The relationship between the optical properties and the kinetic behaviour of ascorbate-inhibited alkaline phosphatase.
Ontology highlight
ABSTRACT:
SUBMITTER: Martorana GE
PROVIDER: S-EPMC1147472 | biostudies-other | 1986 Dec
REPOSITORIES: biostudies-other
Similar Datasets
Project description:1. The effects of varying pH, ionic strength and temperature on the parameters K(m) and V(max.) for a purified alkaline phosphatase from calf intestinal mucosa with a new fluorogenic substrate, 4-methylumbelliferyl phosphate monoester disodium salt, and an ammediol-hydrochloric acid buffer system were determined. 2. It was found that, under varying conditions, a relationship exists between K(m) and V(max.) such that V(max.)=beta/(1+alpha/K(m)), where alpha and beta are constants, temperature- and ionic strength-dependent, but pH-independent. It is shown that this relationship accounts satisfactorily for the well-known effect of varying substrate concentration on optimum pH and velocity. 3. The various results are interpreted in terms of a pH-dependent conformational equilibrium between two forms of the enzyme, E(1) and E(2). Only E(1) combines with substrate, and only E(2) reacts to give inorganic phosphate. 4. To account for the pH-variation of K(m) and V(max.) in terms of this theory, it is postulated that the conformational change is associated with a change in pK of two basic groups in the enzyme.
| S-EPMC1264548 | biostudies-other
Project description:Studies on phosphatase activity of Streptomyces fradiae 3535 grown in three different media indicate that neomycin formation varies directly with enzyme activity, sodium nitrate-maltose-mineral salts medium giving the highest yields of alkaline phosphatase and neomycin. S. fradiae contains more than one alkaline phosphatase and the phosphatase responsible for hydrolysis of neomycin phosphate appears to be substrate specific. The same enzyme apparently hydrolyses both the N-P and P-O-P bonds of neomycin pyrophosphate. The enzyme is stimulated by Ca(2+), is inactive at a pH below 7 and is inhibited by EDTA. Enzymic activity increases when mycelia are incubated in mineral salts medium, but decreases when phosphate or glucose is included in the medium, although the latter is more effective. The inhibitory effect of EDTA on neomycin formation by resting mycelia is completely reversed by Ca(2+).
| S-EPMC1176793 | biostudies-other
Project description:1. The phosphorylation of milk alkaline phosphatase was studied under various conditions: maximum incorporation occurred at pH5.0 and 50% incorporation at pH6.6-7.0. 2. The phosphorylation was shown to be specific and the results suggest that the active centre of the enzyme is involved in the process. 3. Phosphoryl-enzyme is rapidly hydrolysed at alkaline pH. at pH7.0 the results suggest that a phosphoryl-enzyme could occur as a transient intermediate in the hydrolysis of phosphate esters by the phosphatase. 4. The catalytic-centre activity of the enzyme was found to be 2700sec.(-1) at pH10.0 and 25 degrees with p-nitrophenyl phosphate as substrate.
| S-EPMC1270129 | biostudies-other
Project description:Renal hyperfiltration, which is associated with renal injury, occurs in diabetic or obese individuals. Serum alkaline phosphatase (ALP) level is also elevated in patients with diabetes (DM) or metabolic syndrome (MS), and increased urinary excretion of ALP has been demonstrated in patients who have hyperfiltration and tubular damage. However, little was investigated about the association between hyperfiltration and serum ALP level. A retrospective observational study of the 21,308 adults in the Korea National Health and Nutrition Examination Survey IV-V databases (2008-2011) was performed. Renal hyperfiltration was defined as exceeding the age- and sex-specific 97.5th percentile. We divided participants into 4 groups according to their estimated glomerular filtration rate (eGFR): >120, 90-119, 60-89, and <60 mL/min/1.73 m2. The participants with eGFR >120 mL/min/1.73 m2 showed the highest risk for MS, in the highest ALP quartiles (3.848, 95% CI, 1.876-7.892), compared to the lowest quartile. Similarly, the highest risk for DM, in the highest ALP quartiles, was observed in participants with eGFR >120 ml/min/1.73 m2 (2.166, 95% CI, 1.084-4.329). ALP quartiles were significantly associated with albuminuria in participants with eGFR ? 60 ml/min/1.73m2. The highest ALP quartile had a 1.631-fold risk elevation for albuminuria with adjustment of age and sex. (95% CI, 1.158-2.297, P = 0.005). After adjustment, the highest ALP quartile had a 1.624-fold risk elevation, for renal hyperfiltration (95% CI, 1.204-2.192, P = 0.002). In addition, hyperfiltration was significantly associated with hemoglobin, triglyceride, white blood cell count, DM, smoking, and alcohol consumption (P<0.05). The relationship between serum ALP and metabolic disorders is stronger in participants with an upper-normal range of eGFR. Higher ALP levels are significantly associated with renal hyperfiltration in Korean general population.
