Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The mechanism of hydrolysis of beta-glycerophosphate by kidney alkaline phosphatase.

ABSTRACT: 1. To identify the functional groups that are involved in the conversion of beta-glycerophosphate by alkaline phosphatase (EC 3.1.3.1) from pig kidney, the kinetics of alkaline phosphatase were investigated in the pH range 6.6-10.3 at substrate concentrations of 3 muM-30 mM. From the plots of log VH+ against pH and log VH+/KH+m against pH one functional group with pK = 7.0 and two functional groups with pK = 9.1 were identified. These groups are involved in substrate binding. Another group with pK = 8.8 was found, which in its unprotonated form catalyses substrate conversion. 2. GSH inhibits the alkaline phosphatase reversibly and non-competitively by attacking the bound Zn(II). 3. The influence of the H+ concentration on the activation by Mg2+ ions of alkaline pig kidney phosphate was investigated between pH 8.4 and 10.0. The binding of substrate and activating Mg2+ ions occurs independently at all pH values between 8.4 and 10.0. The activation mechanism is not affected by the H+ concentration. The Mg2+ ions are bound by a functional group with a pK of 10.15. 4. A scheme is proposed for the reaction between enzyme, substrate, Mg2+ and H+ and the overall rate equation is derived. 5. The mechanism of substrate binding and splitting by the functional groups of the active centre is discussed on the basis of a model. Mg2+ seems to play a role as an autosteric effector.

SUBMITTER: Ahlers J

PROVIDER: S-EPMC1165659 | biostudies-other | 1975 Sep

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Kinetics of alkaline phosphatase from pig kidney. Mechanism of activation by magnesium ions.

Project description:The mechanism of activation of alkaline phosphatase (EC 3.1.3.1) from pig kidney by Mg(2+) ions was investigated with the aid of kinetic measurements. Mg(2+) ions are essential for enzyme activity. The following model (Scheme 1 of the text) for the reaction of enzyme, substrate and Mg(2+) ions was derived: [Formula: see text] The binding of the substrate to the enzyme is independent of the binding of the activator, and vice versa. Mg(2+) must therefore play a part in the substrate decomposition. It is not possible to determine whether the Mg(2+) ions are involved directly in the catalytic process, or whether they act as regulatory effectors. Because of the strong affinity existing between the alkaline phosphatase and Mg(2+), it is necessary to adjust the metal-ion concentration with the aid of a metal buffer. In the Appendix the necessary equations are derived for calculating the concentration of free metal ions in a system with several different metal ions. A FORTRAN IV program for solving these equations and for graphic presentation of the results has been deposited as Supplementary Publication SUP 50030 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS 23 7 BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1973), 131, 5.

| S-EPMC1168072 | biostudies-other

Pharmacologic epigenetic modulators of alkaline phosphatase in chronic kidney disease.

Project description:PURPOSE OF REVIEW:In chronic kidney disease (CKD), disturbance of several metabolic regulatory mechanisms cause premature ageing, accelerated cardiovascular disease (CVD), and mortality. Single-target interventions have repeatedly failed to improve the prognosis for CKD patients. Epigenetic interventions have the potential to modulate several pathogenetic processes simultaneously. Alkaline phosphatase (ALP) is a robust predictor of CVD and all-cause mortality and implicated in pathogenic processes associated with CVD in CKD. RECENT FINDINGS:In experimental studies, epigenetic modulation of ALP by microRNAs or bromodomain and extraterminal (BET) protein inhibition has shown promising results for the treatment of CVD and other chronic metabolic diseases. The BET inhibitor apabetalone is currently being evaluated for cardiovascular risk reduction in a phase III clinical study in high-risk CVD patients, including patients with CKD (ClinicalTrials.gov Identifier: NCT02586155). Phase II studies demonstrate an ALP-lowering potential of apabetalone, which was associated with improved cardiovascular and renal outcomes. SUMMARY:ALP is a predictor of CVD and mortality in CKD. Epigenetic modulation of ALP has the potential to affect several pathogenetic processes in CKD and thereby improve cardiovascular outcome.

| S-EPMC6903384 | biostudies-literature

Catalytic properties of alkaline phosphatase from pig kidney.

