Bovine milk acid phosphatase: II. Binding to casein substrates and heat-inactivation studies

1974 ◽  
Vol 41 (2) ◽  
pp. 229-237 ◽  
Author(s):  
A. T. Andrews
Keyword(s):  
Acid Phosphatase ◽  
Protein Denaturation ◽  
Bovine Milk ◽  
Raw Milk ◽  
Heat Inactivation ◽  
Heat Stable ◽  
First Order Kinetics ◽  
Nitrophenyl Phosphate ◽  
Substrate Protection ◽  
Hydrolysis Of

SummaryThe acid phosphatase of bovine milk was further purified to yield enzyme with an activity of about 2 units/mg. This was almost 105 times the activity present in milk and enabled a detailed study of heat inactivation to be made, together with further measurements on binding to casein substrates.The effectiveness of caseins as inhibitors of the hydrolysis of p-nitrophenyl phosphate by acid phosphatase paralleled the phosphate content of the casein molecules, so that αs1-casein A was a more potent inhibitor with a K1 of 1·7 mM than β-casein A1A2 (K1 = 4·3 mM), which in turn was more inhibitory than κ-casein A (K1 = 5·9 mM).The heat inactivation of acid phosphatase followed first-order kinetics at pH 4·9, 5·2 and 6·7 and values of E, the activation energy, were between 2·4×105 and 3·0×105 J mole −1 in all cases, consistent with simple protein denaturation. The presence of 1% αs1-casein A, 1% β-casein A1A2, 1% κ-casein A, 1% isoelectrically precipitated ‘whole’ casein and 1% fresh raw milk provided no substrate protection at pH 5·2 or 6·7. Acid phosphatase was somewhat less heat stable at pH 6·7 than at pH 4·9, but may be expected to survive typical milk pasteurization conditions almost completely. However, conventional milk sterilization or ultra-high-temperature (UHT) processes would be expected to give total inactivation.

Development of a monoclonal antibody-based immunoassay for specific quantification of bovine milk alkaline phosphatase

2007 ◽  
Vol 74 (3) ◽  
pp. 290-295 ◽  
Author(s):  
Nathalie Geneix ◽  
Eric Dufour ◽  
Annie Venien ◽  
Didier Levieux
Keyword(s):  
Monoclonal Antibody ◽  
Alkaline Phosphatase ◽  
Monoclonal Antibodies ◽  
Dairy Products ◽  
Indirect Elisa ◽  
Bovine Milk ◽  
Raw Milk ◽  
Heat Stable ◽  
Nitrophenyl Phosphate ◽  
Enzymatic Methods

The detection of alkaline phosphatase (ALP) activity is used as a legal test to determine whether milk has been adequately pasteurized or recontaminated with raw milk. However, a wide variety of microorganisms produce both heat labile and heat stable ALPs which cannot be differentiated from the milk ALP by current enzymatic methods. Monoclonal antibodies specific of the bovine milk ALP were obtained in mice from a raw bovine milk ALP preparation. Coated in microtitre plates, these antibodies specifically capture the bovine milk ALP from dairy products. After washing, the enzymatic activity of the captured ALP is revealed by adding p-nitrophenyl-phosphate as a substrate. This simple immunoassay does not react with ALPs of intestinal or bacterial origin and, once optimized, was found to be the first immunoassay suitable to detect raw milk in boiled milk down to a 0·02% dilution. Moreover, in contrast with competitive indirect ELISA formats, the capture immunoassay does not require purified ALP.

scholarly journals Phosphatase synthesis in Klebsiella (Aerobacter) aerogenes growing in continuous culture

1972 ◽  
Vol 127 (1) ◽  
pp. 87-96 ◽  
Author(s):  
P. G. Bolton ◽  
A. C. R. Dean
Keyword(s):  
Alkaline Phosphatase ◽  
Acid Phosphatase ◽  
Continuous Culture ◽  
Activity Profile ◽  
Glucose Effect ◽  
Ph Values ◽  
Nitrophenyl Phosphate ◽  
Wide Range ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

