Specific Detection and Properties of Enzyme Hydrolyzing Phosphonate Ester in Serum

1992 ◽  
Vol 38 (3) ◽  
pp. 377-380 ◽  
Author(s):  
G Y Han ◽  
X H Fan ◽  
X B Jin ◽  
D P Wang
Keyword(s):  
Alkaline Phosphatase ◽  
Human Serum ◽  
Maximum Activity ◽  
Enzymic Activity ◽  
Specific Detection ◽  
Phenylphosphonic Acid ◽  
Nitrophenyl Phosphate ◽  
Optimum Ph ◽  
Km Value ◽  
C 30

Abstract An enzyme capable of hydrolyzing 4-methylumbelliferyl phenylphosphonate to 4-methylumbelliferone and phenylphosphonic acid has been detected in human serum. It has a Km value of 1.72 x 10(-4) mol/L, has an optimum pH of 8.8-9.1 in Tris buffer, and shows maximum activity at 60 degrees C (30 min). The enzymic activity can be inhibited by Na3PO4, EDTA, and cysteine. We saw no effect of CuSO4, adenosine, thymidine, NaN3, diethyl p-nitrophenyl phosphate, p-chloromercuribenzoate, isopropyl fluorophosphate, or eserine on the enzymic activity. The enzyme cannot hydrolyze substrates of phosphodiesterase I or alkaline phosphatase. The enzyme is considered a phosphonate esterase.

A reference method for measurement of alkaline phosphatase activity in human serum.

1983 ◽  
Vol 29 (5) ◽  
pp. 751-761 ◽  
Author(s):  
N W Tietz ◽  
C A Burtis ◽  
P Duncan ◽  
K Ervin ◽  
C J Petitclerc ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Human Serum ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Zinc Sulfate ◽  
Metal Ion ◽  
Reference Method ◽  
Reaction Conditions ◽  
Factors Affecting ◽  
Nitrophenyl Phosphate

Abstract We present an official AACC reference method for the measurement of alkaline phosphatase, the culmination of optimization experiments conducted by a group of independent laboratories. The details of this method and evaluation of factors affecting the measurement are described. A metal ion buffer has been incorporated that maintains optimal and constant concentrations of zinc(II) and magnesium(II) ions. Final reaction conditions are: pH (30 degrees C), 10.40 +/- 0.05; 2-amino-2-methyl-1-propanol buffer, 0.35 mol/L; 4-nitrophenyl phosphate, 16.0 mmol/L; magnesium acetate, 2.0 mmol/L; zinc sulfate, 1.0 mmol/L; and N-(2-hydroxyethyl)ethylenediaminetriacetic acid, 2.0 mmol/L.

scholarly journals A Novel Alkaline Phosphatase/Phosphodiesterase, CamPhoD, from Marine Bacterium Cobetia amphilecti KMM 296

Marine Drugs ◽  
2019 ◽  
Vol 17 (12) ◽  
pp. 657 ◽  
Author(s):  
Yulia Noskova ◽  
Galina Likhatskaya ◽  
Natalia Terentieva ◽  
Oksana Son ◽  
Liudmila Tekutyeva ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Alkaline Phosphatase ◽  
Marine Bacterium ◽  
Inorganic Phosphorus ◽  
Maximum Activity ◽  
Appreciable Effect ◽  
Structural Protein ◽  
Calculated Molecular Weight ◽  
Nitrophenyl Phosphate ◽  
Menten Constant

A novel extracellular alkaline phosphatase/phosphodiesterase from the structural protein family PhoD that encoded by the genome sequence of the marine bacterium Cobetia amphilecti KMM 296 (CamPhoD) has been expressed in Escherichia coli cells. The calculated molecular weight, the number of amino acids, and the isoelectric point (pI) of the mature protein’s subunit are equal to 54832.98 Da, 492, and 5.08, respectively. The salt-tolerant, bimetal-dependent enzyme CamPhoD has a molecular weight of approximately 110 kDa in its native state. CamPhoD is activated by Co2+, Mg2+, Ca2+, or Fe3+ at a concentration of 2 mM and exhibits maximum activity in the presence of both Co2+ and Fe3+ ions in the incubation medium at pH 9.2. The exogenous ions, such as Zn2+, Cu2+, and Mn2+, as well as chelating agents EDTA and EGTA, do not have an appreciable effect on the CamPhoD activity. The temperature optimum for the CamPhoD activity is 45 °C. The enzyme catalyzes the cleavage of phosphate mono- and diester bonds in nucleotides, releasing inorganic phosphorus from p-nitrophenyl phosphate (pNPP) and guanosine 5′-triphosphate (GTP), as determined by the Chen method, with rate approximately 150- and 250-fold higher than those of bis-pNPP and 5′-pNP-TMP, respectively. The Michaelis–Menten constant (Km), Vmax, and efficiency (kcat/Km) of CamPhoD were 4.2 mM, 0.203 mM/min, and 7988.6 S−1/mM; and 6.71 mM, 0.023 mM/min, and 1133.0 S−1/mM for pNPP and bis-pNPP as the chromogenic substrates, respectively. Among the 3D structures currently available, in this study we found only the low identical structure of the Bacillus subtilis enzyme as a homologous template for modeling CamPhoD, with a new architecture of the phosphatase active site containing Fe3+ and two Ca2+ ions. It is evident that the marine bacterial phosphatase/phosphidiesterase CamPhoD is a new structural member of the PhoD family.

