BIOCi-IEMiSTRY 52, 30&310 (19%) A Sensitive Assay for the Determination of Propionyl-CoA in Biological Material Propionyl-CoA is a regular intermediate in the metabolism of the branched chain amino acid isoleucine in higher organisms and a metabolite in the catabolism of L-valine in Pseudomonas aeruginosa (1). Moreover, propionyl-CoA is an important intermediate in the formation of glucose from propionate in ruminants. From experiments of Scrutton and Utter (2) it is known that liver pyruvate carboxylase is allosterically activated not only by acetyl-CoA but also by propionyl-Coil.
We therefore developed an assay which allows the measurement of tissue concentrations of propionyl-CoA as low as 1 nmole/g wet tissue. This assay can easily be adapted to even higher sensitivity. '
Methods and Results.1 (1) Preparation of propionyl-CoA carboxylase (EC 6.4.1.3.). This enzyme was prepared from fresh beef liver by the method described by Giorgio and Plaut (3) with some minor modifications: The acetone powder was extracted for 120 min. For the first DEAE-cellulose step 2 g of DEAE-cellulose per g acetone powder, and for the second step 1 g of DEAE-cellulose per g of acetone powder, were used. After ammonium sulfate fractionation and for gel filtration 0.1 M potassium phosphate buffer was used because the enzyme was much more stable with this higher ionic strength.
The purification was between 160-and 200-fold and the final specific activity ranged from 16.6 to 20.0 U/mg protein. Concentrated enzyme solutions in 0.1 M potassium phosphate, pH 7.0 with 0.5 mM Cleland's reagent and 1 m&r MgCl, lost less than 10% of their activity during storage at -20°C over a period of up to 3 mo.
(2) Preparation of propionyl-Coil.
'All biochemicala used were purchased from Boehringer Corp., Mannheim-Waldhof, Germany, all other chemicals from E. Merck A. G., Darmstadt, Germany. H"C0, was ordered from Amersham Buchler G.m.b.H.,