Enzyme saturation and inhibition kinetics studied from multiple progress curves recorded spectrophotometrically from single reaction mixtures for ADP-ribose pyrophosphatase.
Saturation and inhibition kinetics data for rat liver ADP-ribose pyrophosphatase (EC 3.6.1.13) were obtained from progress curves initiated by the addition of substrate and recorded spectrophotometrically until the end point was reached. The hydrolysis of ADP-ribose was coupled to either alkaline phosphatase and adenosine deaminase or AMP deaminase. The validity of the approach was shown because: (i) the coupled hydrolysis of ADP-ribose was essentially irreversible; (ii) ADP-ribose pyrophosphate was stable at 37 degrees C in the conditions needed for the assay; and (iii) accumulated reaction products did not inhibit detectably in the conditions of the assay. In addition, several identical progress curves could be successively recorded by repetition of the addition of substrate. In that way it was possible to carry out complete inhibition studies by increasing the inhibitor concentration between successive substrate additions. Studying the inhibition by high D-ribose concentrations, meaningful results could be obtained at four different inhibitor concentrations in a single reaction mixture, which represented a great saving of enzyme preparation with respect to what would be needed in an equivalent initial rate study.
Miró
A
A
Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, Badajoz, Spain.
Hernández
M T
MT
Costas
M J
MJ
Cameselle
J C
JC
D000659:AMP Deaminase / Q000378:metabolism; D000243:Adenosine Deaminase / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D013056:Spectrophotometry, Ultraviolet / Q000295:instrumentation
D000246:Adenosine Diphosphate Ribose; D000469:Alkaline Phosphatase; D000243:Adenosine Deaminase; D000659:AMP Deaminase; D011755:Pyrophosphatases; C019620:ADPribose pyrophosphatase