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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 -
PloS One 2016Decreased alkaline phosphatase activity after infant cardiac surgery is associated with increased post-operative cardiovascular support requirements. In adults...
RATIONALE
Decreased alkaline phosphatase activity after infant cardiac surgery is associated with increased post-operative cardiovascular support requirements. In adults undergoing coronary artery bypass grafting, alkaline phosphatase infusion may reduce inflammation. Mechanisms underlying these effects have not been explored but may include decreased conversion of extracellular adenine nucleotides to adenosine.
OBJECTIVES
1) Evaluate the association between alkaline phosphatase activity and serum conversion of adenosine monophosphate to adenosine after infant cardiac surgery; 2) assess if inhibition/supplementation of serum alkaline phosphatase modulates this conversion.
METHODS AND RESEARCH
Pre/post-bypass serum samples were obtained from 75 infants <4 months of age. Serum conversion of 13C5-adenosine monophosphate to 13C5-adenosine was assessed with/without selective inhibition of alkaline phosphatase and CD73. Low and high concentration 13C5-adenosine monophosphate (simulating normal/stress concentrations) were used. Effects of alkaline phosphatase supplementation on adenosine monophosphate clearance were also assessed. Changes in serum alkaline phosphatase activity were strongly correlated with changes in 13C5-adenosine production with or without CD73 inhibition (r = 0.83; p<0.0001). Serum with low alkaline phosphatase activity (≤80 U/L) generated significantly less 13C5-adenosine, particularly in the presence of high concentration 13C5-adenosine monophosphate (10.4μmol/L vs 12.9μmol/L; p = 0.0004). Inhibition of alkaline phosphatase led to a marked decrease in 13C5-adenosine production (11.9μmol/L vs 2.7μmol/L; p<0.0001). Supplementation with physiologic dose human tissue non-specific alkaline phosphatase or high dose bovine intestinal alkaline phosphatase doubled 13C5-adenosine monophosphate conversion to 13C5-adenosine (p<0.0001).
CONCLUSIONS
Alkaline phosphatase represents the primary serum ectonucleotidase after infant cardiac surgery and low post-operative alkaline phosphatase activity leads to impaired capacity to clear adenosine monophosphate. AP supplementation improves serum clearance of adenosine monophosphate to adenosine. These findings represent a potential therapeutic mechanism for alkaline phosphatase infusion during cardiac surgery.
NEW AND NOTEWORTHY
We identify alkaline phosphatase (AP) as the primary soluble ectonucleotidase in infants undergoing cardiopulmonary bypass and show decreased capacity to clear AMP when AP activity decreases post-bypass. Supplementation of AP ex vivo improves this capacity and may represent the beneficial therapeutic mechanism of AP infusion seen in phase 2 studies.
Topics: Adenosine; Adenosine Monophosphate; Alkaline Phosphatase; Analysis of Variance; Animals; Cardiopulmonary Bypass; Cattle; Chi-Square Distribution; Extracellular Space; Female; Humans; Infant; Infant, Newborn; Male; Postoperative Period; Prospective Studies; Solubility
PubMed: 27384524
DOI: 10.1371/journal.pone.0158981 -
Journal of Nutritional Science and... 2022Intestinal-type alkaline phosphatase (IAP) is expressed at a high concentration in the brush border membrane of intestinal epithelial cells and is known to be a gut...