1. Phosphatase synthesis was studied in Klebsiella aerogenes grown in a wide range of continuous-culture systems. 2. Maximum acid phosphatase synthesis was associated with nutrient-limited, particularly carbohydrate-limited, growth at a relatively low rate, glucose-limited cells exhibiting the highest activity. Compared with glucose as the carbon-limiting growth material, other sugars not only altered the activity but also changed the pH–activity profile of the enzyme(s). 3. The affinity of the acid phosphatase in glucose-limited cells towards p-nitrophenyl phosphate (Km 0.25–0.43mm) was similar to that of staphylococcal acid phosphatase but was ten times greater than that of the Escherichia coli enzyme. 4. PO43−-limitation derepressed alkaline phosphatase synthesis but the amounts of activity were largely independent of the carbon source used for growth. 5. The enzymes were further differentiated by the effect of adding inhibitors (F−, PO43−) and sugars to the reaction mixture during the assays. In particular, it was shown that adding glucose, but not other sugars, stimulated the rate of hydrolysis of p-nitrophenyl phosphate by the acid phosphatase in carbohydrate-limited cells at low pH values (<4.6) but inhibited it at high pH values (>4.6). Alkaline phosphatase activity was unaffected. 6. The function of phosphatases in general is discussed and possible mechanisms for the glucose effect are outlined.

Polyphosphate body and acid phosphatase localization in Nostoc sp.

1984 ◽  
Vol 30 (1) ◽  
pp. 8-15 ◽  
Author(s):  
John D. DuBois ◽  
Keith R. Roberts ◽  
Lawrence A. Kapustka
Keyword(s):  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Acid Phosphatase Activity ◽  
Energy Stress ◽  
Nitrophenyl Phosphate ◽  
Carbon Compounds ◽  
Electron And Light Microscopy ◽  
Polyphosphate Bodies ◽  
Hydrolysis Of

Polyphosphate bodies and acid phosphatase activity were characterized in Nostoc sp. to determine if the hydrolysis of polyphosphate bodies occurs during dark (energy stress) periods. Electron and light microscopy were used to locate polyphosphate bodies. Acid phosphatase activity was measured using p-nitrophenyl phosphate as the substrate to determine net changes in the level of the enzyme activity. To induce energy stress, Nostoc sp. cells were kept in the dark for 72 h to deplete stored carbon compounds. Cells incubated in the light for 72 h (controls) showed acid phosphatase activity localized around the perimeter of polyphosphate bodies. When cells were incubated in the dark, acid phosphatase activity occurred throughout the polyphosphate body matrix. However, complete hydrolysis of the polyphosphate body did not occur and the rate of acid phosphatase activity was not affected.

Acid phosphatase activity in cheese and starters

1975 ◽  
Vol 42 (2) ◽  
pp. 327-339 ◽  
Author(s):  
A. T. Andrews ◽  
E. Alichanidis
Keyword(s):  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Acid Phosphatase Activity ◽  
Bovine Milk ◽  
Cheddar Cheese ◽  
Starter Cultures ◽  
Minor Importance ◽  
Activity Levels ◽  
Kinetic Measurements ◽  
Nitrophenyl Phosphate

SummaryThe acid phosphatase activity levels in a number of Greek cheeses and in Cheddar cheeses were found to be unaffected by storage for up to 18 months and 12 months respectively. In Cheddar cheese, starter organisms made an insignificant contribution to this activity. Studies of acid phosphatase prepared from Streptococcus cremoris-lactis NCDO 762 starter cultures showed that the enzyme was of high molecular weight and largely particle-bound. The pH of optimum activity was 5·2 and the enzyme was inhibited by F−, Al3+, a number of heavy metals, oxidizing agents and sulphydryl-modifying reagents. Kinetic measurements at pH 5·2 gave a Km value for p-nitrophenyl phosphate of 1·2 mM. Orthophosphate, pyrophosphate and isoelectrically precipitated casein behaved as competitive inhibitors to the hydrolysis of p-nitrophenyl phosphate with K1 values of 1·2 mM, 1·0 mM and 1·1 mM respectively. In spite of this binding to the enzyme, casein provided a very poor substrate for the starter acid phosphatase. The properties of acid phosphatase present in Cheddar cheese made with Str. cremoris NCDO 924 starter were consistent with the enzyme being exclusively of milk origin and small differences between this and the acid phosphatase previously isolated from bovine milk were attributable to the binding of peptides produced during the cheese maturation to the enzyme molecules. It was concluded that in cheese, phosphatase action was due largely to the enzyme of milk origin, with that provided by the starter being of minor importance.

Biochemical properties of tartrate-resistant acid phosphatase in serum of adults and children.

