Conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active complex by the phosphate reaction in heart mitochondria is inhibited by alloxan-diabetes or starvation in the rat.

1. The conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active (dephosphorylated) complex by pyruvate dehydrogenase phosphate phosphatase is inhibited in heart mitochondria prepared from alloxan-diabetic or 48h-starved rats, in mitochondria prepared from acetate-perfused rat hearts and in mitochondria prepared from normal rat hearts incubated with respiratory substrates for 6 min (as compared with 1 min). 2. This conclusion is based on experiments with isolated intact mitochondria in which the pyruvate dehydrogenase kinase reaction was inhibited by pyruvate or ATP depletion (by using oligomycin and carbonyl cyanide m-chlorophenylhydrazone), and in experiments in which the rate of conversion of inactive complex into active complex by the phosphatase was measured in extracts of mitochondria. The inhibition of the phosphatase reaction was seen with constant concentrations of Ca2+ and Mg2+ (activators of the phosphatase). The phosphatase reaction in these mitochondrial extracts was not inhibited when an excess of exogenous pig heart pyruvate dehydrogenase phosphate was used as substrate. It is concluded that this inhibition is due to some factor(s) associated with the substrate (pyruvate dehydrogenase phosphate complex) and not to inhibition of the phosphatase as such. 3. This conclusion was verified by isolating pyruvate dehydrogenase phosphate complex, free of phosphatase, from hearts of control and diabetic rats an from heart mitochondria incubed for 1min (control) or 6min with respiratory substrates. The rates of re-activation of the inactive complexes were then measured with preparations of ox heart or rat heart phosphatase. The rates were lower (relative to controls) with inactive complex from hearts of diabetic rats or from heart mitochondria incubated for 6min with respiratory substrates. 4. The incorporation of 32Pi into inactive complex took 6min to complete in rat heart mitocondria. The extent of incorporation was consistent with three or four sites of phosphorylation in rat heart pyruvate dehydrogenase complex. 5. It is suggested that phosphorylation of sites additional to an inactivating site may inhibit the conversion of inactive complex into active complex by the phosphatase in heart mitochondria from alloxan-diabetic or 48h-starved rats or in mitochondria incubated for 6min with respiratory substrates.     

Pyruvate dehydrogenase kinase/activator in rat heart mitochondria, Assay, effect of starvation, and effect of protein-synthesis inhibitors of starvation.

Purified pig heart pyruvate dehydrogenase complex is denuded of its intrinsic pyruvate dehydrogenase kinase activity by sedimentation from dilute solution (60 munits/ml). Kinase activity is restored by a supernatant fraction prepared by high-speed centrifugation of rat heart mitochondrial extracts; the factor responsible is referred to as kinase/activator. Kinase/activator was also assayed by its ability to accelerate NgATP-induced inactivation in dilute solutions of unprocessed complex (50 munits/ml). With this assay it has been shown that the activity of kinase/activator in heart mitochondria is increased 3-6 fold by starvation of rats for 48 h. This increase was prevented completely by cycloheximide treatment and prevented partially by puromycin treatment of rats during starvation. The concentration of kinase/activator in heart mitochondria fell during 20 h of re-feeding of 48 h-starved rats; this fall was correlated with an increase in the proportion of complex in the active form. Kinase/activator was also extracted from ox kidney mitochondria, and on gel filtration (Sephadex G-100, superfine grade) was eluted close to the void volume. Kinase/activator (ox kidney or rat heart) was thermolabile, non-diffusable on dialysis, and inactivated by trypsin. The results of this study appear to show increased cytoplasmic synthesis in starvation of pyruvate dehydrogenase kinase and/or of an activator of the kinase.     

