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M F Utter

Publications and source records attributed to M F Utter.

At least 19 recordsLinked to original sources
PubMed1984

Stereochemical course of thiophosphoryl group transfer catalyzed by mitochondrial phosphoenolpyruvate carboxykinase.

Guinea pig liver mitochondrial phosphoenolpyruvate carboxykinase catalyzes the conversion of (Rp)-guanosine 5'-(3-thio[3-18O]triphosphate) and oxalacetate to (Sp)-[18O] thiophosphoenolpyruvate , GDP, and CO2 by a mechanism that involves overall inversion in the configuration of the chiral [18O]thiophosphate group. This result is most consistent with a single displacement mechanism in which the [18O]thiophosphoryl group is transferred from (Rp)-guanosine 5'-(3-thio[3-18O]triphosphate) bound at the active site directly to enolpyruvate generated at the active site by the decarboxylation of oxalacetate. In particular, this result does not indicate the involvement of a covalent thiophosphoryl-enzyme on the reaction pathway.

Animals↗
PubMed1983

Activation of yeast pyruvate carboxylase: interactions between acyl coenzyme A compounds, aspartate, and substrates of the reaction.

Chicken liver pyruvate carboxylase has an absolute requirement for short-chain acyl coenzyme A (CoA), whereas the same enzyme from yeast has less stringent requirements. The yeast enzyme has now been studied in an effort to elucidate the mechanism by which acyl-CoA stimulates pyruvate carboxylase activity. Yeast pyruvate carboxylase has an apparent basal level of activity above which CoA and acyl-CoAs of 2-20 carbons activate; the concentration of acyl-CoA required for half-maximum activation (K0.5) decreases as the chain length of the acyl moiety increases to 16 carbons. Activation of yeast pyruvate carboxylase by acyl-CoA is brought about in part by increasing the affinity of pyruvate carboxylase for two substrates, bicarbonate and pyruvate. The affinity of pyruvate carboxylase for bicarbonate is also increased by potassium ions. The observation of only low levels of activity in the absence of acyl-CoA or potassium ion leads to the conclusion that the basal activity so frequently referred to is probably due to the presence of activating monovalent cations. Pyruvate carboxylase from yeast probably has an absolute requirement for monovalent cations or acyl-CoA with a combination of the two being required for optimum conditions for maximal activity. Stimulation by acyl-CoA and inhibition by aspartate are mutually antagonistic with each affecting the activation or inhibition constant and the degree of cooperativity brought about by the other. The enzyme from liver is unaffected by aspartate.

Acyl Coenzyme A↗
PubMed1983

Regulation of the synthesis and degradation of pyruvate carboxylase in 3T3-L1 cells.

The differentiation of mouse 3T3-L1 cells is characterized by an accumulation of cytosolic triglyceride and marked increase in many enzymatic activities involved in triglyceride biosynthesis. The specific activity of one such enzyme, pyruvate carboxylase, increases at least 20-fold and is due to a parallel increase in the intracellular concentration of the protein. Pulse-labeling experiments demonstrated that the increase in the specific activity of pyruvate carboxylase was due to an increase in the rate of enzyme synthesis. In the differentiated cell, pyruvate carboxylase represented 1.9% of the total cellular protein and 1% of the protein radiolabeled during a 1-h pulse. This was 35-and 28-fold higher than in the undifferentiated cell, respectively. The turnover of pyruvate carboxylase in the differentiated cell was similar to that in the undifferentiated cell with the enzyme having a half-life of 28-35 h. The half-life of apopyruvate carboxylase in avidin-treated 3T3-L1 cells was 24 h, indicating that the turnover of the apoenzyme was not significantly different than that of the holoenzyme. Radiolabeling pyruvate carboxylase with [14C]biotin and [3H]leucine demonstrated that the turnover of biotin associated with the enzyme was identical to the turnover of the enzymatic protein.

Animals↗
PubMed1983

Induction of pyruvate dehydrogenase in 3T3-L1 cells during differentiation.