Criteria for establishing a standardized method for determining alkaline phosphatase activity in human serum.

1977 ◽  
Vol 23 (12) ◽  
pp. 2263-2274 ◽  
Author(s):  
J P Bretaudière ◽  
A Vassault ◽  
L Amsellem ◽  
M L Pourci ◽  
H Thieu-Phung ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Human Serum ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Volume Fraction ◽  
Zinc Ion ◽  
Magnesium Ion ◽  
Ion Temperature ◽  
Nitrophenyl Phosphate ◽  
Standardized Method

Abstract We investigated factors influencing alkaline phosphatase activity in the course of developing criteria for the establishment of a standardized method for its determination in human serum at 30 degrees C. The effects of pH, phosphorylatable acceptor (2-amino-2-methyl-1-propanol and diethanolamine), 4-nitrophenyl phosphate, magnesium ion, zinc ion, temperature, volume fraction of specimen, and details of initiation of the reaction have been studied, with use of partly purified enzymes from bone, intestine, liver, and placenta, and sera from patients with a predominant characterized isoenzyme. The purity of the diethanolamine was examined and contaminant monoethanolamine was characterized as a competitive inhibitor. Two sets of recommended conditions are: 2-amino-2-methyl-1-propanol, 0.9 mol/liter; 4-nitrophenyl phosphate, 16 mmol/liter; magnesium ion, 1 mmol/liter; volume fraction of specimen 1/30, and pH30 degrees C 10.5; diethanolamine, 1.8 mol/liter; 4-nitrophenyl phosphate, 18 mmol/liter; magnesium ion, 1 mmol/liter; volume fraction of specimen 1/60, and pH30 degrees C 10.1. Serum is preincubated with all reagents but 4-nitrophenyl phosphate, which is used as the reaction-initiating substrate.

4-nitrophenyl phosphate--characterization of high-purity materials for measuring alkaline phosphatase activity in human serum.

1981 ◽  
Vol 27 (1) ◽  
pp. 135-143 ◽  
Author(s):  
G N Bowers ◽  
R B McComb ◽  
A Upretti
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Inorganic Phosphate ◽  
Molar Absorptivity ◽  
Maximum Activity ◽  
Comparative Testing ◽  
Total Uncertainty ◽  
Nitrophenyl Phosphate

Abstract We studied 53 lots of 4-nitrophenyl phosphate (I), obtained from 20 different commercial suppliers, and used this information to set specifications for it. Using these well-defined specifications, we classified 21 lots of I as "unacceptable," 26 lots as "borderline," and six as "acceptable." All lots were shown to contain some 4-nitrophenol and inorganic phosphate. However, "acceptable" I had < 0.3 mmol of 4-nitrophenol and < 10 mmol of inorganic phosphate per mole of I. The mole concentration of I (based on disodium hexahydrate, formula weight 371) was determined by enzymic conversion to 4-nitrophenol in five lots of "acceptable" materials. The mole fraction of I ranged from 0.982 to 0.998. From these measurements and from estimates of impurities that absorb at 311 nm, as determined by liquid chromatography and spectrophotometry at other wavelengths, our best estimate of the molar absorptivity of I at 311 nm in 10 mmol/L NaOH at 25 degrees C was 9867 L x mol-1 x cm-1, with a total uncertainty of 76 L x mol-1 x cm-1. We recommend that I used in clinical laboratories for measurement of alkaline phosphatase activity in serum meet the specifications given in this paper: I content > 98%, maximum activity > 98% in comparative testing with other "acceptable" lots of I, and impurities not to exceed the values cited above.

scholarly journals Immobilization of Naringinase from Penicillium decumbens on Chitosan Microspheres for Debittering Grapefruit Juice

Molecules ◽  
2019 ◽  
Vol 24 (23) ◽  
pp. 4234 ◽  
Author(s):  
Joanna Bodakowska-Boczniewicz ◽  
Zbigniew Garncarek
Keyword(s):  
Grapefruit Juice ◽  
Immobilized Enzyme ◽  
Maximum Activity ◽  
Chitosan Microspheres ◽  
Optimum Ph ◽  
Potential Applicability ◽  
Km Value ◽  
Hydrolysis Of ◽  
Thermal Stability Of