Purification and characterization of a divalent cation-independent, spermine-stimulated protein phosphatase from bovine kidney mitochondria

A divalent cation-independent and spermine-stimulated phosphatase (protein phosphatase SP) that is active toward the phosphorylated pyruvate dehydrogenase complex has been purified about 15,000-fold to near homogeneity from extracts of bovine kidney mitochondria. Half-maximal stimulation, 1.5- to 3-fold at pH 7.0-7.3, occurred at 0.5 mM spermine. Protein phosphatase SP exhibited an apparent Mr = 140,000-170,000 as estimated by gel-filtration chromatography on Sephacryl S-300. Two major subunits, with apparent Mr = 60,000 and 34,000, were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel-permeation chromatography of protein phosphatase SP on Sephacryl S-200 in the presence of 6 M urea and 1.4 M NaCl increased its activity 3- to 6-fold and was accompanied by conversion to the catalytic subunit with an apparent Mr = approximately 34,000. Protein phosphatase SP was inactive with p-nitrophenyl phosphate and was not inhibited by protein phosphatase inhibitor 1, inhibitor 2, or the protein inhibitor of branched-chain alpha-keto acid dehydrogenase phosphatase. Protein phosphatase SP was inhibited by sheep antibody to the catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle. It appears that protein phosphatase SP is related to protein phosphatase 2A.     

Purification and properties of a protamine kinase and a type II casein kinase from bovine kidney mitochondria

Bovine kidney mitochondrial extracts contain an inactive protamine kinase and an inactive casein kinase. The protamine kinase was activated by chromatography on poly(L-lysine)-agarose. Two forms of this soluble mitochondrial protamine kinase were separated by chromatography on protamine-agarose. Both forms were purified about 80,000-fold to apparent homogeneity. Both forms of the protamine kinase consist of a single polypeptide chain with an apparent Mr approximately 45,000. Both enzyme forms underwent autophosphorylation without significant effect on activity, and both forms exhibited identical substrate specificities. The protamine kinase showed little activity toward branched-chain alpha-keto acid dehydrogenase (less than 3%), and it was essentially inactive (less than 0.1%) with pyruvate dehydrogenase, casein, and ovalbumin. The enzyme was active with histone H1 and with bovine serum albumin. Protamine kinase activity was unaffected by heparin (up to 100 micrograms/ml), by the protein inhibitor of cyclic AMP-dependent protein kinase, by Ca2+ and calmodulin, and by monoclonal antibody to the catalytic domain of protein kinase C from rat brain. The casein kinase was activated in the presence of spermine or by chromatography of the extract on DEAE-cellulose or poly(L-lysine)-agarose. The enzyme was purified about 80,000-fold to apparent homogeneity. It exhibited an apparent Mr 130,000 as determined by gel-permeation chromatography on Sephacryl S-300 in the presence of 0.5 M NaCl. Two subunits, with apparent Mr's 36,000 (alpha) and 28,000 (beta) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The kinase underwent autophosphorylation of its beta-subunit, without significant effect on activity. Casein kinase activity was inhibited 50% by 1.5 micrograms/ml of heparin. Spermine (1.0 mM) stimulated activity of the purified kinase two- to three-fold at 1.5 mM Mg2+. Half-maximal stimulation occurred at 0.1 mM spermine. The kinase utilized both ATP and GTP as substrates. The casein kinase showed little activity (less than 1%) toward pyruvate dehydrogenase and branched-chain alpha-keto acid dehydrogenase from kidney mitochondria, and the kinase was essentially inactive with glycogen synthase a. The properties of this soluble mitochondrial kinase indicate that it is a type II casein kinase.     

Rat liver mitochondria contain two immunologically distinct dihydrolipoamide dehydrogenases