The activity of the pyruvate dehydrogenase complex and the content and turnover of the pyruvate dehydrogenase component were measured during the differentiation of 3T3-L1 preadipocytes into 3T3-L1 adipocytes. The specific activity of "total" pyruvate dehydrogenase complex increased approximately 7-fold in 3T3-L1 adipocytes differentiated with a treatment of insulin plus dexamethasone plus 1-methyl-3-isobutyl xanthine. The ratio of "active" pyruvate dehydrogenase complex to total pyruvate dehydrogenase complex remained unaltered in both 3T3-L1 preadipocytes and adipocytes. A specific goat antibody to bovine kidney pyruvate dehydrogenase quantitatively precipitated both alpha and beta subunits of pyruvate dehydrogenase from solubilized 3T3-L1 adipocytes. Using immunoprecipitation and gel electrophoresis techniques, we demonstrated an approximate 6-fold increase in pyruvate dehydrogenase content in 3T3-L1 adipocytes as compared to 3T3-L1 preadipocytes. Pulse labeling experiments revealed an approximately 5-fold increase in the rates of synthesis of both alpha and beta subunits of pyruvate dehydrogenase in 3T3-L1 adipocytes after 6 days of the hormonal treatment compared to those observed in 3T3-L1 preadipocytes. In contrast, the half-lives of alpha and beta subunits of pyruvate dehydrogenase were not significantly altered in 3T3-L1 preadipocytes (41 h) and adipocytes (49 h). The 6-8-fold increment in the specific activity of the pyruvate dehydrogenase complex in 3T3-L1 adipocytes therefore results from increased rates of synthesis of alpha and beta subunits of pyruvate dehydrogenase.

Adipose Tissue↗
PubMed1983

Hypoglycemia, hepatic dysfunction, muscle weakness, cardiomyopathy, free carnitine deficiency and long-chain acylcarnitine excess responsive to medium chain triglyceride diet.

Fraternal twins who had fasting hypoglycemia, hypoketonemia, muscle weakness, and hepatic dysfunction are reported. The hepatic dysfunction occurred only during periods of caloric deprivation. The surviving patient developed a cardiomyopathy. In this sibling, muscle weakness and cardiomyopathy were markedly improved by a diet high in medium chain triglycerides. There was a marked deficiency of muscle total carnitine and a mild deficiency of hepatic total carnitine. Unlike patients with systemic carnitine deficiency, serum and muscle long-chain acylcarnitine were elevated and renal reabsorption of carnitine was normal. It was postulated that the defect in long-chain fatty acid oxidation in this disorder is caused by an abnormality in the mitochondrial acylcarnitine transport. Detailed studies of the cause of the hypoglycemia revealed that insulin, growth hormone, cortisol, and glucagon secretion were appropriate and that it is unlikely that there was a major deficiency of a glycolytic or gluconeogenic enzyme. Glucose production and alanine conversion to glucose were in the low normal range when compared to normal children in the postabsorptive state. The hypoglycemia in our patients was probably due to a modest increase in glucose consumption, secondary to the decreased oxidation of fatty acids and ketones, alternate fuels which spare glucose utilization, plus a modest decrease in hepatic glucose production secondary to decreased available hepatic energy substrates.

Cardiomyopathies↗
PubMed1982

The mitochondrial and cytosolic forms of avian phosphoenolpyruvate carboxykinase (GTP) are encoded by different messenger RNAs.

Previous work from our laboratory (Watford, M., Hod, Y., Chiao, Y. B., Utter M. F., and Hanson R. W. (1981) J. Biol. Chem. 256, 10023-10027) indicated that in the chicken, hepatic phosphoenolpyruvate carboxykinase is in the mitochondria, whereas kidney contains both a mitochondrial and cytosolic form of the enzyme. In the present study the two forms of phosphoenolpyruvate carboxykinase were purified and shown to be distinct proteins which differ in size, charge, and immunochemical properties. Using a cell-free protein synthesis system, we demonstrate that the cytosolic isozyme is encoded only by kidney mRNA and that its translation form has similar properties to that of the mature protein. On the other hand, the mitochondrial enzyme is encoded by liver and kidney mRNA and synthesized as a protein about 2000 daltons larger than the mature form. The putative precursor of the mitochondrial phosphoenolpyruvate carboxykinase is processed to a mature size by isolated, respiring mitochondria. We further show that the mRNA species for the cytosolic and mitochondrial forms are separable and are about 3 and 4 kilobases, respectively. It is concluded that the two forms of phosphoenolpyruvate carboxykinase of the chicken are encoded by distinct mRNA species.

