Ralph Paxton
Indiana University
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Archives of Biochemistry and Biophysics | 1984
Ralph Paxton; Robert A. Harris
Isolated rabbit liver branched-chain α-ketoacid dehydrogenase was inhibited in a mixed manner relative to ATP by α-ketoisocaproate, α-keto-β-methylvalerate, α-ketoisovalerate, α-ketocaproate, α-ketovalerate, and α-chloroisocaproate with I40 values mm), respectively, of 0.065, 0.49, 2.5, 0.2, 0.5, and 0.08. The concentration (mm) of α-ketoisocaproate, α-keto-β-methylvalerate, and α-ketoisovalerate needed to activate branched-chain α-ketoacid dehydrogenase in the perfused rat heart to 50% of total activity was 0.07, 0.10, and 0.25, respectively. Isolated branched-chain α-ketoacid dehydrogenase kinase was inhibited (I40 values, mm) by octanoate (0.5), acetoacetyl-CoA (0.01), methylmalonyl-CoA (0.2), NADP+ (1.5), and heparin (12 μg/ml). The kinase activity, in the presence or absence of ADP, was inhibited approximately 30% by 0.1 mm isobutyryl-CoA, isovaleryl-CoA, and malonyl-CoA, while not affected by NAD+ and NADH (1 mm), CoA, acetyl-CoA, methylcrotonyl-CoA, crotonyl-CoA, β-hydroxy-β-methyl-glutaryl-CoA, octanoyl-CoA, succinyl-CoA, and propionyl-CoA (0.1 mm). The following compounds at 2 mm also did not inhibit branched-chain α-ketoacid dehydrogenase kinase; acetate, propionate, β-hydroxybutyrate, lactate, acetoacetate, malonate, α-ketomalonate, succinate, citrate, oxaloacetate, FAD, and NADPH. These findings help explain the unique effects of Leu compared with Val and Ile on branched-chain amino acid metabolism and the differences between control of the kinases associated with pyruvate dehydrogenase and branched-chain α-ketoacid dehydrogenase.
Biochemical and Biophysical Research Communications | 1983
Sarah E. Gillim; Ralph Paxton; George A. Cook; Robert A. Harris
The proportion of active (unphosphorylated) branched chain alpha-ketoacid dehydrogenase was determined in tissues from rats in different metabolic states. Hearts from normal, high-protein, and low-protein fed rats contained about 45% of the enzyme in the active form. Only 10-20% of the enzyme was active in hearts of fasted and diabetic rats. Virtually all of the liver enzyme was in the active form in fed, fasted, diabetic and high-protein fed animals. Protein starved rats, however, exhibited a dramatic decrease in both the % active form and total amount of liver enzyme. Kidneys from normal, fasted, diabetic and high-protein fed rats contained 70-80% of the enzyme in the active form. The % active form of the kidney enzyme decreased in protein starved rats, but less dramatically than in liver. Covalent modification is concluded to be important for in vivo regulation of the branched chain alpha-ketoacid dehydrogenase complex.
Archives of Biochemistry and Biophysics | 1986
Ralph Paxton; Martha J. Kuntz; Robert A. Harris
Branched-chain alpha-ketoacid dehydrogenase complex was isolated from rat heart, bovine kidney, and rabbit liver, heart, kidney, brain, and skeletal muscle. Phosphorylation to approximately 1 mol Pi/mol alpha-subunit of the alpha-ketoacid decarboxylase component was linearly associated with 90-95% inactivation. The complex from some tissues (i.e., from rabbit kidney and heart, and rat heart) showed 30-40% more phosphate incorporation for an additional 5-10% inactivation. Reverse-phase HPLC analysis of tryptic digests of 32P-labeled complexes from all of the above tissues revealed two major (peaks 1 and 2) and one minor (peak 3) phosphopeptide which represent phosphorylation sites 1, 2, and a combination of 1 and 2, respectively. These phosphopeptides, numbered according to the order of elution from reverse-phase HPLC, had the same elution time regardless of the tissue or animal source of the complex. The amino acid sequence of site 1 from rabbit heart branched-chain alpha-ketoacid dehydrogenase was Ile-Gly-His-His-Ser(P)-Thr-Ser-Asp-Asp-Ser-Ser-Ala-Tyr-Arg. Regardless of the source of the complex, both sites were almost equally phosphorylated until total phosphorylation was approximately 1 mol Pi/mol of alpha-subunit and the rate of inactivation was correlated with the rate of total, site 1, or site 2 phosphorylation. Phosphorylation beyond this amount was associated with greater site 2 than site 1 phosphorylation. alpha-Chloroisocaproate, a potent inhibitor of branched-chain alpha-ketoacid dehydrogenase kinase activity, greatly reduced total phosphorylation and inactivation; however, phosphorylation of site 2 was almost abolished and inactivation was directly correlated with phosphorylation of site 1. Thus, the complex isolated from different tissues and mammals had an apparent conservation of amino acid sequence adjacent to the phosphorylation sites. Both sites were phosphorylated to a similar extent temporally although site 1 phosphorylation was directly responsible for inactivation.