Naringinase is an enzyme complex which exhibits α-l-rhamnosidase and β-d-glucosidase activity. This enzymatic complex catalyzes the hydrolysis of naringin (4′,5,7-trihydroxy flavanone 7-rhamnoglucoside), the main bittering component in grapefruit. Reduction of the level of this substance during the processing of juice has been the focus of many studies. The aim of the study was the immobilization of naringinase on chitosan microspheres activated with glutaraldehyde and, finally, the use of such immobilized enzyme for debittering grapefruit juice. The effect of naringinase concentration and characterization of the immobilized enzyme compared to the soluble enzyme were investigated. The maximum activity was observed at optimum pH 4.0 for both free and immobilized naringinase. However, the optimum temperature was shifted from 70 to 40 °C upon immobilization. The KM value of the immobilized naringinase was higher than that of soluble naringinase. The immobilization did not change the thermal stability of the enzyme. The immobilized naringinase had good operational stability. This preparation retained 88.1 ± 2.8% of its initial activity after ten runs of naringin hydrolysis from fresh grapefruit juice. The results indicate that naringinase immobilized on chitosan has potential applicability for debittering and improving the sensory properties of grapefruit juices.

scholarly journals An Exonuclease I-Aided Turn-Off Fluorescent Strategy for Alkaline Phosphatase Assay Based on Terminal Protection and Copper Nanoparticles

Biosensors ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 139
Author(s):  
Yan Wang ◽  
Ying Yan ◽  
Xinfa Liu ◽  
Changbei Ma
Keyword(s):  
Alkaline Phosphatase ◽  
Human Serum ◽  
Clinical Diagnosis ◽  
Copper Nanoparticles ◽  
Cost Effective ◽  
Fast Method ◽  
Exonuclease I ◽  
Alkaline Phosphatase Assay ◽  
Phosphatase Assay ◽  
Phosphatase Alkaline

As an important DNA 3′-phosphatase, alkaline phosphatase can repair damaged DNA caused by replication and recombination. It is essential to measure the level of alkaline phosphatase to indicate some potential diseases, such as cancer, related to alkaline phosphatase. Here, we designed a simple and fast method to detect alkaline phosphatase quantitively. When alkaline phosphatase is present, the resulting poly T-DNA with a 3′-hydroxyl end was cleaved by exonuclease I, prohibiting the formation of fluorescent copper nanoparticles. However, the fluorescent copper nanoparticles can be monitored with the absence of alkaline phosphatase. Hence, we can detect alkaline phosphatase with this turn-off strategy. The proposed method is able to quantify the concentration of alkaline phosphatase with the LOD of 0.0098 U/L. Furthermore, we utilized this method to measure the effects of inhibitor Na3VO4 on alkaline phosphatase. In addition, it was successfully applied to quantify the level of alkaline phosphatase in human serum. The proposed strategy is sensitive, selective, cost effective, and timesaving, having a great potential to detect alkaline phosphatase quantitatively in clinical diagnosis.

Alkaline phosphatase. I. Kinetics and inhibition by levamisole of purified isoenzymes from humans.

1976 ◽  
Vol 22 (7) ◽  
pp. 972-976 ◽  
Author(s):  
H Van Belle
Keyword(s):  
Alkaline Phosphatase ◽  
Substrate Concentration ◽  
Human Liver ◽  
Alkaline Phosphatases ◽  
Mechanism Of Inhibition ◽  
Optimum Ph ◽  
Alkaline Phosphatase Isoenzymes

Abstract I studied the kinetics and sensitivity toward inhibition by levamisole and R 8231 of the most important human alkaline phosphatase isoenzymes. N-Ethylaminoethanol proved superior to the now widely used diethanolamine buffer, especially for the enzymes from the intestine and placenta, behaving as an uncompetitive activator. The optimum pH largely depends on the substrate concentration. The addition of Mg2+ has no effect on the activities. The meaning of Km-values for alkaline phosphatases is questioned. Isoenzymes from human liver, bone, kidney, and spleen are strongly inhibited by levamisole or R 8231 at concentrations that barely affect the enzymes from intestine or placenta. The inhibition is stereospecific, uncompetitive, and not changed by Mg2+. Inhibition is counteracted by increasing concentrations of N-ethylaminoethanol. The mechanism of inhibition is suggested to be formation of a complex with the phosphoenzyme.

4-Nitrophenol in 4-nitrophenyl phosphate, a substrate for alkaline phosphatase, as measured by paired-ion high-performance liquid chromatography.

1977 ◽  
Vol 23 (12) ◽  
pp. 2288-2291 ◽  
Author(s):  
P H Culbreth ◽  
I W Duncan ◽  
C A Burtis
Keyword(s):  
Alkaline Phosphatase ◽  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Flow Rate ◽  
Mobile Phase ◽  
High Performance ◽  
Reversed Phase ◽  
Nitrophenyl Phosphate ◽  
Phase Column ◽  
Reversed Phase Column

Abstract We used paired-ion high-performance liquid chromatography to determine the 4-nitrophenol content of 4-nitrophenyl phosphate, a substrate for alkaline phosphatase analysis. This was done on a reversed-phase column with a mobile phase of methanol/water, 45/55 by vol, containing 3 ml of tetrabutylammonium phosphate reagent per 200 ml of solvent. At a flow rate of 1 ml/min, 4-nitrophenol was eluted at 9 min and monitored at 404 nm; 4-nitrophenyl phosphate was eluted at 5 min and could be monitored at 311 nm. Samples of 4-nitrophenyl phosphate obtained from several sources contained 0.3 to 7.8 mole of 4-nitrophenol per mole of 4-nitrophenyl phosphate.

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