Project description:The enzymic properties of alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes were studied. 1. It hydrolyses ortho- and pyro-phosphate esters, the rate limiting step (V(max.)) being independent of the substrate. It transphosphorylates to Tris at concentrations above 0.1m-Tris. 2. The pH optimum for hydrolysis was between 9.8 and 10. The pK of the enzyme-substrate complex is 8.7 for p-nitrophenyl phosphate and beta-glycerophosphate. Excess of substrate inhibits the enzymic activity with decreasing pH. The pK of the substrate-inhibited enzyme-substrate complex, 8.7, is very similar to that for the enzyme-substrate complex. The pK values of the free enzyme appear to be 8.7 and 7.9. 3. Inactivation studies suggest that there is an essential tyrosine residue at the active centre of the enzyme. 4. The energy of activation (E) and the heat of activation (DeltaH) at pH9.5 showed a transition at 24.8 degrees C that was unaffected by Mg(2+). 5. Kinetic and atomic-absorption analysis indicated the essential role of two Zn(2+) ions/tetrameric enzyme for an ordered association of the monomers. Zn(2+) in excess and other bivalent ions compete for a second site with Mg(2+). Mg(2+) enhances only the rate-limiting step of substrate hydrolysis. 6. Amino acid inhibition studies classified the pig kidney enzyme as an intermediate type of previously described alkaline phosphatases. It has more similarity with the enzyme from liver and bone than with that from placenta.

| S-EPMC1168075 | biostudies-other

A carbon dot-based Co-nanozyme with alkaline phosphatase - mechanism and application.

Project description:Elevated levels of alkaline phosphatase (ALP) are associated with bone metastasis, liver cancer, prostate cancer, breast cancer, and many other diseases or stem cell marker. It is therefore of great significance to quantitatively detect the ALP levels by a rapid, highly sensitive, and easy-to-use strip paper test. In the present work, we discovered an enhancement of ALP activity upon the addition of cauliflower-derived carbon dots (CFCDs), which can be applied as a sensor for ALP. The mixed ALP and CFCDs exhibited a typical Michaelis Menten mechanism with increased V max and reduced K m compared to ALP alone. High-Resolution Atomic Force Microscopy (HR-AFM) reveals the dimensions of ALP, the CFCDs, and the phosphatase substrate para-nitrophenyl phosphate (pNPP), as well as the potential interactions among them. The role of the CFCDs was identified as the addition of reaction centers to ALP; in other words, a competitive activator. Besides the improved kinetics, the yield of dephosphorylated product was also increased by at least twice upon the addition of CFCDs. Taking advantage of this effect, a portable CFCD-based paper strip assay was developed to achieve sensitive detection of abnormally elevated ALP levels and visualization of cancer stem cells or proteins by phosphatase-conjugated antibodies. Our findings show great promise for disease diagnosis and bioassays related to ALP enhancement that may be used for protein or cell detection.

| S-EPMC9042285 | biostudies-literature

Nucleoside pyrophosphatase activity associated with pig kidney alkaline phosphatase.

Project description:1. A study was made of the hydrolysis, at pH9.0, of ATP and ADP catalysed by pig kidney alkaline phosphatase. Both of these nucleoside pyrophosphates are substrates for the enzyme; K(m) values are 4x10(-5)m for ATP and 6.3x10(-5)m for ADP. V(max.) for ADP is approximately double that of ATP. 2. Above 0.1mm approximately, both ATP and ADP are inhibitory, but the inhibition is reversible by the addition of Mg(2+) ions to form MgATP(2-) or MgADP(-) complexes. The complexes, besides being non-inhibitory, are also substrates for the enzyme with K(m) values identical with those of the respective free nucleotides. 3. Mg(2+) ions are inhibitory when present in excess of ATP or ADP. The degree of inhibition is greater with ATP as substrate, but with both ATP and ADP a mixed competitive-non-competitive type of inhibition is observed. 4. It is suggested that under normal conditions the enzyme is inhibited by cellular concentrations of ATP plus ADP but that an increase in the concentration of Mg(2+) ions stimulates activity by relieving nucleoside pyrophosphate inhibition. The properties may be of importance in the regulation of the transport of bivalent cations.

| S-EPMC1177276 | biostudies-other

Alkaline phosphatase: an overview.

