Comparison of two methods for the measurement of rat liver methylmalonyl-coenzyme A mutase activity: HPLC and radioisotopic assays.
Methylmalonyl-coenzyme A mutase (MCM) is a 5'-deoxyadenosylcobalamin-linked mitochondrial enzyme that catalyzes the isomerization of L-methylmalonyl-coenzyme A to succinyl-coenzyme A. In vitro assays of total and holo-MCM activities are important tools for investigating the cobalamin pathway. Several methods have been described for measuring MCM activity. The most commonly-used method is a radioassay based on the permanganate oxidation of DL[CH(3)-(14)C]methylmalonyl-coenzyme A, but radiometric methods are insensitive, laborious, and time-consuming. Therefore, we have compared this method with a nonradiometric assay, potentially most sensitive, based on the separation of methylmalonyl-coenzyme A and succinyl-coenzyme A by high performance liquid chromatography (HPLC). We determined the optimal assay conditions and the reproducibility and sensitivity of each technique. The results obtained by the two techniques were very different: the specific activities obtained by the permanganate oxidation method (0.039 +/- 0.013 nmol/min/mg protein for the holo-MCM activity and 1.90 +/- 0.69 nmol/min/mg protein for the total-MCM activity) were threefold lower than those obtained with the HPLC method (0.124 +/- 0.011 nmol/min/mg protein for the holo-MCM activity and 6.15 +/- 0.76 nmol/min/mg protein for the total-MCM activity). The coefficients of variation for the radiometric method (18.4-40.6%) were three to five times greater than those for the HPLC assay (3.5-12.2%). This demonstrates the lack of sensibility and reproducibility of the permanganate radioassay. Thus, the radiometric method is not suitable for measuring low mutase activities such as the holo activities in tissues. The intrinsic inconvenience of the radiometric assay indicates that the HPLC method is a method of choice for measuring MCM activity.
Gaire
D
D
INSERM U. 308, Faculté de Médecine, Avenue de la Forêt de Haye, Vandoeuve Cedex, France.
Sponne
I
I
Droesch
S
S
Charlier
A
A
Nicolas
J P
JP
Lambert
D
D