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M F Utter

Publications and source records attributed to M F Utter.

18 recordsLinked to original sources
PubMed1979

Effect of thyroid hormone on the turnover of rat liver pyruvate carboxylase and pyruvate dehydrogenase.

Immunochemical techniques have been utilized to study the effect of thyroid status on the content and rates of synthesis and degradation of pyruvate carboxylase and pyruvate dehydrogenase in rat liver. Liver from hyperthyroid rats had twice the pyruvate carboxylase activity of normal rats while thyroidectomized rats had about two-thirds of normal activity. Pyruvate dehydrogenase complex activity was unchanged in the hyperthyroid state but was significantly reduced (by a third) in hypothyroid rats. Changes in catalytic activity during altered thyroid status were by immunochemical means to be closely related to the amount of the hepatic enzymes present. Isotopic studies showed that the changes in the content of pyruvate carboxylase and pyruvate dehydrogenase reflected alterations in the rate of the synthesis of the enzymes with the degradation rates little affected by thyroid status. The half-life for pyruvate carboxylase was 4.6 days, and that for pyruvate dehydrogenase, 8.1 days. In both cases, the turnover time was slower than that of the average mitochondrial protein (t1/2 = 3.8 days) for the control animals.

Animals↗
PubMed1979

Quaternary structure of pyruvate carboxylase from Pseudomonas citronellolis.

Physical-chemical studies of pyruvate carboxylase from Pseudomonas citronellolis demonstrate that the enzyme has an alpha 4 beta 4 structure. The individual polypeptides, alpha (Mr = 65,000) and beta (Mr = 54,000), were separated and isolated by preparative gel electrophoresis. Analysis of the relationship between Coomassie blue staining and protein quantity for each polypeptide indicated that the alpha and beta subunits are present in a 1:1 stoichiometry in the native enzyme. Determinations of the molecular weight of the protein by sedimentation equilibrium (Mr = 454,000), gel filtration analysis (Mr = 510,000), disc gel electrophoresis (Mr = 530,000), and mass measurement from the Scanning Transmission Electron Microscope (Mr = 530,000) are consistent with the proposed alpha 4 beta 4 structure. Disc gel electrophoresis studies revealed that under certain circumstances the enzyme may dissociate to a smaller molecular weight species (Mr = 228,000). This dissociation phenomenon could explain the earlier reported observation of Taylor et al. ((1972) J. Biol. Chem 22, 7388-8390) that the enzyme had a molecular weight of 265,000. Evidence from electron microscopic studies shows that the three-dimensional structure of this enzyme is quite distinct from other species of pyruvate carboxylase. The enzyme does not show the typical rhombic appearance which has been noted for chicken liver, sheep liver, and yeast pyruvate carboxylase.

Cross-Linking Reagents↗
PubMed1979

A re-examination of the electron microscopic appearance of pyruvate carboxylase from chicken liver.

Electron microscopic studies of chicken liver pyruvate carboxylase conducted under a variety of conditions show that this enzyme has an overall rhombic appearance and is comprised of four nonspherical subunits. The square planar tetramers originally identified by Valentine et al. (Valentine, R.C., Wrigley, N.G., Scrutton, M.C., Irias, J.J., and Utter, M.F. (1966) Biochemistry 5, 3111-3116) as pyruvate carboxylase have been shown to represent a minor protein contaminant found in many impure preparations of this enzyme. The contaminating protein has been separated from pyruvate carboxylase and further purified. It does not contain biotin and its constituent polypeptides are smaller than those of pyruvate carboxylase. This protein, whose function is not yet identified, shows a strong tendency to aggregate and is highly visible under many conditions of electron microscopy. Several lines of evidence support the thesis that the nonsquare tetramers are pyruvate carboxylase. When essentially homogeneous material is examined with a variety of different negative stains, numbers of intact molecules represent 20 to 70% of the visible protein. These tetramers, like pyruvate carboxylase, are very cold-labile and are protected from dissociation under these conditions by acetyl-CoA, a specific activator of this enzyme. Also, the structures form complexes with avidin and antibiotin antibody and thus, like pyruvate carboxylase, contain biotin.

