scholarly journals Nucleoside pyrophosphatase activity associated with pig kidney alkaline phosphatase

1971 ◽  
Vol 124 (5) ◽  
pp. 891-896 ◽  
Author(s):  
Milica Wass ◽  
P. J. Butterworth
Keyword(s):  
Alkaline Phosphatase ◽  
Pig Kidney ◽  
Bivalent Cations ◽  
Free Nucleotides ◽  
Pyrophosphatase Activity ◽  
Kidney Alkaline Phosphatase

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; Km values are 4×10-5m for ATP and 6.3×10-5m for ADP. Vmax. 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 Mg2+ ions to form MgATP2- or MgADP- complexes. The complexes, besides being non-inhibitory, are also substrates for the enzyme with Km values identical with those of the respective free nucleotides. 3. Mg2+ 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 Mg2+ ions stimulates activity by relieving nucleoside pyrophosphate inhibition. The properties may be of importance in the regulation of the transport of bivalent cations.

scholarly journals Evidence for the importance of arginine residues in pig kidney alkaline phosphatase

1979 ◽  
Vol 181 (1) ◽  
pp. 137-142 ◽  
Author(s):  
M N Woodroofe ◽  
P J Butterworth
Keyword(s):  
Alkaline Phosphatase ◽  
Active Site ◽  
Competitive Inhibitor ◽  
Arginine Residue ◽  
Pig Kidney ◽  
Close Proximity ◽  
Arginine Residues ◽  
Km Value ◽  
Uncompetitive Inhibitor ◽  
Kidney Alkaline Phosphatase

The arginine-specific reagents 2,3-butanedione and phenylglyoxal inactivate pig kidney alkaline phosphatase. As inactivation proceeds there is a progressive fall in Vmax. of the enzyme, but no demonstrable change in the Km value for substrate. Pi, a competitive inhibitor, and AMP, a substrate of the enzyme, protect alkaline phosphatase against the arginine-specific reagents. These effects are explicable by the assumption that the enzyme contains an essential arginine residue at the active site. Protection is also afforded by the uncompetitive inhibitor NADH through a partially competive action against the reagents. Enzyme that has been exposed to the reagents has a decreased sensitivity to NADH inhibition. It is suggested that an arginine residue is important for NADH binding also, although this residue is distinct from that at the catalytic site. The protection given by NADH against loss of activity is indicative of the close proximity of the active and NADH sites.

Identification of Functional Groups of Pig Kidney Alkaline Phosphatase by Specific Inhibitors

1975 ◽  
Vol 30 (11-12) ◽  
pp. 829-831 ◽  
Author(s):  
Jan Ahlers
Keyword(s):  
Alkaline Phosphatase ◽  
Functional Groups ◽  
Pig Kidney ◽  
Kidney Alkaline Phosphatase ◽  
Specific Inhibitors ◽  
Regulatory Sites

Abstract Inactivation studies with 17 group-specific inhibitors showed that amino, hystidyl and tyrosyl residues probably are components of the active and/or regulatory sites of pig kidney alkaline phosphatase.

scholarly journals The reversible inactivation of pig kidney alkaline phosphatase at low pH

1968 ◽  
Vol 108 (2) ◽  
pp. 243-246 ◽  
Author(s):  
P. J. Butterworth
Keyword(s):  
Alkaline Phosphatase ◽  
Acid Treatment ◽  
Gel Filtration ◽  
Reversible Inactivation ◽  
Pig Kidney ◽  
Reactivation Mechanism ◽  
Increasing Temperature ◽  
Filtration Properties ◽  
Kinetics Of ◽  
Kidney Alkaline Phosphatase

1. Pig kidney alkaline phosphatase is inactivated by treatment with acid at 0°. 2. Inactivated enzyme can be partially reactivated by incubation at 30° in neutral or alkaline buffer. The amount of reactivation that occurs depends on the degree of acid treatment; enzyme that has been inactivated below pH3·3 shows very little reactivation. 3. Studies of the kinetics of reactivation indicate that the process is greatly accelerated by increasing temperature and proceeds by a unimolecular mechanism. The reactivated enzyme has electrophoretic and gel-filtration properties identical with those of non-treated enzyme. 4. The results can be best explained by assuming that a lowering of the pH causes a reversible conformational change of the alkaline phosphatase molecule to a form that is no longer enzymically active but is very susceptible to permanent denaturation by prolonged acid treatment. A reactivation mechanism involving sub-unit recombination seems unlikely.

scholarly journals The pyrophosphatase activity of pig kidney alkaline phosphatase and its inhibition by magnesium ions and excess of pyrophosphate

1968 ◽  
Vol 110 (4) ◽  
pp. 671-675 ◽  
Author(s):  
P. J. Butterworth
Keyword(s):  
Metal Ion ◽  
Total Concentration ◽  
Alkaline Phosphatases ◽  
Inorganic Pyrophosphate ◽  
Pig Kidney ◽  
A Value ◽  
Pyrophosphatase Activity ◽  
Concentration Curves ◽  
Kidney Alkaline Phosphatase ◽  
Complex Ion

1. Pig kidney enzyme resembles other non-specific alkaline phosphatases in its ability to hydrolyse inorganic pyrophosphate (PPi). 2. Studies of enzyme velocity as a function of PPi concentration show that Michaelis–Menten kinetics are obeyed when a constant PPi/Mg2+ concentration ratio is maintained, but velocity–substrate concentration curves are sigmoid when the concentration of PPi is increased but that of Mg2+ is kept constant. The enzyme is inhibited when the total PPi concentration is greater than the total concentration of Mg2+. Pyrophosphatase activity is activated by Mg2+, but if the concentration of the metal ion is increased to a value in excess of the total PPi concentration Mg2+ is then strongly inhibitory. 4. It appears that the enzyme is most active towards the complex ion MgPPi2−. The enzyme probably hydrolyses PPi4− also, but this is a poorer substrate and its competition with MgPPi2− leads to inhibition. At high Mg2+ concentrations Mg2PPi is formed. This complex appears to be a potent inhibitor. 5. Sigmoid plots of v against s and of v against i result from interactions occurring between Mg2+ and PPi4− leading to MgPPi2− and Mg2PPi, and are not indicative of allosteric behaviour.

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