We have raised antisera against dihydrolipoamide dehydrogenase. One antigen was isolated from purified bovine kidney pyruvate dehydrogenase complex (PDC). The other antigen was a commercial preparation of porcine heart dihydrolipoamide dehydrogenase (E3) which did not first involve purification of the alpha-keto acid dehydrogenase complex(es). Both antibody preparations cross-reacted with the E3 components of PDC, alpha-ketoglutarate dehydrogenase complex, and branched-chain keto acid dehydrogenase complex. This demonstrates the immunological identity of the E3 components. These sera totally precipitated E3 activity from the purified complexes, from purified preparations of E3, and from extracts of rat heart and kidney mitochondria. The two sera vary in their reaction with rat liver mitochondrial extracts: the anti PDC-E3 serum left residual E3 activity (approximately 50% of the original) that was precipitable by the anti-E3 anti-serum. This indicates that liver contains two immunologically distinct forms of E3. Metabolic assays measuring the differential effects of the two sera on the glycine decarboxylation reaction suggest that the form which is immunologically nonreactive with the anti-PDC-E3 serum could represent the E3 involved in the glycine cleavage system.     

Role of branched-chain 2-oxo acid dehydrogenase and pyruvate dehydrogenase in 2-oxobutyrate metabolism.

Purified branched-chain 2-oxo acid dehydrogenase (BCODH) and pyruvate dehydrogenase (PDH) had apparent Km values (microM) for 2-oxobutyrate of 26 and 114, with a relative Vmax. (% of Vmax. for 3-methyl-2-oxobutyrate and pyruvate) of 38 and 45% respectively. The phosphorylation state of both complexes in extracts of mitochondria from rat liver, kidney, heart and skeletal muscle was shown to influence oxidative decarboxylation of 2-oxobutyrate. Inhibitory antibodies to BCODH and an inhibitor of PDH (3-fluoropyruvate) were used with mitochondrial extracts to determine the relative contribution of both complexes to oxidative decarboxylation of 2-oxobutyrate. Calculated rates of 2-oxobutyrate decarboxylation in mitochondrial extracts, based on the kinetic constants given above and the activities of both complexes, were the same as the measured rates. Hydroxyapatite chromatography of extracts of mitochondria from rat liver revealed only two peaks of oxidative decarboxylation of 2-oxobutyrate, with one peak associated with PDH and the other with BCODH. Competition studies with various 2-oxo acids revealed a different inhibition pattern with mitochondrial extracts from liver compared with those from heart or skeletal muscle. We conclude that both intramitochondrial complexes are responsible for oxidative decarboxylation of 2-oxobutyrate. However, the BCODH is probably the more important complex, particularly in liver, on the basis of kinetic analyses, activity or phosphorylation state of both complexes, competition studies, and the apparent physiological concentration of pyruvate, 2-oxobutyrate and the branched-chain 2-oxo acids.     

Effects of dietary protein intake on branched-chain keto acid dehydrogenase activity of the rat. Immunochemical analysis of the enzyme complex

Polyclonal antibodies directed against the dihydrolipoyl transacylase (E2) and alpha subunit of branched-chain alpha-keto acid decarboxylase (E1 alpha) components of the bovine branched-chain keto acid dehydrogenase complex were shown to cross-react with the E2 and E1 alpha polypeptides of the enzyme complex of different rat tissues. Phosphorylation of the branched-chain keto acid dehydrogenase complex resulted in inhibition of enzyme activity concomitant with phosphate incorporation into the E1 alpha polypeptide. Phosphorylation of E1 alpha slowed its rate of migration through sodium dodecyl sulfate-polyacrylamide gels. This permitted resolution of the phosphorylated and unphosphorylated forms of E1 alpha on immunoblots. Liver and skeletal muscle mitochondria were prepared from rats consuming 6, 20, or 50% casein diets. The enzyme complex in mitochondria was measured by radioisotopic enzyme assay and immunoassay. Liver branched-chain keto acid dehydrogenase was 25% active in rats consuming 6% casein diets; whereas in rats consuming 20 or 50% casein diets, the liver enzyme was 82 or 100% active, respectively. Branched-chain keto acid dehydrogenase of muscle was 10, 13, and 22% active, respectively, in rats consuming 6, 20, and 50% casein diets. The amount of protein consumed by rats did not affect the total amount of the enzyme complex per unit of mitochondrial protein as measured by either the radioisotopic assay (enzyme activity) or the immunoassay. However, the protein intake of rats did affect activity of the enzyme kinase in liver. Liver branched-chain keto acid dehydrogenase kinase was more active in rats consuming 6% casein than in those fed chow or 50% casein diets. The amount of protein consumed by rats thus influences the enzyme activity in liver and muscle by affecting the reversible phosphorylation mechanism and not by induction of branched-chain keto acid dehydrogenase.     