Advances in Enzyme Regulation | 1986
Robert A. Harris; Ralph Paxton; Stephen M. Powell; Gary W. Goodwin; Martha J. Kuntz; Amy Han
Abstract The branched-chain α-ketoacid dehydrogenase complex, like the pyruvate dehydrogenase complex, is an intramitochondrial enzyme subject to regulation by covalent modification. Phosphorylation causes inactivation and dephosphorylation causes activation of both complexes. The branched-chain α-ketoacid dehydrogenase kinase, believed distinct from pyruvate dehydrogenase kinase, is an integral component of the branched-chain α-ketoacid dehydrogenase complex and is sensitive to inhibition by branched-chain α-ketoacids, α-chloroisocaproate, phenylpyruvate, clofibric acid, octanoate and dichloroacetate. Phosphorylation of branched-chain α-ketoacid dehydrogenase occurs at two closely-linked serine residues (sites 1 and 2) of the α-subunit of the decarboxylase. HPLC and sequence data suggest homology of the amino acid sequence adjacent to phosphorylation sites 1 and 2 of complexes isolated from several different tissues. Stoichiometry for phosphorylation of all of the complexes studies was about 1 mol P/mol α-subunit for 95% inactivation and 1.5 mol P/mol α-subunit for maximally phosphorylated complex. Site 1 and site 2 were phosphorylated at similar rates until total phosphorylation exceeded 1 mol P/mol α-subunit. The complexes from rabbit kidney, rabbit heart, and rat heart showed 30–40% additional phosphorylation of the α-subunit beyond 95% inactivation. Site specificity studies carried out with the kinase partially inhibited with α-chloroisocaproate suggest that phosphorylation of site 1 is primarily responsible for regulation of the complex. The capacity of the branched-chain α-ketoacid dehydrogenase to oxidize pyruvate ( K m = 0.8 m m , V max = 20% of that of α-ketoisovalerate) interferes with the estimation of activity state of the hepatic pyruvate dehydrogenase complex. The disparity between the activity states of the two complexes in most physiologic states contributes to this interference. An inhibitory antibody for branched-chain α-ketoacid dehydrogenase can be used to prevent interference with the pyruvate dehydrogenase assay. Almost all of the hepatic branched-chain α-ketoacid dehydrogenase in chow-fed rats is active (> 90% dephosphorylated). In contrast, almost all of the hepatic enzyme of rats fed a low-protein (8%) diet is inactive (> 85% phosphorylated). Fasting of chow-fed rats has no effect on the activity state of hepatic branched-chain α-ketoacid dehydrogenase, i.e. > 90% of the enzyme remains in the active state. However, fasting of rats maintained on low-protein diets greatly activates the hepatic enzyme. Thus, dietary protein deficiency results in inactivation of hepatic branched-chain α-ketoacid dehydrogenase, presumably because of low hepatic levels of branched-chain α-ketoacids, established inhibitors of branched-chain α-ketoacid dehydrogenase kinase. With rats fed a low-protein diet and subsequently fasted, inhibition of branched-chain α-ketoacid dehydrogenase kinase by branched-chain α-ketoacids generated from branched-chain amino acid produced by proteolysis of endogenous protein most likely accounts for the greater activity state of the branched-chain