Methods

Enzyme Assays. The overall activity of the pyruvate dehydrogenase complex was determined by monitoring NADH formation at 340 nm and 30 C (Linn et al., 1972). Activity is based on the initial rate. The activity of the pyruvate dehydrogenase component was determined after reconstituting the pyruvate dehydrogenase complex (Butler et al., 1977). Measurement of protein-bound radioactivity on filter paper disks was carried out as described by Linn et al.(1972). Phosphorylation of Pyruvate Dehydrogenase. Crystalline pyruvate dehydrogenase (154 mg; 1.0^ mol) was incubated at 23 C with 1.5 mg of kinase-dihydrolipoyl transacetylase subcomplex from bovine kidney (Linn et al., 1972), 0.2 mM [t-32P] ATP (20 000 cpm/nmol), 2 mM dithiothreitol, 0.1 mM EDTA, 1*** 1 mM MgCl2, and 20 mM potassium phosphate buffer (pH 7.0) in a final volume of 20 mL. After 3 h of incu-bation, the mixture was dialyzed for 40 h at 4 C against two changes of 0.2 M NH4HCO3 to remove ATP. Time Course of Phosphorylation and Inactivation of the Pyruvate Dehydrogenase Complex. Bovine heart pyruvate dehydrogenase complex (6.0 mg) was incubated at 23 C with 0.1 mM 1T-32P] ATP, 2 mM dithiothreitol, 0.1 mM EDTA, 1.0 mM MgCl2, and 10 mM NaF in 2.0 mL of 20 mM phosphate buffer (pH 7.0). NaF was included to inhibit trace amounts of PDHb phosphatase. Phosphorylation was catalyzed by the endogenous PDHa kinase, and the reaction was started by the addition of [t-32P] ATP. Since the activity of endoge-nous kinase in the bovine kidney pyruvate dehydrogenase complex was about tenfold higher than that of endogenous kinase in the heart complex, the experiments with the kidney complex were carried out in the presence of ADP (ADP/ATP ratio, 4: 1) toreduce the rate of phosphorylation. Aliquots (0.2 mL) were removed at the specified time intervals and added to 300 mM (final concentration) glucoseand 10 qg of hexokinase in 0.1 mL to scavenge the ATP. A small aliquot (1 or 2 mL) was analyzed for NAD-reduction activity, duplicate aliquots (50 qL) were assayed for protein-bound phosphoryl groups, and the remainder of the sample was dialyzed for 40 h at 4 C against 0.2 M NH4HCO3. Controlexperiments showed that under the conditions used hexokinase and glucose rapidly degraded the radioactive ATP and did not affect the enzymatic activity or the 32P content of the complex. The di-alyzed material was digested with trypsin (50: 1, w/w) for 6 h at 23 C, lyophilized, and then subjected to high-voltage paper electrophoresis at pH 1.9 for 40 min. Radioactive peptides were located with Kodak No Screen X-ray film, the spots were cut out, and radioactivity was determined in a Beckman LS-230 scintillation counter with 5-mL portions of ACS cocktail (Amersham-Searle). The content of pyruvate dehydrogenasetetramers in the pyruvate dehydrogenase complex was determined by titrating the thiamin pyrophosphate binding sites, two per tetramer (Butler et al., 1977; Walsh et al., 1976), with thiamin thiazo-lone pyrophosphate (Gutowski and Lienhard, 1976; Butler et al., 1977). The bovine kidney pyruvate dehydrogenase complex contains about 20 pyruvate dehydrogenase tetramers per molecule of complex of Mr about 7 X 106 (L. Hamilton, P. Munk and L. J. Reed, unpublished data), and the bovine heart complex (Mr about 8.5 X 106) contains about 30 pyruvate dehydrogenase tetramers.