Branched-chain alpha-ketoacid dehydrogenase kinase. Molecular cloning, expression, and sequence similarity with histidine protein kinases.

A cDNA for branched-chain alpha-ketoacid dehydrogenase kinase was cloned from a rat heart cDNA library. The cDNA had an open reading frame encoding a protein of 382 amino acid residues with a calculated molecular weight of 43,280. The clone codes for the branched-chain alpha-ketoacid dehydrogenase kinase based on the following: 1) the deduced amino acid sequence contained the partial sequence of the kinase determined by direct sequencing; 2) expression of the cDNA in Escherichia coli resulted in synthesis of a 43,000-Da protein that was recognized specifically by kinase antibodies; and 3) enzyme activity that phosphorylated and inactivated the branched-chain alpha-ketoacid dehydrogenase complex was found in extracts of E. coli expressing the protein. Northern blot analysis indicated the mRNA for the branched-chain alpha-ketoacid dehydrogenase kinase was more abundant in rat heart than in rat liver, as expected from the relative amounts of kinase activity expressed in these tissues. The deduced sequence of the kinase aligned with a high degree of similarity within subdomains characteristic of procaryotic histidine protein kinases. This first mitochondrial protein kinase to be cloned appears more closely related in sequence to procaryotic histidine protein kinases than to eucaryotic serine/threonine protein kinases.     

Mitochondrial 2-oxoacid dehydrogenase complexes of animal tissues

The pyruvate dehydrogenase and branched-chain 2-oxoacid dehydrogenase complexes of animal mitochondria are inactivated by phosphorylation of serine residues, and reactivated by dephosphorylation. In addition, phosphorylated branched-chain complex is reactivated, apparently without dephosphorylation, by a protein or protein-associated factor present in liver and kidney mitochondria but not in heart or skeletal muscle mitochondria. Interconversion of the branched-chain complex may adjust the degradation of branched-chain amino acids in different tissues in response to supply. Phosphorylation is inhibited by branched-chain ketoacids, ADP and TPP. The pyruvate dehydrogenase complex is almost totally inactivated (99%) by starvation or diabetes, the kinase reactions being accelerated by products of fatty acid oxidation and by a protein or protein-associated factor induced by starvation or diabetes. There are three sites of phosphorylation, but only sites 1 and 2 are inactivating. Site 1 phosphorylation accounts for 98% of inactivation except during dephosphorylation when its contribution falls to 93%. Sites 2 and 3 are only fully phosphorylated when the complex is fully inactivated (starvation, diabetes). Phosphorylation of sites 2 and 3 inhibits reactivation by phosphatase. The phosphatase reaction is activated by Ca2+ (which may mediate effects of muscle work) and possibly by uncharacterized factors mediating insulin action in adipocytes.     

The subcellular location of isozymes of NADP-isocitrate dehydrogenase in tissues from pig, ox and rat