Animals↗
PubMed1979

Pyruvate carboxylase and phosphoenolpyruvate carboxykinase activity in leukocytes and fibroblasts from a patient with pyruvate carboxylase deficiency.

Normal values are given for the activities of pyruvate carboxylase (E.C.6.4.1.1), mitochondrial phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32, PEPCK), and citrate synthase (E.C. 4.1.3.7) in fibroblasts, lymphocytes, and leukocytes. Also given are values for these enzymes in the leukocytes and fibroblasts from a severely mentally and developmentally retarded patient with proximal renal tubular acidosis and hepatic, cerebral, and renal cortical pyruvate carboxylase deficiency. In normals, virtually all of the mitochondrial PEPCK and pyruvate carboxylase activity was present in the mononuclear leukocyte fraction of whole venous blood. Cellular fractionation studies with human lymphocytes and fibroblasts demonstrated that all of the PEPCK activity in these cells is mitochondrial. Normal values for pyruvate carboxylase in leukocytes were 0.092 (0.070--0.208) mU/mg protein (n=5), in lymphocytes 0.154 (0.092--0.262) mU/mg protein (n=5), and in fibroblasts 1.36 (0.778--2.19) mU/mg protein (n=5). The patient with hepatic, renal, and cerebral pyruvate carboxylase deficiency had no detectable activity (less than 0.009 mU/mg protein) in his leukocytes and 0.018 mU/mg protein in his fibroblasts. Data from an assay for pyruvate carboxylase activity in the patient's fibroblasts show that the activity observed is significant but very close to the lower limits of the assay. Values for PEPCK in normal lymphocytes were 1.42 (0.824--1.88) mU/mg protein (n=5), in leukocytes 1.68 (1.64--1.72) mU/mg protein (n=2), and in fibroblasts 5.49 (3.94--6.33) mU/mg protein (n=6).

Acidosis, Renal Tubular↗
PubMed1979

Pyruvate carboxylase deficiency and lactic acidosis in a retarded child without Leigh's disease.

A child with lactic acidosis, severe mental and developmental retardation, and proximal renal tubular acidosis is presented. Biopsy and autopsy studies show severe hepatic, renal cortical, and cerebral deficiencies in pyruvate carboxylase (EC 6.4.1.1) activity. The patient had 1.81 +/- 0.20 units/g fresh weight at biopsy and 0.75 +/- 0.07 units/g fresh weight hepatic pyruvate carboxylase activity at autopsy compared with 10.9, 11.3, and 9.5 units/g fresh weight in two autopsy and one biopsy controls, respectively. The patient's renal cortical pyruvate carboxylase activity at autopsy was 0.008 +/- 0.004 units/g fresh weight compared with 5.05 units/g in the autopsy control. The patient had no detectable (less than 0.018 units/g fresh weight) cerebral pyruvate carboxylase activity at autopsy compared with 0.44, 0.53, and 0.695 units/g in the autopsy cerebrum of one human and two rhesus monkeys, respectively. Pyruvate dehydrogenase complex, phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32), and fructose-1,6-bisphosphatase (EC 3.1.3.11) activities were in the normal range. The patient's urine pH was above 7.9 when the total serum CO2 was greater than 7.8 mM. However, the patient was able to acidify the urine to pH 5.1 when the total serum CO2 was 1.6 mM. The neuropathologic examination of the brain at autopsy revealed no sign of Leigh's disease, although developmental and degenerative lesions were observed. This is the first reported patient with a primary deficiency in hepatic, renal, and cerebral pyruvate carboxylase deficiency in whom the neuropathologic lesions, distinct from those of Leigh's disease, and proximal renal tubular acidosis have both been documented.

Acidosis↗
PubMed1977

Novel enzymic machinery for the metabolism of oxalacetate, phosphoenolpyruvate, and pyruvate in Pseudomonas citronellolis.

