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R Paxton

Publications and source records attributed to R Paxton.

At least 55 records · Page 3Linked to original sources
PubMed1987

Purification and partial characterization of chicken liver acetyl coenzyme A: arylamine N-acetyltransferase.

Arylamine acetyltransferase (EC 2.3.1.5) was purified 120-fold from chicken liver. The enzyme showed a rise in activity from pH 6.5 to 7.7 followed by a constant activity to about pH 8.6. The relative molecular weight of the enzyme was about 34,000. The apparent Km for acetyl-CoA was 13 microM with 4-nitroaniline as acetyl-acceptor. CoA was a noncompetitive inhibitor relative to acetyl-CoA with apparent Ki value of 110 microM. With 4-methylaniline as substrate, arylamine acetyltransferase activity in pigeon liver was about 8 times greater than in chicken liver, and about 40 times greater than in rabbit.

Acetyltransferases↗
PubMed1986

Branched-chain 2-oxo acid dehydrogenase interferes with the measurement of the activity and activity state of hepatic pyruvate dehydrogenase.

Oxidative decarboxylation of pyruvate by branched-chain 2-oxo acid dehydrogenase can result in overestimation of the expressed and total activity of hepatic pyruvate dehydrogenase. Pyruvate is a poor substrate for branched-chain 2-oxo acid dehydrogenase relative to the branched-chain oxo acids; however, the comparable total activities of the two complexes in liver, the much greater activity state of branched-chain 2-oxo acid dehydrogenase compared with pyruvate dehydrogenase in most physiological states, and the use of high pyruvate concentrations, explain the interference that can occur in conventional radiochemical or indicator-enzyme linked assays of pyruvate dehydrogenase. Goat antibody that specifically inhibited branched-chain 2-oxo acid dehydrogenase was used in this study to provide a more specific assay for pyruvate dehydrogenase.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1986

Regulation of the branched-chain 2-oxo acid dehydrogenase complex in hepatocytes isolated from rats fed on a low-protein diet.

Hepatocytes isolated from rats fed on a chow diet or a low-protein (8%) diet were used to study the effects of various factors on flux through the branched-chain 2-oxo acid dehydrogenase complex. The activity of this complex was also determined in cell-free extracts of the hepatocytes. Hepatocytes isolated from chow-fed rats had greater flux rates (decarboxylation rates of 3-methyl-2-oxobutanoate and 4-methyl-2-oxopentanoate) than did hepatocytes isolated from rats fed on the low-protein diet. Oxidizable substrates tended to inhibit flux through the branched-chain 2-oxo acid dehydrogenase, but inhibition was greater with hepatocytes isolated from rats fed on the low-protein diet. 2-Chloro-4-methylpentanoate (inhibitor of branched-chain 2-oxo acid dehydrogenase kinase), dichloroacetate (inhibitor of both pyruvate dehydrogenase kinase and branched-chain 2-oxo acid dehydrogenase kinase) and dibutyryl cyclic AMP (inhibitor of glycolysis) were effective stimulators of branched-chain oxo acid decarboxylation with hepatocytes from rats fed on a low-protein diet, but had little effect with hepatocytes from rats fed on chow diet. Activity measurements indicated that the branched-chain 2-oxo acid dehydrogenase complex was mainly (96%) in the active (dephosphorylated) state in hepatocytes from chow-fed rats, but only partially (50%) in the active state in hepatocytes from rats fed on a low-protein diet. Oxidizable substrates markedly decreased the activity state of the enzyme in hepatocytes from rats fed on a low-protein diet, but had much less effect in hepatocytes from chow-fed rats. 2-Chloro-4-methylpentanoate and dichloroacetate increased the activity state of the enzyme in hepatocytes from rats fed on a low-protein diet, but had no effect on the activity state of the enzyme in hepatocytes from chow-fed rats. The results indicate that protein starvation greatly increases the sensitivity of the hepatic branched-chain 2-oxo acid dehydrogenase complex to regulation by covalent modification.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1986

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.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1986

Phosphorylation sites and inactivation of branched-chain alpha-ketoacid dehydrogenase isolated from rat heart, bovine kidney, and rabbit liver, kidney, heart, brain, and skeletal muscle.