α-ketoacid dehydrogenase complex. Hepatocytes isolated from rats fed a chow diet or a low-protein (8%) diet were used to study the activity state and flux through the branched-chain α-ketoacid dehydrogenase complex. Alpha-chloroisocaproate stimulated α-ketoisovalerate decarboxylation with hepatocytes from rats fed a low-protein diet but had no effect with hepatocytes from rats fed chow diet. Activity measurements indicated that branched-chain α-ketoacid dehydrogenase was mainly in the inactive (phosphorylated) state in hepatocytes from low-protein-fed rats but mainly in the active (dephosphorylated) state in hepatocytes from chow-fed rats. Furthermore, α-ketoisocaproate greatly activated ( A 50 = 20 μ m ) α-ketoisovalerate oxidation by hepatocytes isolated from low-protein-fed rats but had no effect with hepatocytes isolated from chow-fed rats. The dietary studies and the hepatocyte experiments, taken together, suggest that portal blood levels of branched-chain α-ketoacids, particularly α-ketoisocaproate,are important determinants of the activity state of the hepatic branched-chain α-ketoacid dehydrogenase complex.
Archives of Biochemistry and Biophysics | 1985
Robert A. Harris; Steven M. Powell; Ralph Paxton; Sarah E. Gillim; Hidetoshi Nagae
Abstract A radiochemical assay was developed for measuring branched-chain α-ketoacid dehydrogenase activity of Triton X-100 extracts of freeze-clamped rat liver. The proportion of active (dephosphorylated) enzyme was determined by measuring enzyme activities before and after activation of the complex with a broad-specificity phosphoprotein phosphatase. Hepatic branched-chain α-ketoacid dehydrogenase activity in normal male Wistar rats was 97% active but decreased to 33% active after 2 days on low-protein (8%) diet and to 13% active after 4 days on the same diet. Restricting protein intake of lean and obese female Zucker rats also caused inactivation of hepatic branched-chain α-ketoacid dehydrogenase complex. Essentially all of the enzyme was in the active state in rats maintained for 14 days on either 30 or 50% protein diets. This was also the case for rats maintained on a commercial chow diet (minimum 23% protein). However, maintaining rats on 20, 8, and 0% protein diets decreased the percentage of the active form of the enzyme to 58, 10, and 7% of the total, respectively. Fasting of chow-fed rats for 48 h had no effect on the activity state of hepatic branched-chain α-ketoacid dehydrogenase, i.e., 93% of the enzyme remained in the active state compared to 97% for chow-fed rats. However, hepatic enzyme of rats maintained on 8% protein diet was 10% active before starvation and 83% active after 2 days of starvation. Thus, dietary protein deficiency results in inactivation of hepatic branched-chain α-ketoacid dehydrogenase complex, presumably as a consequence of low hepatic levels of branched-chain α-ketoacids, established inhibitors of branched-chain α-ketoacid dehydrogenase kinase. With rats fed a low-protein diet and subsequently starved, inhibition of branched-chain α-ketoacid dehydrogenase kinase by branched-chain α-ketoacids generated as a consequence of endogenous proteolysis most likely promotes the greater branched-chain α-ketoacid dehydrogenase activity state.