| S-EPMC4390244 | biostudies-literature
Project description:1. The stability of the tetrameric form of Escherichia coli alkaline phosphatase was examined by analytical ultracentrifugation. 2. The stopped-flow technique was used to study the hydrolysis of nitrophenyl phosphates by the alkaline phosphatase tetramer at pH7.5 and 8.3. In both cases transient product formation was observed before the steady state was attained. Both transients consisted of the liberation of 1mol of nitrophenol/2mol of enzyme subunits within the dead-time of the apparatus. The steady-state rates were identical with those observed with the dimer under the same conditions. 3. The binding of 2-hydroxy-5-nitrobenzyl phosphonate to the alkaline phosphatase tetramer was studied by the temperature-jump technique. The self-association of two dimers to form the tetramer is linked to a conformation change within the dimer. This accounts for the differences between the transient phases in the reactions of the dimer and the tetramer with substrate. 4. Addition of P(i) to the alkaline phosphatase tetramer caused it to dissociate into dimers. The tetramer is unable to bind this ligand. It is suggested that the tetramer undergoes a compulsory dissociation before the completion of its first turnover with substrate. 5. On the basis of these findings a mechanism is proposed for the involvement of the alkaline phosphatase tetramer in the physiology of E. coli.
| S-EPMC1178530 | biostudies-other
Project description:Intestinal alkaline phosphatase (IAP) plays an essential role in intestinal homeostasis and health through interactions with the resident microbiota, diet and the gut. IAP's role in the intestine is to dephosphorylate toxic microbial ligands such as lipopolysaccharides, unmethylated cytosine-guanosine dinucleotides and flagellin as well as extracellular nucleotides such as uridine diphosphate. IAP's ability to detoxify these ligands is essential in protecting the host from sepsis during acute inflammation and chronic inflammatory conditions such as inflammatory bowel disease. Also important in these complications is IAP's ability to regulate the microbial ecosystem by forming a complex relationship between microbiota, diet and the intestinal mucosal surface. Evidence reveals that diet alters IAP expression and activity and this in turn can influence the gut microbiota and homeostasis. IAP's ability to maintain a healthy gastrointestinal tract has accelerated research on its potential use as a therapeutic agent against a multitude of diseases. Exogenous IAP has been shown to have beneficial effects when administered during ulcerative colitis, coronary bypass surgery and sepsis. There are currently a handful of human clinical trials underway investigating the effects of exogenous IAP during sepsis, rheumatoid arthritis and heart surgery. In light of these findings IAP has been marked as a novel agent to help treat a variety of other inflammatory and infectious diseases. The purpose of this review is to highlight the essential characteristics of IAP in protection and maintenance of intestinal homeostasis while addressing the intricate interplay between IAP, diet, microbiota and the intestinal epithelium.
| S-EPMC4229529 | biostudies-literature
Project description:Behaviour is derived from complex molecular interactions within the brain, in response to specific environmental stimuli. In some rare cases, the direct causes of behaviour have been attributed to the interactions of a single or small group of gene transcripts and proteins. We conducted two experiments with the hope of defining some of the molecular interactors for four separate behaviours: sugar feeding, locomotor activity in a novel field, and acoustic startle reflex, and prepulse inhibition of the acoustic startle reflex, which have been linked to prefrontal cortex dopaminergic function or as predictors of sensorimotor gating in diseases such as schizophrenia. Rats with high and low response phenotypes were selected to determine the differences between these two extremes of behaviour. From our analyses, transcripts of Homer1, a neuronal scaffolding protein which interacts with group1 metabotropic glutamate receptors, were found to be significantly correlated with array data in both experiments, and with behaviour data across three separate tests in the second experiment, indicating that this gene's transcripts and probably downstream protein interactions have a significant correlation with behaviour phenotype in the inbred Lewis rat. Future areas of pursuit for this data should involve modification of the expression of Homer1 in an isolated fashion to determine a pharmacological threshold for differences in behaviour. This SuperSeries is composed of the SubSeries listed below.
2009-11-24 | GSE14904 | GEO
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
Project description:Alkaline phosphatase (ALP), which catalyzes the dephosphorylation process of proteins, nucleic acids, and small molecules, can be found in a variety of tissues (intestine, liver, bone, kidney, and placenta) of almost all living organisms. This enzyme has been extensively used as a biomarker in enzyme immunoassays and molecular biology. ALP is also one of the most commonly assayed enzymes in routine clinical practice. Due to its close relation to a variety of pathological processes, ALP's abnormal level is an important diagnostic biomarker of many human diseases, such as liver dysfunction, bone diseases, kidney acute injury, and cancer. Therefore, the development of convenient and reliable assay methods for monitoring ALP activity/level is extremely important and valuable, not only for clinical diagnoses but also in the area of biomedical research. This paper comprehensively reviews the strategies of optical and electrochemical detection of ALP and discusses the electrochemical techniques that have been addressed to make them suitable for ALP analysis in cell culture.
| S-EPMC6784369 | biostudies-literature
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