Antibodies against purified NADP-isocitrate dehydrogenase from pig liver cytosol and pig heart were raised in rabbits. The purified enzymes from these sources are different proteins, as demonstrated by differences in electrophoretic mobility and absence of crossreactivity by immunotitration and immunodiffusion. The NADP-isocitrate dehydrogenase in the soluble supernatant homogenate fraction from pig liver, kidney cortex, brain and erythrocyte hemolyzate was identical with the purified enzyme from pig liver cytosol, as determined by electrophoretic mobility and immunological techniques. The enzyme in extracts of mitochondria from pig heart, kidney, liver and brain was identical with the purified pig heart enzyme by the same criteria. However, the 'mitochondrial' isozyme was the major component also in the soluble supernatant fraction of pig heart homogenate. The 'cytosolic' isozyme accounted for only 1-2% of total NADP-isocitrate dehydrogenase in pig heart, as determined by separation of the isozymes with agarose gel electrophoresis and immunotitration. The mitochondrial isozyme was also the predominant NADP-isocitrate dehydrogenase in porcine skeletal muscle. The ratio of cytosolic/mitochondrial isozyme for porcine whole tissue extract, determined by immunotitration, was about 2 for liver and 1 for kidney cortex and brain. The distribution of isozymes in cell homogenate fractions from ox and rat tissues corresponded to that observed in organs of porcine origin. The mitochondrial and cytosolic isozymes from ox and rat tissues exhibited crossreactivity with the antibodies against the pig heart and pig liver cytosol enzyme, respectively, and the electrophoretic migration patterns were similar qualitatively to those found for the isozymes in porcine tissues. Nevertheless, there were species specific differences in the characteristics of each of the corresponding isozymes. NAD-isocitrate dehydrogenase was not inhibited by the antibodies, confirming that the protein is distinct from that of either isozyme of NADP-isocitrate dehydrogenase.     

Low immunogenicity of the common lipoamide dehydrogenase subunit (E3) of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes.

The production of high-titre monospecific polyclonal antibodies against the purified pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart is described. The specificity of these antisera and their precise reactivities with the individual components of the complexes were examined by immunoblotting techniques. All the subunits of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes were strongly antigenic, with the exception of the common lipoamide dehydrogenase component (E3). The titre of antibodies raised against E3 was, in both cases, less than 2% of that of the other subunits. Specific immunoprecipitation of the dissociated N-[3H]ethylmaleimide-labelled enzymes also revealed that E3 alone was absent from the final immune complexes. Strong cross-reactivity with the enzyme present in rat liver (BRL) and ox kidney (NBL-1) cell lines was observed when the antibody against ox heart pyruvate dehydrogenase was utilized to challenge crude subcellular extracts. The immunoblotting patterns again lacked the lipoamide dehydrogenase band, also revealing differences in the apparent Mr of the lipoate acetyltransferase subunit (E2) from ox kidney and rat liver. The additional 50 000-Mr polypeptide, previously found to be associated with the pyruvate dehydrogenase complex, was apparently not a proteolytic fragment of E2 or E3, since it could be detected as a normal component in boiled sodium dodecyl sulphate extracts of whole cells. The low immunogenicity of the lipoamide dehydrogenase polypeptide may be attributed to a high degree of conservation of its primary sequence and hence tertiary structure during evolution.     

Purification and properties of the catalytic subunit of the branched-chain alpha-keto acid dehydrogenase phosphatase from bovine kidney mitochondria

The catalytic subunit of the branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase (Damuni, Z., Merryfield, M.L., Humphreys, J.S., and Reed, L.J., (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4335-4338) has been purified over 50,000-fold from extracts of bovine kidney mitochondria. The apparently homogeneous protein consists of a single polypeptide chain with an apparent Mr = approximately 33,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. BCKDH phosphatase, with an apparent Mr = 460,000, was dissociated to its catalytic subunit with no apparent change in activity, at an early stage in the purification procedure by treatment with 6 M urea. The specific activity of the catalytic subunit was 1,500-2,500 units/mg. The catalytic subunit exhibited approximately 10% maximal activity with 32P-labeled pyruvate dehydrogenase complex but was inactive with phosphorylase a and with p-nitrophenyl phosphate. The catalytic subunit, like the Mr = 460,000 species, was inhibited by nanomolar concentrations of BCKDH phosphatase inhibitor protein, was unaffected by protein phosphatase inhibitor 1 and inhibitor 2, and was inhibited by nucleoside tri- and diphosphates but not by nucleoside monophosphates.     

Effects of diet and of alloxan-diabetes on the activity of branched-chain 2-oxo acid dehydrogenase complex and of activator protein in rat tissues.