The metabolic pathways for the interconversion of oxalacetate, phosphoenolpyruvate, and pyruvate in Pseudomonas citronellolis form an interlocking system (Scheme 1) that would appear to require complex regulatory mechanisms to permit a proper flow of metabolites through the pathways and to prevent futile cycling. Oxalacetate decarboxylase (I in Scheme 1), P-enolpyruvate synthase (II), P-enolpyruvate carboxylase (III), and pyruvate kinase (V) are constitutive enzymes in this organism. Pyruvate carboxylase (VI) is inducible and has its highest activity in cells grown on glucose or lactate, moderate activity in cells grown on acetate, citrate, or glutamate, and virtually no activity in aspartate-grown cells. P-enolpyruvate carboxykinase (IV) was not detected. The presence of these five enzymes in a single cell has not been previously reported. In Scheme 1, three futile cycles are possible: the simultaneous operation of Reactions I and VI; of Reactions II and V; or of I, II, and III. An examination of the regulatory properties of the individual enzymes after partial purification offers support for the hypothesis of an intricate regulatory system. Oxalacetate decarboxylase (I) is inhibited by acetyl-CoA; phosphoenolpyruvate carboxylase (III) is activated by acetyl-CoA and ADP and inhibited by aspartate; phosphoenolpyruvate synthase (II) is inhibited by 5'-AMP and phosphoenolpyruvate; and pyruvate kinase (V) is activated by 5'-AMP and 2 keto, 3-deoxy,6-phosphogluconate and inhibited by ATP. The presence of metabolites with reciprocal but reinforcing functions is noteworthy. As an example, acetyl-CoA both inhibits the breakdown of oxalacetate and stimulates its formation. Only pyruvate carboxylase appears to be regulated by the carbon substrates of the growth medium.

Acetyl Coenzyme A↗
PubMed1977

Binding of acetyl-CoA to chicken liver pyruvate carboxylase.

Pyruvate carboxylase from chicken liver is a tetramer whose catalytic activity is completely dependent on the presence of acetyl-CoA. However, no direct evidence concerning the nature of the binding of the activator to the enzyme has been available. This is due in part to the instability of the enzyme and its ability to hydrolyze acetyl-CoA at an appreciable rate. The present studies on binding of the activator with the rapid flow dialysis method of Colowick and Womack ((1969) J. Biol. Chem. 244, 774-777) as modified by Klapper ((1970) Biochem. Biophys. Res. Commun. 38, 172-179) show four binding sites for acetyl-CoA. The binding dissociation constant at pH 7.2 is 13.9 muM as compared with an activation constant of 13.3 muM for the catalytic reaction at this pH. The relationship between acetyl-CoA concentration and catalytic activity is highly cooperative (nH = 2.9). The binding process also exhibits positive cooperativity but to a lower degree (nH = 1.9). Pyruvate carboxylase from chicken liver is rapidly inactivated and dissociated in the cold (0 degrees). The inactive protomeric form of the enzyme has been shown to be unable to bind acetyl-CoA at 0 degrees although the tetrameric species can do so. These results provide a plausible explanation for the catalytic inactivity of the protomer. The presence of acetyl-CoA results in an ultraviolet difference spectrum for the enzyme with a maximum at 280 nm. Half-maximal optical density difference is observed at an acetyl-CoA concentration of 9 muM, in reasonable agreement with the binding and activation constants.

Acetyl Coenzyme A↗
PubMed1975

Decarboxylation of oxalacetate to pyruvate by purified avian liver phosphoenolpyruvate carboxykinase.