Animals↗
PubMed1981

Characterization of the subunit structure of pyruvate carboxylase from Pseudomonas citronellolis.

Pyruvate carboxylase from Pseudomonas citronellolis is composed of non-identical subunits which include a larger biotin-containing polypeptide (alpha) of Mr = 65,000, and a smaller biotin-free polypeptide (beta) of Mr = 54,000. We have investigated these two polypeptides by analyzing their amino acid composition, cyanogen bromide peptide maps, and immunochemistry. The results showed that the subunits of the enzyme have quite different properties. Antibodies prepared against the polypeptides were used as probes of the catalytic functions of the subunits. Immunotitration studies indicated that only anti-alpha inhibited enzyme activity. The antibiotin fraction of this antibody population was removed by passage through biotin-Sepharose (anti-alpha'). Titration curves using anti-alpha' showed identical inhibition when total pyruvate carboxylase activity, ATP/Pi exchange activity, and pyruvate/oxalacetate exchange activity were measured, suggesting that both active sites are located on the alpha polypeptide. The arrangement of the subunits in the quaternary structure was investigated by means of the surface probe carbonic anhydrase linked to toluene isocyanate, and by partial digestion experiments with trypsin, chymotrypsin, and pronase. The results indicated that the alpha polypeptides are on the outside of the molecule and the beta polypeptides are the internal subunits.

Amino Acid Sequence↗
PubMed1981

Comparative inhibition of mitochondrial and cytosolic phosphoenolpyruvate carboxykinases by stereospecific substrate analogues.

Phosphoenolpyruvate carboxykinase [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] was purified from the liver of several species; the mitochondrial enzyme and the cytosolic enzyme were separated from each other. The species included guinea pig, monkey, rat, and chicken. Each enzyme was assayed for inhibition by the substrate analogues E-2-phosphoenolbutyrate and Z-2-phosphoenolbutyrate. Each enzyme tested displayed the same stereospecificity: the E diastereoisomer was the more potent inhibitor than the Z. These results suggest an active site homology for all carboxykinases. The absolute values for Ki measured show that in almost every case the mitochondrial enzyme is more susceptible to inhibition by these analogues than is the cytosolic enzyme. The Ki values are one-fifth those for the mitochondrial enzymes. These results imply subtle differences in ligand interactions at the active sites of these enzymes.

Animals↗
PubMed1981

Pyruvate dehydrogenase complex activity in normal and deficient fibroblasts.

Pyruvate dehydrogenase complex (PDC) activity in human skin fibroblasts appears to be regulated by a phosphorylation-dephosphorylation mechanism, as is the case with other animal cells. The enzyme can be activated by pretreating the cells with dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, before they are disrupted for measurement of PDC activity. With such treatment, the activity reaches 5-6 nmol/min per mg of protein at 37 degrees C with fibroblasts from infants. Such values represent an activation of about 5-20-fold over those observed with untreated cells. That this assay, based on [1-(14)C]pyruvate decarboxylation, represents a valid measurement of the overall PDC reaction is shown by the dependence of (14)CO(2) production on the presence of thiamin-PP, coenzyme A (CoA), Mg(++), and NAD(+). Also, it has been shown that acetyl-CoA and (14)CO(2) are formed in a 1:1 ratio. A similar degree of activation of PDC can also be achieved by adding purified pyruvate dehydrogenase phosphatase and high concentrations of Mg(++) and Ca(++), or in some cases by adding the metal ions alone to the cell homogenate after disruption. These results strongly suggest that activation is due to dephosphorylation. Addition of NaF, which inhibits dephosphorylation, leads to almost complete loss of PDC activity. Assays of completely activated PDC were performed on two cell lines originating from patients reported to be deficient in this enzyme (Blass, J. P., J. Avigan, and B. W. Ublendorf. 1970. J. Clin. Invest. 49: 423-432; Blass, J. P., J. D. Schuman, D. S. Young, and E. Ham. 1972. J. Clin. Invest. 51: 1545-1551). Even after activation with DCA, fibroblasts from the patients showed values of only 0.1 and 0.3 nmol/min per mg of protein. A familial study of one of these patients showed that both parents exhibited activity in fully activated cells about half that of normal values, whereas cells from a sibling appeared normal. These results demonstrate the inheritance nature of PDC deficiency, and that the present assay is sufficient to detect the heterozygous carriers of the deficiency. Application of the same procedures to fibroblasts obtained from 16 individuals who were believed to have normal PDC activities showed a range from about 2-2.5 nmol/min per mg protein for adults to 5-6 nmol/min per mg protein for cells from infants.

