Assay of BCKDH activity
Livers were removed, frozen in liquid nitrogen, and stored at -80°C. At the time of enzyme assay, livers were thawed, and homogenized (1:9, w/v) in 0.25 M sucrose, 10 mM Tris-HC1, pH 7.4. Liver homogenates were centrifuged at 600 × g for 10 min at 4°C and the supernatant fraction was saved to determine the BCKDH activity. The use of tissue homogenates was necessitated by the limited availability of liver tissue, particularly from newborn pups. BCKDH activity was determined by measuring the release of 14CO2 from α-keto [1-14C] isocaproate as described previously [31]. The complete reaction mixture contained (final volume 1 ml) 30 mM potassium phosphate buffer, pH 7.4, 0.20 mM α-ketoisocaproate, 0.40 mM CoASH, 0.40 mM thiamin pyrophosphate, 2 mM NAD+, 2 mM dithiothreitol, 5 mM Mg2+, approximately 250,000 DPM of α-keto [1-14C] isocaproate, and 0.10 ml of liver homogenate (2–3 mg protein). Assays were carried out for 15 min at 37°C, 14CO2 was trapped in hydroxide of Hyamine, and radioactivity was determined by liquid scintillation spectrometry.