The total activities (sum of active and inactive forms) of branched-chain 2-oxo acid dehydrogenase complex in tissues of normal rats fed on a standard diet were (unit/g wet wt.): liver, 0.82; kidney, 0.77; heart, 0.57; hindlimb skeletal muscles, 0.034. Total activity was decreased in liver by 9%- or 0%-casein diets and by 48 h starvation, but not by alloxan-diabetes. Total activities were unchanged in kidney and heart. The amount of active form of the complex (in unit/g wet wt. and as % of total) in tissues of normal rats fed on standard diet was: liver, 0.45, 55%; kidney, 0.55, 71%; heart, 0.03, 5%; skeletal muscle less than 0.007, less than 20% (below lower limit of assay). The concentration of the active form of the complex was decreased in liver and kidney, but not in heart, by low-protein diets, 48 h starvation and alloxan-diabetes. In heart muscle alloxan-diabetes increased the concentration of active complex. The concentration of activator protein (which activates phosphorylated complex without dephosphorylation) in liver and kidney was decreased by 70-90% by low-protein diets and 48 h starvation. Alloxan-diabetes decreased activator protein in liver, but not in kidney. Evidence is given that in tissues of rats fed on a normal diet approx. 70% of whole-body active branched chain complex is in the liver and that the major change in activity occasioned by low-protein diets is also in the liver.     

Dephosphorylation and reactivation of phosphorylated purified ox-kidney branched-chain dehydrogenase complex by co-purified phosphatase

The branched-chain 2 oxoacid dehydrogenase complex has been purified from well-washed ox-kidney mitochondria together with branched-chain dehydrogenase kinase. The complex was inactivated and phosphorylated by ATP in about 5 min at 30 degrees C. After hydrolysis of ATP by a contaminating ATPase (5-10 min) the complex was dephosphorylated and reactivated. Dephosphorylation and reactivation were linearly correlated. Reactivation was dependent upon Mg2+ (K0.5 greater than 1 mM) and inhibited completely by 50 mM fluoride. Reactivation and dephosphorylation are attributed to a mitochondrial branched-chain dehydrogenase phosphatase.     

Purification and partial characterization of branched-chain alpha-ketoacid dehydrogenase kinase from rat liver and rat heart

Branched-chain alpha-ketoacid dehydrogenase kinase was purified to homogeneity from rat liver and rat heart. The initial step was the purification of rat liver and heart branched-chain alpha-ketoacid dehydrogenase complex with high kinase activity by a modification of a method described previously. Preservation of high kinase activity during purification of the complex required the presence of fresh dithiothreitol throughout the procedure. The kinase was released from the complex by oxidation of dithiothreitol with potassium ferricyanide and purified by high-speed centrifugation, immunoadsorption chromatography, and DEAE-Sephacel chromatography. Both kinase preparations gave only one polypeptide band with a molecular weight of 44,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase complex by the purified kinase was inhibited by alpha-chloroisocaproate and dichloroacetate, established inhibitors of the phosphorylation of the branched-chain alpha-ketoacid dehydrogenase complex. The kinase did not exhibit autophosphorylation and does not correspond to the same protein as pyruvate dehydrogenase kinase. The kinase phosphorylated histone (type II-S), but this reaction was slow relative to the phosphorylation of the branched-chain alpha-ketoacid dehydrogenase complex and was not inhibited by alpha-chloroisocaproate.     

Phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in isolated adipocytes. Effects of 2-oxo acids.

Isolated adipocytes from rat epididymal fat-pads were incubated with [32P]Pi, and intracellular phosphoproteins were then analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography. A phosphorylated polypeptide of apparent Mr 46,000 was identified as the alpha-subunit of branched-chain 2-oxo acid dehydrogenase complex by immunoprecipitation using antiserum raised against the homogeneous E1 component of branched-chain 2-oxo acid dehydrogenase complex. Immunoprecipitation of this phosphoprotein is blocked in a competitive manner by purified branched-chain 2-oxo acid dehydrogenase complex. Peptide mapping of the isolated phosphoprotein indicates that two sites on the polypeptide are phosphorylated in the intact cells. Addition of branched-chain 2-oxo acids to the incubation medium causes diminution in the extent of labelling of both phosphorylation sites on the alpha-subunit, an effect presumably mediated via their known inhibitory action on branched-chain 2-oxo acid dehydrogenase kinase. These observations provide direct evidence for phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in intact cells.     

