scholarly journals The significance of inhibitor-resistant alkaline phosphatase in the cytochemical demonstration of transport adenosine triphosphatase.

1975 ◽  
Vol 23 (8) ◽  
pp. 571-574 ◽  
Author(s):  
J A Firth ◽  
B Y Marland
Keyword(s):  
Alkaline Phosphatase ◽  
Adenosine Triphosphatase ◽  
Renal Cortex ◽  
Sodium Potassium ◽  
Cytochemical Demonstration ◽  
Nitrophenyl Phosphate ◽  
Atpase Cytochemistry ◽  
Strontium Ions ◽  
Dependent Transport ◽  
Hydrolysis Of

The hydrolysis of disodium p-nitrophenyl phosphate at pH 9.0 by slices of formaldehydee-fixed rat renal cortex was investigated by colorimetric estimation of the nitrophenol liberated. It was found that three types of activity could be identified on the basis of their responses to inhibitors and cations: (a) alkaline phosphatase sensitive to inhibition by L-tetramisole; (b) potassium-dependent phosphatase, probably identifiable with the phosphatase component of sodium-potassium-dependent transport adenosine triphosphatase (?Na-K-ATPase); and (c) alkaline phosphatase insensitive to L-tetramisole. It was found that in the presence of strontium ions, as used in Na-K-ATPase cytochemistry, the activities of the second and third types of enzyme were approximately equal. The implications of these findings for the cytochemical demonstration of Na-K-ATPase are discussed.

scholarly journals Renal sodium-potassium adenosine triphosphatase. Optical localization and x-ray microanalysis.

1975 ◽  
Vol 23 (11) ◽  
pp. 828-839 ◽  
Author(s):  
R Beeuwkes ◽  
S Rosen
Keyword(s):  
Atpase Activity ◽  
Electron Probe ◽  
Sequential Analysis ◽  
Relative Sensitivity ◽  
Initial Reaction ◽  
Initial Product ◽  
Sodium Potassium ◽  
Nitrophenyl Phosphate ◽  
Hydrolysis Of ◽  
Convoluted Tubules

The distribution of sodium-potassium adenosine triposphatase (Na-K-ATPase) activity in kidney sections has been studied by a method based on the hydrolysis of p-nitrophenyl phosphate in alkaline medium containing dimethyl sulfoxide. The products at each stage in the reaction sequence have been subjected to electron probe microanalysis. The initial product was identified as a mixture of KMgPO4 and Mg(PO4)2, and sequential analysis demonstrated the linearity of conversion of this product to a visible form. In human, rabbit and rat kidneys the distribution of activity was found to be essentially identical, with highest levels located in thick ascending limbs and distal convoluted tubules. The initial reaction was completely potassium dependent and was inhibited by ouabain in concentrations reflecting the relative sensitivity of microsomal Na-K-ATPase in each species. Measurement of initial product phosphorus by means of the electron probe is presented as a practical technique for direct quantitation of Na-K-ATPase activity in identified tubule segments.

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.

A Continuous Spectrophotometric Method for Measuring the Activity of Serum Alkaline Phosphatase

1966 ◽  
Vol 12 (2) ◽  
pp. 70-89 ◽  
Author(s):  
George N Bowers ◽  
Robert B McComb
Keyword(s):  
Alkaline Phosphatase ◽  
Standard Deviation ◽  
Sample Size ◽  
Spectrophotometric Method ◽  
Serum Alkaline Phosphatase ◽  
Serum Alkaline Phosphatase Activity ◽  
Factors Affecting ◽  
Nitrophenyl Phosphate ◽  
Relative Standard ◽  
Hydrolysis Of

Abstract A continuous spectrophotometric method for measuring serum alkaline phosphatase activity is described. The effects of temperature, pH, substrate concentration, type and molarity of the buffer, sample size, cofactors, and inhibitors on the enzymatic hydrolysis of p-nitrophenyl phosphate were studied. The optimal conditions for assay of serum alkaline phosphatase at 30° were found to be 0.75 M 2-amino-2-methyl-1-propanol buffer, pH30° 10.15, 4 mmole substrate, and 100 µl. or less sample size. Studies of the factors affecting analytical precision-i.e., control of reaction temperature, of reagent manufacture, and of standardization-are discussed. The precision of this method was 2.3% (relative standard deviation) on 10 within day replicates and 5.0% on day-to-day replicates spread over a 5-week period. The range of activity for 258 apparently healthy adult blood donors was 6-110 mU./ml. (International milliunits per milliliter), with a mean of 49 and a standard deviation of 14.