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.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1986

Regulation of branched-chain alpha-ketoacid dehydrogenase complex by covalent modification.

The branched-chain alpha-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 alpha-ketoacid dehydrogenase kinase, believed distinct from pyruvate dehydrogenase kinase, is an integral component of the branched-chain alpha-ketoacid dehydrogenase complex and is sensitive to inhibition by branched-chain alpha-ketoacids, alpha-chloroisocaproate, phenylpyruvate, clofibric acid, octanoate and dichloroacetate. Phosphorylation of branched-chain alpha-ketoacid dehydrogenase occurs at two closely-linked serine residues (sites 1 and 2) of the alpha-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 alpha-subunit for 95% inactivation and 1.5 mol P/mol alpha-subunit for maximally phosphorylated complex. Site 1 and site 2 were phosphorylated at similar rates until total phosphorylation exceeded 1 mol P/mol alpha-subunit. The complexes from rabbit kidney, rabbit heart, and rat heart showed 30-40% additional phosphorylation of the alpha-subunit beyond 95% inactivation. Site specificity studies carried out with the kinase partially inhibited with alpha-chloroisocaproate suggest that phosphorylation of site 1 is primarily responsible for regulation of the complex. The capacity of the branched-chain alpha-ketoacid dehydrogenase to oxidize pyruvate (Km = 0.8 mM, Vmax = 20% of that of alpha-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 alpha-ketoacid dehydrogenase can be used to prevent interference with the pyruvate dehydrogenase assay. Almost all of the hepatic branched-chain alpha-ketoacid dehydrogenase in chow-fed rats is active (greater than 90% dephosphorylated). In contrast, almost all of the hepatic enzyme of rats fed a low-protein (8%) diet is inactive (greater than 85% phosphorylated). Fasting of chow-fed rats has no effect on the activity state of hepatic branched-chain alpha-ketoacid dehydrogenase, i.e. greater than 90% of the enzyme remains in the active state. However, fasting of rats maintained on low-protein diets greatly activates the hepatic enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1985

Physiological covalent regulation of rat liver branched-chain alpha-ketoacid dehydrogenase.

A radiochemical assay was developed for measuring branched-chain alpha-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 alpha-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 alpha-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 alpha-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 alpha-ketoacid dehydrogenase complex, presumably as a consequence of low hepatic levels of branched-chain alpha-ketoacids, established inhibitors of branched-chain alpha-ketoacid dehydrogenase kinase. With rats fed a low-protein diet and subsequently starved, inhibition of branched-chain alpha-ketoacid dehydrogenase kinase by branched-chain alpha-ketoacids generated as a consequence of endogenous proteolysis most likely promotes the greater branched-chain alpha-ketoacid dehydrogenase activity state.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1985

Nutritional control of branched chain alpha-ketoacid dehydrogenase in rat hepatocytes.

Branched chain alpha-ketoacid dehydrogenase (EC 1.2.4.4) complex, the rate-limiting enzyme of branched chain amino acid catabolism in most tissues, is subject to regulation by covalent modification, with phosphorylation inactivating and dephosphorylation activating the complex. The enzyme complex from liver of chow-fed rats is mainly in the active form but that from liver of rats fed a low-protein diet is mainly in the inactive form. Isolated hepatocytes were used to identify factors that affect interconversion of branched chain alpha-ketoacid dehydrogenase. The enzyme present in hepatocytes of rats fed a low-protein diet appears much more responsive to regulation by covalent modification than the branched chain alpha-ketoacid dehydrogenase present in hepatocytes of normal chow-fed rats. alpha-Chloroisocaproate, a specific inhibitor of the kinase responsible for phosphorylation and inactivation of the complex, greatly stimulates oxidation of alpha-keto[1-14C]isovalerate by hepatocytes prepared from rats fed a low-protein diet but not from normal chow-fed rats. Oxidizable substrates are also much more effective inhibitors of branched chain alpha-ketoacid oxidation with hepatocytes from rats fed a low-protein diet than from normal chow-fed rats. Activity measurements with cell-free extracts suggest that changes in flux through the dehydrogenase with intact hepatocytes prepared from rats fed a low-protein diet are explained in large part by changes in the proportion of the enzyme in the active, dephosphorylated form. Regulation of liver branched chain alpha-ketoacid dehydrogenase by covalent modification functions to conserve branched chain amino acids for protein synthesis during periods of restricted dietary protein intake.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1984

Regulation of branched-chain alpha-ketoacid dehydrogenase kinase.