Intestinal-type alkaline phosphatase (IAP) is expressed at a high concentration in the brush border membrane of intestinal epithelial cells and is known to be a gut mucosal defense factor. In humans, a single gene (ALPI) for IAP has been isolated, and its transcription produces two kinds of alternatively spliced mRNAs (aAug10 and bAug10). Recently, we discovered that vitamin D up-regulated the expression of both types of human IAP alternative splicing variants in Caco-2 cells. However, the functional difference of protein encoded by the mRNA variants has remained elusive. In the present study, we aimed to provide further insight into the characterization and structure of IAP isoforms. To analyze the protein translated from the ALPI gene, we constructed two kinds of cDNA expression plasmids (aAug10 and bAug10), and the transfected cells were homogenized and assayed for alkaline phosphatase (ALP) activity. We also designed the homology-modeled 3D structures of the protein encoded by the mRNA variants (ALPI-aAug10 and ALPI-bAug10). The levels of ALP activity of COS-1 cells transfected with the aAug10 plasmid were increased significantly, while cells transfected with the bAug10 plasmid had undetectable ALP activity. The homology-modeled 3D structures revealed that the variant bAug10 lacks the central N-terminal α-helix and residue corresponding to Asp-42 of ALPI-aAug10 near the active site. This is the first report on the characterization and structure of alternatively spliced transcript variants of the human ALPI gene. Further studies on the regulation of aAug10 and/or bAug10 mRNA expression may identify novel physiological functions of IAP.
Topics: Alkaline Phosphatase; Alternative Splicing; Caco-2 Cells; Epithelial Cells; GPI-Linked Proteins; Humans; Intestines; RNA, Messenger
PubMed: 36047100
DOI: 10.3177/jnsv.68.284 -
Current Opinion in Structural Biology Apr 2016Catalytic promiscuity, that is, the ability of single enzymes to facilitate the turnover of multiple, chemically distinct substrates, is a widespread phenomenon that... (Review)
Review
Catalytic promiscuity, that is, the ability of single enzymes to facilitate the turnover of multiple, chemically distinct substrates, is a widespread phenomenon that plays an important role in the evolution of enzyme function. Additionally, such pre-existing multifunctionality can be harnessed in artificial enzyme design. The members of the alkaline phosphatase superfamily have served extensively as both experimental and computational model systems for enhancing our understanding of catalytic promiscuity. In this Opinion, we present key recent computational studies into the catalytic activity of these highly promiscuous enzymes, highlighting the valuable insight they have provided into both the molecular basis for catalytic promiscuity in general, and its implications for the evolution of phosphatase activity.
Topics: Alkaline Phosphatase; Catalysis; Catalytic Domain; Static Electricity; Structure-Activity Relationship
PubMed: 26716576
DOI: 10.1016/j.sbi.2015.11.008 -
International Journal of Clinical... Feb 2020To assess the prognostic value of alkaline phosphatase in patients with hormone-sensitive prostate cancer. (Meta-Analysis)
Meta-Analysis
PURPOSE
To assess the prognostic value of alkaline phosphatase in patients with hormone-sensitive prostate cancer.
METHODS
A systematic review and meta-analysis was performed using the PUBMED, Web of Science, Cochrane Library, and Scopus in April 2019 according to the Preferred Reporting Items for Systematic Review and Meta-analysis statement. Studies were deemed eligible if they compared hormone-sensitive prostate cancer patients with high vs. low alkaline phosphatase to determine its predictive value for overall survival, cancer-specific survival, and progression-free survival. We performed a formal meta-analysis of these outcomes.
RESULTS
42 articles with 7938 patients were included in the systematic review and 28 studies with 5849 patients for the qualitative assessment. High alkaline phosphatase was associated with worse overall survival (pooled HR 1.72; 95% CI 1.37-2.14) and progression-free survival (pooled HR 1.30; 95% CI 1.10-1.54). In subgroup analyses of patients with "high-volume" and "low-volume", alkaline phosphatase was associated with the overall survival (pooled HR 1.41; 95% CI 1.21-1.64 and pooled HR 1.64; 95% CI, 1.06-2.52, respectively).
CONCLUSIONS
In this meta-analysis, elevated serum levels of alkaline phosphatase were associated with an increased risk of overall mortality and disease progression in patients with hormone-sensitive prostate cancer. In contrast, those were not associated with an increased risk of cancer-specific mortality. Alkaline phosphatase was independently associated with overall survival in both patients with "high-volume" and "low-volume" hormone-sensitive prostate cancer. Alkaline phosphatase may be useful for being integrated into prognostic tools that help guide treatment strategy, thereby facilitating the shared decision making process.
