Free ADP levels in transgenic mouse liver expressing creatine kinase. Effects of enzyme activity, phosphagen type, and substrate concentration.
ADP is an important regulator of hepatic metabolism. Despite its importance the level of free ADP in the liver remains controversial. Recently, we engineered transgenic mice which express high levels of creatine kinase in liver. The reaction catalyzed by creatine kinase was assumed to be at equilibrium and used to calculate a free ADP level of 0.059 mumol/g wet weight. In this report we test the equilibrium assumption by studying the free ADP level as a function of enzyme activity or substrate content. Over a 5-fold range of creatine kinase activity, from 150-800 mumol/min/g wet weight, there was no change in the free ADP level. The average value of ADP for these mice was 0.061 +/- 0.016 mumol/g wet weight. Similarly, altering hepatic creatine content from 1.6 to 30 mumol/g wet weight had no effect on the calculated total free ADP level. The average value of ADP for the creatine levels was 0.048 +/- 0.015 mumol/g wet weight. Finally, the free ADP level was calculated using the equilibrium with cyclocreatine rather than creatine as substrate. The equilibrium of the reaction with cyclocreatine lies 30 times more toward phosphorylation than does the equilibrium with creatine. A free ADP level of 0.063 +/- 0.031 mumol/g wet weight was calculated using cyclocreatine. This value is not different from that found with creatine. These results show that the equilibrium assumption used to calculate free ADP levels in transgenic mouse liver is valid, and the presence of creatine kinase does not affect ADP levels.
Brosnan
M J
MJ
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
Chen
L
L
Van Dyke
T A
TA
Koretsky
A P
AP
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D003401:Creatine / Q000378:metabolism / Q000494:pharmacology; D003402:Creatine Kinase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D051379:Mice; D008822:Mice, Transgenic; D010725:Phosphocreatine / Q000378:metabolism
D010725:Phosphocreatine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D003402:Creatine Kinase; D003401:Creatine