Phosphoenolpyruvate carboxykinase, which has been isolated from chicken liver mitochondria in essentially homogenous form, carries out the irreversible decarboxylation of oxalacetate to pyruvate in the presence of catalytic amounts of GDP or IDP, as well as the reversible decarboxylation of oxalacetate to phosphoenolpyruvate in the presence of substrate amounts of GTP or ITP. The pyruvate- and phosphoenolpyruvate-forming reactions are similar in their nucleoside specificity and appear to be carried out by the same protein. However, the two activities vary markedly in their response to added metal ions and sulfhydryl reagents. Phosphoenolpyruvate formation is completely dependent on the presence of a divalent metal ion, with Mn2+ the most effective species. This reaction is also stimulated by sulfhydryl reagents such as 2-mercaptoethanol. In contrast, the pyruvate-forming reaction is strongly inhibited by divalent metal ions, including Mn2+, and also by moderate concentrations of sulfhydryl reagents. These observations and the demonstration that pyruvate kinase-like activity is very low or absent make it unlikely that pyruvate formation proceeds via phosphoenolpyruvate as an intermediate. Although the pyruvate-forming reaction is inhibited by added metal ions, the reaction is also inhibited by metal-chelating agents such as 8-hydroxyquinoline and o-phenanthroline, suggesting that the reaction is dependent on the presence of a metal ion. It has not been possible, however, to demonstrate that the enzyme is a metalloprotein.

2,2'-Dipyridyl↗
PubMed1975

The control of the synthesis of pyruvate carboxylase in Pseudomonas citronellolis. Evience from double labeling studies.

The level of pyruvate carboxylase in Pseudmonas citronellolis is controlled by the carbon source of the growth medium. The activity of the enzyme is highest in cells grown on lactate or glucose and virtually absent in cells grown on malate or aspartate. Double labeling studies with 3H- and 14C-labeled leucine confirm that pyruvate carboxylase is synthesized in the presence of lactate but not in the presence of aspartate. The studies also show that coordinated regulation occurs at the level of the synthesis of the two polypeptides which make up pyruvate carboxylase in P. citronellolis, rather than at the stages of their assembly into protomers or the biotinylation of the apoenzyme. There is no evidence for control of the catalytic acitivity of the holoenzyme via effectors. In all other varieties of pyruvate carboxylase examined thus far, the enzyme appears to be constitutive with regulation accomplished either through effector modulation of holoenzyme activity (pyruvate carbox-lase from animal sources, yeast, several species of bacteria) or through control of the biotinylation of the apoenzyme by holocarboxylase synthetase (Bacillus stearothermophilus, yeast).

Aspartic Acid↗
PubMed1975

Structural properties of pyruvate carboxylases from chicken liver and other sources.

Varieties of pyruvate carboxylase [pyruvate: CO2 ligase (ADP-forming), EC 6.4.1.1] obtained from the livers of several species of vertebrates, including humans, all show the same basic structure. They are composed of large polypeptide chains of molecular weights ranging from 1.2 to 1.3 X 10(5) for the different varieties of the enzyme. The native form of the enzyme appears to be a tetramer with a molecular weight of about 5 X 10(5). In the case of pyruvate carboxylase from chicken liver each polypeptide chain contains a biotin moiety, thus supporting the thesis that the tetramer contains four identical polypeptide chains. Pyruvate carboxylase from yeast appears to be basically similar to those from the vertebrate species and has a tetrameric structure. Each protomer contains a single polypeptide chain with a molecular weitht of 1.25 X 10(5). In contrast, pyruvate carboxylase from two bacterial species, Pseudomonas citronellolis and Axotobacter vinelandii, appears to be a dimer with a molecular weight (2.5 X 10(5)) about half that of the animal and yeast species. As a further difference, each of the protomers of the bacterial enzymes contain two polypeptides of 6.5 and 5.4 X 10(5) molecular weight in case of the Pseudomonas enzyme. The larger of the two polypeptides contains the biotin moiety. The functional units of the bacterial enzyme thus appear to contain two polypeptides while that of the liver and yeast enzymes is made up of a single chain. Neither of these arrangements corresponds with those of other biotin enzymes whose structure has been extensively studied (acetyl-CoA carboxylases from liver or Excherichia coli, and transcarboxylase from Propionibacterium).

Animals↗