Topics: Alkaline Phosphatase; Biomarkers, Tumor; Hormones; Humans; Male; Prognosis; Prostatic Neoplasms; Survival Analysis
PubMed: 31768692
DOI: 10.1007/s10147-019-01578-9 -
American Journal of Physiology.... May 2014
Topics: Alkaline Phosphatase; Animals; Intestines
PubMed: 24674777
DOI: 10.1152/ajpgi.00435.2013 -
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 -
The ISME Journal Jul 2021Adaptation of cell populations to environmental changes is mediated by phenotypic variability at the single-cell level. Enzyme activity is a key factor in cell phenotype...
Adaptation of cell populations to environmental changes is mediated by phenotypic variability at the single-cell level. Enzyme activity is a key factor in cell phenotype and the expression of the alkaline phosphatase activity (APA) is a fundamental phytoplankton strategy for maintaining growth under phosphate-limited conditions. Our aim was to compare the APA among cells and species revived from sediments of the Bay of Brest (Brittany, France), corresponding to a pre-eutrophication period (1940's) and a beginning of a post-eutrophication period (1990's) during which phosphate concentrations have undergone substantial variations. Both toxic marine dinoflagellate Alexandrium minutum and the non-toxic dinoflagellate Scrippsiella acuminata were revived from ancient sediments. Using microfluidics, we measured the kinetics of APA at the single-cell level. Our results indicate that all S. acuminata strains had significantly higher APA than A. minutum strains. For both species, the APA in the 1990's decade was significantly lower than in the 1940's. For the first time, our results reveal both inter and intraspecific variabilities of dinoflagellate APA and suggest that, at a half-century timescale, two different species of dinoflagellate may have undergone similar adaptative evolution to face environmental changes and acquire ecological advantages.
Topics: Alkaline Phosphatase; Dinoflagellida; Eutrophication; France; Phytoplankton
PubMed: 33568788
DOI: 10.1038/s41396-021-00904-2 -
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 -
International Journal of Molecular... Jan 2021Tissue nonspecific alkaline phosphatase (TNAP/) is associated with cell stemness; however, the function of TNAP in mesenchymal progenitor cells remains largely unknown....
Tissue nonspecific alkaline phosphatase (TNAP/) is associated with cell stemness; however, the function of TNAP in mesenchymal progenitor cells remains largely unknown. In this study, we aimed to establish an essential role for TNAP in bone and muscle progenitor cells. We investigated the impact of TNAP deficiency on bone formation, mineralization, and differentiation of bone marrow stromal cells. We also pursued studies of proliferation, mitochondrial function and ATP levels in TNAP deficient bone and muscle progenitor cells. We find that TNAP deficiency decreases trabecular bone volume fraction and trabeculation in addition to decreased mineralization. We also find that mice (global TNAP knockout mice) exhibit muscle and motor coordination deficiencies similar to those found in individuals with hypophosphatasia (TNAP deficiency). Subsequent studies demonstrate diminished proliferation, with mitochondrial hyperfunction and increased ATP levels in TNAP deficient bone and muscle progenitor cells, plus intracellular expression of TNAP in TNAP+ cranial osteoprogenitors, bone marrow stromal cells, and skeletal muscle progenitor cells. Together, our results indicate that TNAP functions inside bone and muscle progenitor cells to influence mitochondrial respiration and ATP production. Future studies are required to establish mechanisms by which TNAP influences mitochondrial function and determine if modulation of TNAP can alter mitochondrial respiration in vivo.
Topics: Adenosine Triphosphate; Alkaline Phosphatase; Animals; Bone and Bones; Calcification, Physiologic; Cell Differentiation; Cell Respiration; Male; Mesenchymal Stem Cells; Mice; Mice, Knockout; Mitochondria; Muscle, Skeletal; Osteogenesis; Skull
PubMed: 33498907
DOI: 10.3390/ijms22031140