1978 ◽  
Vol 24 (7) ◽  
pp. 1105-1108 ◽  
Author(s):  
W K Lam ◽  
D T Eastlund ◽  
C Y Li ◽  
L T Yam
Keyword(s):  
Acid Phosphatase ◽  
Catalytic Properties ◽  
Biochemical Properties ◽  
Tartrate Resistant Acid Phosphatase ◽  
Total Acid ◽  
Average Value ◽  
Nitrophenyl Phosphate ◽  
Adults And Children ◽  
Major Acid ◽  
Hydrolysis Of

Abstract Spectrophotometry of total acid phosphatase activity in children's sera showed an average value of 22.4 +/- 2.9 and 7.4 +/- 0.8 U/liter, for the hydrolysis of p-nitrophenyl phosphate and alpha-naphthyl phosphate, respectively. Analyses of "band 5b", after electrophoresis on acrylamide gel, gave even higher values. The values for children's sera were much higher than those for sera from adults. The multiplicity of acid phosphatases in sera of children and adults was studied by electrophoresis on acrylamide gel and by chromatography on CM-Sepharose. Both methods showed the major acid phosphatase in children's sera to be an acid pyrophosphatase, band 5b. Its catalytic properties are indistinguishable from the enzyme previously isolated from the spleen of leukemic reticuloendotheliosis.

THE WHEAT LEAF PHOSPHATASES: VII. FURTHER STUDIES ON INHIBITORS AT pH 5.7

1963 ◽  
Vol 41 (1) ◽  
pp. 1727-1731 ◽  
Author(s):  
D. W. A. Roberts
Keyword(s):  
Acid Phosphatase ◽  
Enzymatic Hydrolysis ◽  
Inhibitory Effect ◽  
Substrate Specificities ◽  
Nitrophenyl Phosphate ◽  
Wheat Leaf ◽  
Hydrolysis Of

A survey of the inhibitory effect of various ribonucleosides, deoxyribonucleosides, sugars, phenols, and vitamins on the hydrolysis of 17 phosphatase substrates has been made. The more soluble ribonucleosides and deoxyribonucleosides inhibited the enzymatic hydrolysis of adenosine 5′-phosphate, phenolphthalein diphosphate, phenylphosphate, p-nitrophenyl phosphate, and in some cases the hydrolysis of adenosine 3′-phosphate, adenosine 2′-phosphate, and riboflavin 5′-phosphate. A 0.02 M concentration of orthophosphate inhibited the hydrolysis of all the compounds tested except adenosine 5′-phosphate and phenolphthalein diphosphate.These results, together with earlier findings, are discussed in terms of the concept that wheat leaf press juice contains two types of acid phosphatase, namely, β-glycerophosphatase and adenosine 5′-phosphatase. These two types of enzyme appear to have partially overlapping substrate specificities.

THE WHEAT LEAF PHOSPHATASES: VII. FURTHER STUDIES ON INHIBITORS AT pH 5.7

1963 ◽  
Vol 41 (8) ◽  
pp. 1727-1731 ◽  
Author(s):  
D. W. A. Roberts
Keyword(s):  
Acid Phosphatase ◽  
Enzymatic Hydrolysis ◽  
Inhibitory Effect ◽  
Substrate Specificities ◽  
Nitrophenyl Phosphate ◽  
Wheat Leaf ◽  
Hydrolysis Of

A survey of the inhibitory effect of various ribonucleosides, deoxyribonucleosides, sugars, phenols, and vitamins on the hydrolysis of 17 phosphatase substrates has been made. The more soluble ribonucleosides and deoxyribonucleosides inhibited the enzymatic hydrolysis of adenosine 5′-phosphate, phenolphthalein diphosphate, phenylphosphate, p-nitrophenyl phosphate, and in some cases the hydrolysis of adenosine 3′-phosphate, adenosine 2′-phosphate, and riboflavin 5′-phosphate. A 0.02 M concentration of orthophosphate inhibited the hydrolysis of all the compounds tested except adenosine 5′-phosphate and phenolphthalein diphosphate.These results, together with earlier findings, are discussed in terms of the concept that wheat leaf press juice contains two types of acid phosphatase, namely, β-glycerophosphatase and adenosine 5′-phosphatase. These two types of enzyme appear to have partially overlapping substrate specificities.