scholarly journals Phosphodiesterase activity is a novel property of alkaline phosphatase from osseous plate

1994 ◽  
Vol 301 (2) ◽  
pp. 517-522 ◽  
Author(s):  
A A Rezende ◽  
J M Pizauro ◽  
P Ciancaglini ◽  
F A Leone
Keyword(s):  
Alkaline Phosphatase ◽  
Cyclic Amp ◽  
Catalytic Site ◽  
Hill Coefficient ◽  
Type I ◽  
Phosphodiesterase Activity ◽  
Apparent Dissociation Constant ◽  
Nitrophenyl Phosphate ◽  
Hydrolysis Of ◽  
Osseous Plate

Phosphodiesterase activity is a novel property of the still-enigmatic alkaline phosphatase from osseous plate. Bis-(p-nitrophenyl) phosphate was hydrolysed at both pH 7.5 and 9.4 with an apparent dissociation constant (K0.5) of 1.9 mM and 3.9 mM respectively. The hydrolysis of p-nitrophenyl-5′-thymidine phosphate followed hyberbolic kinetics with a K0.5 of 500 microM. For p-nitrophenyl phenylphosphonate, site-site interactions [Hill coefficient (h) = 1.3] were observed in the range between 0.2 and 100 microM, and K0.5 was 32.8 mM. The hydrolysis of cyclic AMP by the enzyme followed more complex kinetics, showing site-site interactions (h = 1.7) and K0.5 = 300 microM for high-affinity sites. The low-affinity sites, representing 85% of total activity, also showed site-site interactions (h = 3.8) and a K0.5 of about 22 mM. ATP and cyclic AMP were competitive inhibitors of bis-(p-nitrophenyl) phosphatase activity of the enzyme and Ki values (25 mM and 0.6 mM for cyclic AMP and ATP respectively) very close to those of the K0.5 (22 mM and 0.7 mM for cyclic AMP and ATP respectively), determined by direct assay, indicated that a single catalytic site was responsible for the hydrolysis of both substrates. Non-denaturing PAGE of detergent-solubilized enzyme showed coincident bands on the gel for phosphomonohydrolase and phosphodiesterase activities. Additional evidence for a single catalytic site was the similar pKa values (8.5 and 9.7) found for the two ionizing groups participating in the hydrolysis of bis-(p-nitrophenyl) phosphate and p-nitrophenyl phosphate. The alkaline apparent pH optima, the requirement for bivalent metal ions and the inhibition by methylxanthines, amrinone and amiloride demonstrated that rat osseous-plate alkaline phosphatase was a type I phosphodiesterase. Considering that there is still confusion as to which is the physiological substrate for the enzyme, the present results describing a novel property for this enzyme could be of relevance in understanding the mineralization process.

Influence of pH on the Kinetics of Reactions Catalyzed by Alkaline Phosphatase

1973 ◽  
Vol 51 (7) ◽  
pp. 1096-1103 ◽  
Author(s):  
Irwin Hinberg ◽  
Keith J. Laidler
Keyword(s):  
Alkaline Phosphatase ◽  
Intestinal Alkaline Phosphatase ◽  
Nitrophenyl Phosphate ◽  
Wide Range ◽  
Coordinated Water ◽  
Ph Range ◽  
Barbital Buffer ◽  
Influence Of Ph ◽  
Kinetics Of ◽  
Hydrolysis Of

An experimental study has been made of the kinetics of the hydrolysis of p-nitrophenyl phosphate catalyzed by chicken-intestinal alkaline phosphatase. The work was done in barbital buffer (carbonate above pH 9.6), and covered the pH range from 7.0 to 10.0. A sufficiently wide range of substrate concentration was used to allow reliable values of [Formula: see text] and [Formula: see text] to be determined. The results lead to pK values of 8.1 and 8.6 for the free enzyme, and it is concluded that the Michaelis complex and the phosphoryl intermediate ionize only on the acid side, the former also having a pK of 8.1. It is suggested that the group of pK 8.1 is probably an α-amino group and that the group of pK 8.6 probably corresponds to the ionization of a Zn(II)-coordinated water molecule.

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