Kinase activator protein mediates longer-term effects of starvation on activity of pyruvate dehydrogenase kinase in rat liver mitochondria.

Starvation of rats for 48 h increased the activity of PDH (pyruvate dehydrogenase) kinase 2.2-fold in extracts of liver mitochondria, 2.9-fold in PDH complex partially purified therefrom by fractional precipitation, and 5-fold in PDH complex partially purified by gel filtration on Sephacryl S-300. A protein fraction was separated from PDH complex in extracts of rat liver mitochondria by gel filtration or fractional precipitation, which increased the activity of PDH kinase in rat liver and pig heart PDH complexes. The activity of this protein fraction was increased approx. 2.5-fold by 48 h starvation of rats. With highly purified pig heart PDH complex it was shown that the protein fraction increased the Vmax. of the PDH kinase reaction 35-fold (fraction from fed rats) or 82-fold (fraction from starved rats); starvation had no effect on the concentration of protein fraction required to give 0.5 Vmax. Evidence is given that the increase in PDH kinase activity effected in extracts of liver mitochondria by starvation is due to increased activity of kinase activator protein, which is tightly bound by rat liver PDH complex and not removed by a single gel filtration. With pig heart PDH complex, increased PDH kinase activity was retained after gel filtration of an admixture with kinase activator protein from starved rats, but was restored to the control value by a second gel filtration; the alterations in PDH kinase activity were associated with obvious changes in protein bands in SDS gels.     

Evidence for conservation of dietary lipid in the rat during lactation and the immediate period after removal of the litter. Decreased oxidation of oral [1-14C]triolein.

The activity of pyruvate dehydrogenase kinase in extracts of mitochondria from rat hepatocytes cultured for 21 h in medium 199 was increased 2.5-fold by the presence of 55 nM-glucagon and 1 mM-sodium n-octanoate in the culture medium. The change was comparable with that induced in vivo by 48 h starvation. The potential contribution of branched-chain complex to estimates of PDH-complex activity in rat liver mitochondria has been defined.     

The regulation of branched-chain 2-oxo acid dehydrogenase of liver, kidney and heart by phosphorylation.

1. Incubation of mitochondria from heart, liver and kidney with [32P]phosphate allowed 32P incorporation into two intramitochondrial proteins, the decarboxylase alpha-subunit of the pyruvate dehydrogenase complex (mol.wt 42000) and a protein of mol.wt. 48000. 2. This latter protein incorporated 32P more slowly than did pyruvate dehydrogenase, was not precipitated by antibody to pyruvate dehydrogenase and showed behaviour distinct from that of pyruvate dehydrogenase towards high-speed centrifugation and pyruvate dehydrogenase phosphate phosphatase. 3. 32P incorporation into the protein was greatly diminished by the presence of 0.1 mM-4-methyl-2-oxopentanoate, but enhanced by pyruvate (1 mM), hypo-osmotic treatment of mitochondria and, under some conditions, by uncoupler. 4. The activity of branched-chain 2-oxo acid dehydrogenase was assayed in parallel experiments. Under appropriate conditions the enzyme was inhibited when 32P incorporation was increased and activated when incorporation was decreased. The data suggest that the 48000-mol.wt. phosphorylated protein is identical with the decarboxylase subunit of branched-chain 2-oxo acid dehydrogenase and that this enzyme may be controlled by a phosphorylation-dephosphorylation cycle akin to that for pyruvate dehydrogenase. 5. Strict correlation between activity and 32P incorporation was not observed, and a scheme for the regulation of the enzyme is proposed to account for these discrepancies.