Acid phosphatase activity demonstrated by intact Angiostrongylus cantonensis with special reference to its function

Parasitology ◽  
1979 ◽  
Vol 79 (3) ◽  
pp. 417-423 ◽  
Author(s):  
Jun Maki ◽  
Toshio Yanagisawa
Keyword(s):  
Acid Phosphatase ◽  
External Medium ◽  
Reciprocal Inhibition ◽  
Angiostrongylus Cantonensis ◽  
Phosphate Esters ◽  
Glucose Phosphate ◽  
Nitrophenyl Phosphate ◽  
Inhibition Studies ◽  
Hydrolysis Of

SUMMARYIntact Angiostrongylus cantonensis is able to hydrolyse glucose-phosphate esters, mononucleotides and p-nitrophenyl phosphate as well as β-glycerophosphate in vitro. Reciprocal inhibition studies suggest that the hydrolysis of such substrates is due to a non-specific phosphomonoesterase. Molybdate ions, which exert no effect on either the uptake of glucose or the production of lactate, inhibit the hydrolysis of glucose-1- phosphate in the external medium and simultaneously lower the production of lactate by the intact worms in vitro.

The acid phosphatases of bovine leucocytes, plasma and the milk of healthy and mastitic cows

1975 ◽  
Vol 42 (3) ◽  
pp. 391-400 ◽  
Author(s):  
A. T. Andrews ◽  
E. Alichanidis
Keyword(s):  
Molecular Weight ◽  
Acid Phosphatase ◽  
Exchange Resin ◽  
Bovine Milk ◽  
Ion Exchange Resin ◽  
Acid Phosphatases ◽  
Ph Optimum ◽  
Nitrophenyl Phosphate ◽  
Competitive Inhibitors ◽  
Electrophoretic Component

SummarySome of the acid phosphatase isozymes of bovine leucocytes and plasma have been separated and partly characterized. About 80% of the phosphatase activity of leucocytes at pH 4·9 was particle-bound and about 8% was extractable with Amberlite CG-50 ion exchange resin. This extractable enzyme existed as a single electrophoretic component with a mol. wt of about 42000 and with optimum activity at pH 5·8. Km for p-nitrophenyl phosphate was 1·6 mM at pH 5·8 and 0·4 mM at pH 4·9. At pH 5·8 orthophosphate (K1 = 1·5 mM) and pyrophosphate (Ki = 4·1 mM) were competitive inhibitors. The enzyme was also strongly inhibited by F−, Al3+, IO4− and S2032−. The enzyme which was not extractable with Amberlite was very heterogeneous with respect to molecular weight. At the pH optimum (4·9), Km for p-nitrophenyl phosphate was 0·4 mM and orthophosphate (K1 = 2·3 mM) and pyrophosphate (K1 = 2·1 mM) were competitive inhibitors. Other inhibitors included F−, Al3+, Hg2+, IO4− and tartrate. The enzyme extracted from plasma by Amberlite CG-50 treatment had properties similar to that extracted from leucocytes. Normal bovine milk contained a single acid phosphatase, but milk from cows with mastitis showed 3 electrophoretic isozyme bands, one being the same as in normal milk; the 2 additional bands were of leucocyte origin.

Evaluation of a kinetic method for prostatic acid phosphatase with use of self-indicating substrate, 2,6-dichloro-4-nitrophenyl phosphate.

1989 ◽  
Vol 35 (6) ◽  
pp. 939-945 ◽  
Author(s):  
A A Valcour ◽  
G N Bowers ◽  
R B McComb
Keyword(s):  
Acid Phosphatase ◽  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Spectral Characteristics ◽  
Kinetic Method ◽  
Prostatic Acid Phosphatase ◽  
Use Of Self ◽  
Nitrophenyl Phosphate ◽  
Hydrolysis Of

Abstract The purity, spectral characteristics, and rate of nonenzymatic hydrolysis of 2,6-dichloro-4-nitrophenyl phosphate (DCNPP) were determined. Rates of DCNPP hydrolysis by prostatic acid phosphatase (PAP) and erythrocytic acid phosphatase (EAP) (both EC 3.1.3.2) were measured in the absence and in the presence of various alcohols. 1.5-Pentanediol was the most effective transphosphorylation agent for specifically enhancing the activity of PAP. 1,4-Butanediol also enhanced PAP activity but markedly inhibited EAP activity. Bovine and human serum albumin preparations also accelerated the hydrolysis of DCNPP. DCNPP can be used for the continuous or multipoint-rate assay of PAP.

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