Project description:Alkaline phosphatase (ALP; E.C.3.I.3.1.) is an ubiquitous membrane-bound glycoprotein that catalyzes the hydrolysis of phosphate monoesters at basic pH values. Alkaline phosphatase is divided into four isozymes depending upon the site of tissue expression that are Intestinal ALP, Placental ALP, Germ cell ALP and tissue nonspecific alkaline phosphatase or liver/bone/kidney (L/B/K) ALP. The intestinal and placental ALP loci are located near the end of long arm of chromosome 2 and L/B/K ALP is located near the end of the short arm of chromosome 1. Although ALPs are present in many mammalian tissues and have been studied for the last several years still little is known about them. The bone isoenzyme may be involved in mammalian bone calcification and the intestinal isoenzyme is thought to play a role in the transport of phosphate into epithelial cells of the intestine. In this review, we tried to provide an overview about the various forms, structure and functions of alkaline phosphatase with special focus on liver/bone/kidney alkaline phosphatase.

| S-EPMC4062654 | biostudies-literature

Alkaline Phosphatase and Hypophosphatasia.

Project description: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.

| S-EPMC4824800 | biostudies-literature

Molecular aspects of the regulation of rat kidney alkaline phosphatase.

Project description:The mechanisms by which phosphate regulates the activity of alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) in rat kidney were investigated. Measurements of incorporation of [(14)C]leucine into kidney alkaline phosphatase in rats fed on complete or phosphate-free diet provide evidence of a twofold increase in the rate of synthesis of the enzyme in diet-treated animals. Cycloheximide experiments indicated that control and diet-adapted enzyme decreases in activity according to first-order kinetics with a calculated half-life of 10.3 and 6.5h after complete and phosphate-free diet administration respectively. Basal and diet-adapted enzymes exhibit similar K(m) values for several phosphomonoesters and an identical degree of inhibition is produced by cysteine. In addition, the enzyme from both sources is the same with regard to heat inactivation at 45, 56 or 64 degrees C, to the profile of elution from Sephadex and to electrophoretic properties on polyacrylamide gel. A failure of rat kidney alkaline phosphatase to respond to cortisol (hydrocortisone) was also observed.

| S-EPMC1176483 | biostudies-other

Mutationally altered rate constants in the mechanism of alkaline phosphatase.

Project description:The hydrolysis of phosphate esters by a mutationally altered alkaline phosphatase from Escherichia coli was studied by both steady-state and transient-kinetic methods. The difference between the catalytic-centre activities of the mutationally altered and the wild-type alkaline phosphatases was found to vary with pH and at optimal pH values the modified enzyme had the higher activity. Stopped-flow experiments at acidic pH values showed that transient product formation by the mutationally altered enzyme was faster than that with the wild-type enzyme whereas the rate of the steady state was slower. In the alkaline pH region, the transient was observed in the reaction of only the modified enzyme and not the wild type. These observations permit a fuller characterization of the individual steps in the catalytic mechanism of alkaline phosphatase than is possible by study of only the wild-type enzyme.

| S-EPMC1168190 | biostudies-other

Alkaline ceramidase catalyzes the hydrolysis of ceramides via a catalytic mechanism shared by Zn2+-dependent amidases.

Project description:Human alkaline ceramidase 3 (ACER3) is one of three alkaline ceramidases (ACERs) that catalyze the conversion of ceramide to sphingosine. ACERs are members of the CREST superfamily of integral-membrane hydrolases. All CREST members conserve a set of three Histidine, one Aspartate, and one Serine residue. Although the structure of ACER3 was recently reported, catalytic roles for these residues have not been biochemically tested. Here, we use ACER3 as a prototype enzyme to gain insight into this unique class of enzymes. Recombinant ACER3 was expressed in yeast mutant cells that lack endogenous ceramidase activity, and microsomes were used for biochemical characterization. Six-point mutants of the conserved CREST motif were developed that form a Zn-binding active site based on a recent crystal structure of human ACER3. Five point mutants completely lost their activity, with the exception of S77A, which showed a 600-fold decrease compared with the wild-type enzyme. The activity of S77C mutant was pH sensitive, with neutral pH partially recovering ACER3 activity. This suggested a role for S77 in stabilizing the oxyanion of the transition state. Together, these data indicate that ACER3 is a Zn2+-dependent amidase that catalyzes hydrolysis of ceramides via a similar mechanism to other soluble Zn-based amidases. Consistent with this notion, ACER3 was specifically inhibited by trichostatin A, a strong zinc chelator.

| S-EPMC9436119 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data