-based quantitative digital bioassays. This study aims quantitative analysis of single-molecule activities of ALP from Escherichia coli and reveals the static heterogeneity in catalytic activity of ALP with two distinct populations: half-active and fully-active portions. Digital assays with serial buffer exchange uncovered single-molecule Michaelis-Menten kinetics of ALP; half-active molecules have halved values of the catalytic turnover rate, k , and the rate constant of productive binding, k , of the fully active molecules. These findings suggest that half-active ALP molecules are heterogenic dimers composed of inactive and active monomer units, while fully active ALP molecules comprise two active units. Static heterogeneity was also observed for ALP with other origins: calf intestine or shrimp, showing how the findings can be generalized across species. Cell-free expression of ALP with disulfide bond enhancer and spiked zinc ion resulted in homogenous population of ALP of full activity, implying that inactive monomer units of ALP are deficient in correct disulfide bond formation and zinc ion coordination. These findings provide basis for further study on molecular mechanism and biogenesis of ALP, and also offer the way to prepare homogenous and active populations of ALP for highly quantitative and sensitive bioassays with ALP.
Topics: Alkaline Phosphatase; Biological Assay; Cell-Free System; Escherichia coli Proteins; Microscopy, Fluorescence; Single Molecule Imaging
PubMed: 33955095
DOI: 10.1002/pro.4102 -
Biochemistry Jan 2021Recent experimental studies engaging isotopically substituted protein (heavy protein) have revealed that many, but not all, enzymatic systems exhibit altered chemical...
Recent experimental studies engaging isotopically substituted protein (heavy protein) have revealed that many, but not all, enzymatic systems exhibit altered chemical steps in response to an altered mass. The results have been interpreted as femtosecond protein dynamics at the active site being linked (or not) to transition-state barrier crossing. An altered enzyme mass can influence several kinetic parameters (, , and ) in amounts of ≤30% relative to light enzymes. An early report on deuterium-labeled alkaline phosphatase (AP) showed an unusually large enzyme kinetic isotope effect on . We examined steady-state and chemical step properties of native AP, [H]AP, and [H,C,N]AP to characterize the role of heavy enzyme protein dynamics in reactions catalyzed by AP. Both [H]- and [H,C,N]APs showed unaltered steady-state and single-turnover rate constants. These findings characterize AP as one of the enzymes in which mass-dependent catalytic site dynamics is dominated by reactant-linked atomic motions. Two catalytic site zinc ions activate the oxygen nucleophiles in the catalytic site of AP. The mass of the zinc ions is unchanged in light and heavy APs. They are essentially linked to catalysis and provide a possible explanation for the loss of linkage between catalysis and protein mass in these enzymes.
Topics: Alkaline Phosphatase; Binding Sites; Catalysis; Catalytic Domain; Escherichia coli; Escherichia coli Proteins; Kinetics; Models, Molecular; Protein Conformation
PubMed: 33410323
DOI: 10.1021/acs.biochem.0c00917