Isolated rabbit liver branched-chain alpha-ketoacid dehydrogenase was inhibited in a mixed manner relative to ATP by alpha-ketoisocaproate, alpha-keto-beta-methylvalerate, alpha-ketoisovalerate, alpha-ketocaproate, alpha-ketovalerate, and alpha-chloroisocaproate with I40 values (mM), respectively, of 0.065, 0.49, 2.5, 0.2, 0.5, and 0.08. The concentration (mM) of alpha-ketoisocaproate, alpha-keto-beta-methylvalerate, and alpha-ketoisovalerate needed to activate branched-chain alpha-ketoacid dehydrogenase in the perfused rat heart to 50% of total activity was 0.07, 0.10, and 0.25, respectively. Isolated branched-chain alpha-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 micrograms/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, beta-hydroxy-beta-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 alpha-ketoacid dehydrogenase kinase; acetate, propionate, beta-hydroxybutyrate, lactate, acetoacetate, malonate, alpha-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 alpha-ketoacid dehydrogenase.

Adenosine Diphosphate↗
PubMed1984

Clofibric acid, phenylpyruvate, and dichloroacetate inhibition of branched-chain alpha-ketoacid dehydrogenase kinase in vitro and in perfused rat heart.

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.

Acetates↗
PubMed1983

Activity state of the branched chain alpha-ketoacid dehydrogenase complex in heart, liver, and kidney of normal, fasted, diabetic, and protein-starved rats.

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.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1983

Altered activities of branchial and renal Na/K- and Mg-ATPases in cold-acclimated goldfish (Carassius auratus).

1. Cold-acclimated (1 degree C) goldfish (Carassius auratus) branchial Na/K-ATPase activity was elevated 100% while renal Na/K-ATPase activity was not significantly affected compared with warm-acclimated (20 degrees C) goldfish. 2. Cold-acclimated goldfish branchial and renal Mg-ATPase activity was reduced 18 and 30% on a per mg protein basis, respectively. 3. Renal Na/K-ATPase activity was 4.6- and 1.6-fold greater than gill in cold- and warm-acclimated fish, respectively. 4. The elevated branchial Na/K-ATPase activity and the unchanged renal Na/K-ATPase activity are consistent with the maintenance of the reduced blood ion level in cold-acclimated goldfish.

Acclimatization↗
PubMed1982

Inhibition of branched chain alpha-ketoacid dehydrogenase kinase activity by alpha-chloroisocaproate.

alpha-Chloroisocaproate has been shown to inhibit phosphorylation and inactivation of rabbit liver branched chain alpha-ketoacid dehydrogenase complex. Phosphorylation of pyruvate dehydrogenase was also inhibited by this alpha-ketoisocaproate analog, but phosphorylation of branched chain alpha-ketoacid dehydrogenase was much more sensitive than phosphorylation of pyruvate dehydrogenase (I50 values of 7.5 and 675 microM, respectively). Phosphorylation of glycogen synthase by six other protein kinases was not inhibited by alpha-chloroisocaproate (1 mM). Although less sensitive than phosphorylation of the complex, branched chain alpha-ketoacid dehydrogenase was also found inhibited by alpha-chloroisocaproate. The latter inhibition was competitive with respect to the alpha-ketoacid substrates of the dehydrogenase (Ki values of approximately 0.5 mM). alpha-Chloroisocaproate greatly stimulated the capacity of the perfused rat heart to decarboxylate [1-14C]leucine and promoted conversion of practically all (99%) of the complex into the active form.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