Archives of Biochemistry and Biophysics | 1984
Ralph Paxton; Robert A. Harris
Branched-chain alpha-ketoacid dehydrogenase kinase, purified from rabbit liver, was inhibited by clofibric acid, phenylpyruvate, and dichloroacetate in a mixed manner relative to ATP. I40 values relative to 75 microM ATP were 0.33, 1.7, and 3.0 mM, respectively. Inhibition of the kinase by acetate, pyruvate, and lactate was minimal; whereas a p-hydroxyphenyl substitution of these compounds increased their potency as kinase inhibitors, a phenyl substitution gave the most potent inhibitors. Clofibric acid, phenylpyruvate, and dichloroacetate activated branched-chain alpha-ketoacid dehydrogenase in perfused rat hearts. Perfusate concentrations that gave 50% activation (A50) were 0.1, 0.32, and 0.63 mM, respectively. A50 concentrations of clofibric acid and phenylpyruvate also increased flux (decarboxylation of alpha-keto[1-14C]isovalerate) through branched-chain alpha-ketoacid dehydrogenase in perfused rat heart. These findings suggest that, although clofibric acid and phenylpyruvate can inhibit substrate utilization by the branched-chain alpha-ketoacid dehydrogenase complex, the major effect of these compounds on branched-chain amino acid metabolism is due to inhibition of branched-chain alpha-ketoacid dehydrogenase kinase with subsequent activation of and increased flux through the complex.
Biochemical and Biophysical Research Communications | 1982
Robert A. Harris; Ralph Paxton; Rex A. Parker
Summary A broad-specificity protein phosphatase, purified from rat liver, can be used to activate the phosphorylated (inactive) branched-chain α-ketoacid dehydrogenase complex of crude tissue extracts. This enables estimation of the proportion of active (unphosphorylated) complex in a given tissue under different physiological states. Practically all (95 percent) of the complex was found in the active form in rat hearts perfused with leucine as the only oxidizable substrate. In contrast, only 13 percent of the complex was found in the active form when the perfusion medium was supplemented with glucose plus insulin. These findings are consistent with previously measured flux rates through the complex in perfused rat hearts.
Analytical Biochemistry | 1987
Yoshiharu Shimomura; Ralph Paxton; Takayuki Ozawa; Robert A. Harris
Abstract A new method using hydrophobic interaction chromatography on phenyl-Sepharose was developed to purify branched chain α-ketoacid dehydrogenase complex from commercially available frozen rat liver. Yields of greater than 50% were routinely achieved. The purified enzyme, composed of E1α, E1β, and E2 subunits, appeared homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained endogenous kinase activity for phosphorylation and inactivation of the complex.
Analytical Biochemistry | 1987
Gary W. Goodwin; Martha J. Kuntz; Ralph Paxton; Robert A. Harris
A spectrophotometric endpoint assay for determination of branched-chain alpha-keto acids is described. The assay depends on measurement of the NADH produced after addition of branched-chain alpha-keto acid dehydrogenase. Interference by pyruvate and alpha-ketobutyrate was eliminated by pretreating the sample with pyruvate dehydrogenase. The method yielded a peripheral venous plasma value of 59 +/- 5 microM (mean +/- SE) for the branched-chain alpha-keto acids of five overnight fasted healthy humans.
Annals of the New York Academy of Sciences | 1989
Robert A. Harris; Gary W. Goodwin; Ralph Paxton; Paul R. Dexter; Steven M. Powell; Bei Zhang; Amy Han; Yoshiharu Shimomura; Reid Gibson
The hepatic branched-chain alpha-keto acid dehydrogenase complex plays an important role in regulating branched-chain amino acid levels. These compounds are essential for protein synthesis but are toxic if present in excess. When dietary protein is deficient, the hepatic enzyme is present in the inactive, phosphorylated state to allow conservation of branched-chain amino acids for protein synthesis. When dietary protein is excessive, the enzyme is in the active, dephosphorylated state to commit the excess branched-chain amino acids to degradation. Inhibition of protein synthesis by cycloheximide, even when the animal is starving for protein, results in activation of the hepatic branched-chain alpha-keto acid dehydrogenase complex to prevent accumulation of branched-chain amino acids. Likewise, the increase in branched-chain amino acids caused by body wasting during starvation and uncontrolled diabetes is blunted by activation of the hepatic branched-chain alpha-keto acid dehydrogenase complex. The activity state of the hepatic branched-chain alpha-keto acid dehydrogenase complex is regulated in the short term by the concentration of branched-chain alpha-keto acids (inhibitors of branched-chain alpha-keto acid dehydrogenase kinase) and in the long term by alteration in the total branched chain alpha-keto acid dehydrogenase kinase activity.