Acetyl Coenzyme A↗
PubMed1980

Effect of streptozotocin-induced diabetes mellitus on the turnover of rat liver pyruvate carboxylase and pyruvate dehydrogenase.

Immunochemical techniques were used to study the effect of streptozotocin-induced diabetes on the amounts of pyruvate carboxylase and pyruvate dehydrogenase and on their rates of synthesis and degradation. Livers from diabetic rats had twice the pyruvate carboxylase activity of livers from normal rats when expressed in terms of DNA or body weight. The changes in catalytic activity closely paralleled changes in immunoprecipitable enzyme protein. Relative rates of synthesis determined by pulse-labelling studies showed that the ratio of synthesis of pyruvate carboxylase to that of average mitochondrial protein was increased 2.0-2.5 times in diabetic animals over that of control animals. Other radioisotopic studies indicated that the rate of degradation of this enzyme was not altered significantly in diabetic rats, suggesting that the increase in this enzyme was due to an increased rate of synthesis. Similar experiments with pyruvate dehydrogenase, the first component of the pyruvate dehydrogenase complex, showed that livers from diabetic rats had approximately the same amount of immunoprecipitable enzyme protein as the control animals, but a larger proportion of the enzyme was in its inactive state. The rates of synthesis and degradation of pyruvate dehydrogenase were not affected significantly by diabetes.

Animals↗
PubMed1980

Induction of pyruvate carboxylase apoenzyme and holoenzyme in 3T3-L1 cells during differentiation.

The specific activity of pyruvate carboxylase [pyruvate:carbon-dioxide ligase (ADP-forming); EC 6.4.1.1] in 3T3-L1 cells increases approximately 20-fold when these cells differentiate to an adipocyte-like form [Mackall, J. C. & Lane, M. D. (1977) Biochem. Biophys. Res. Commun. 79, 720-725]. A specific antibody to the purified rat liver enzyme quantitatively precipitated pyruvate carboxylase from 3T3-L1 crude homogenates. Use of this immunological technique permitted us to demonstrate that the increase in pyruvate carboxylase activity is due to an increase in the intracellular concentration of the enzyme. The content of pyruvate carboxylase in differentiated 3T3-L1 cells is sufficiently high (1-2% of total protein) that the increase in this large protein (subunit M(r) = 130,000) can be visualized when 3T3-L1 crude extracts are subjected to electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. When 3T3-L1 cells differentiated in the presence of avidin, they contained less than 5% of the pyruvate carboxylase activity of cells that differentiated in the absence of avidin. However, the immunoprecipitable pyruvate carboxylase content of the avidin-treated cells was essentially the same as that of cells that differentiated without avidin. Full activity of the enzyme was rapidly restored in the avidin-treated cells upon the addition of excess biotin. The recovery of activity was closely correlated with the incorporation of [(14)C]biotin into immunoprecipitable pyruvate carboxylase. The rapidity with which the activity was restored and the insensitivity of the process to inhibitors of protein synthesis strongly suggest that the apoenzyme of pyruvate carboxylase accumulates during differentiation in the presence of avidin.

Adipose Tissue↗
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↗