Effect of phenylpyruvate on enzymes involved in fatty acid synthesis in rat brain

1. The effects of phenylpyruvate, a metabolite produced in phenylketonuria, on the pyruvate dehydrogenase-complex activity were investigated in rat brain mitochondria. 2. Pyruvate dehydrogenase activity was measured by two methods, one measuring the release of ¹⁴CO₂ from [1-¹⁴C]pyruvate and the other measuring the acetyl-CoA formed by means of the coupling enzyme, pigeon liver arylamine acetyltransferase (EC 2.3.1.5). In neither case was there significant inhibition of the pyruvate dehydrogenase complex by phenylpyruvate at concentrations below 2mm. 3. However, phenylpyruvate acted as a classical competitive inhibitor of the coupling enzyme arylamine acetyltransferase, with a K_i of 100μm. 4. It was concluded that the inhibition of pyruvate dehydrogenase by phenylpyruvate is unlikely to be a primary enzyme defect in phenylketonuria.     

The stimulating action of low doses of ionizing radiation on glucokinase synthesis in the liver of developing rats

Taking spontaneous glucokinase synthesis as an example the authors showed an increase in the rate of enzymatic differentiation against the insignificantly increased natural radiation background, which might serve as a supporting evidence of the previously obtained data confirming the necessity of the natural radioactive background for the development of mammals.     

Indications of an adaptive response in C57BL mice pre-exposed in vivo to low doses of ionizing radiation

C57BL/6 mice were whole-body irradiated with 5 cGy/day (‘adapting dose’) on 4 consecutive days and their spleens removed on day 1, 3, 7, 12, 19 or 26 after the last irradiation. In vitro UV-light-induced unscheduled DNA synthesis (UDS) and mitomycin C (MMC)-induced sister-chromatid exchanges (SCEs) were scored in lymphocytes (UV-light and MMC being the ‘challenging agents’), yielding higher UDS values and lower frequencies of induced SCEs than cells of non-adapted animals. On day 12 this effect could only be seen in half, on days 19 and 26 in none of the performed experiments. The results support those published by Tuschl et al. (1980, 1983) and Liu et al. (1987), showing that it is possible to induce the adaptive response in vivo.     

Features of DNA synthesis in chromatin from rat blood lymphocytes upon exposure to varying doses of ionizing radiation

Changes in the level of chromatin DNA synthesis after the total X-ray irradiation of rats in the dozes 0.5 Gr, 1 Gr and 7.76 Gr were shown. Interrelations between postradiation correlation of protein and DNA in lymphocytes of peripheral of blood rats were analyzed.     

Effects of thyroid hormones on enzymes involved in fatty acid and glycerolipid synthesis.

The influence of thyroid hormones on lipid biosynthesis was studied after administration of L-thyroxine to rats for 5 days. Their weights remained the same as those of control animals, despite an approximately 3-fold increment in plasma L-thyroxine and L-triiodothyronine concentrations. The activity of acetyl-CoA carboxylase and fatty acid synthetase as well as incorporation of tritium into fatty acids were depressed significantly in epididymal adipose tissue and enhanced significantly in livers of thyroxine-treated rats. Using antibodies specific against rat liver fatty acid synthetase, it was determined that the changes in activity of this multienzymic complex were due to alterations in amount of enzyme protein. In the presence of optimal concentrations of fatty acids, radioactive sn-glycero-3-phosphate, and co-substrates, total glycerolipid synthesis (defined in this study as the sum of newly formed radioactive mono- and diacyl-sn-glycero-3-phosphate, diglyceride, and triglyceride) was decreased significantly in adipose tissue and increased in liver and heart. Thus, administration of thyroid hormone results in tissue-specific alterations in lipid biosynthesis which, at least in the case of fatty acid synthetase, are due to changes in enzyme protein content.     

Inhibitory activity of chlorogenic acid on enzymes involved in the fatty acid synthesis in animals and bacteria

It was found that chlorogenic acid inhibited in vitro animal fatty acid synthase (FAS I) and the ss-ketoacyl-ACP reductase (FabG) from Escherichia coli in a concentration-dependent manner with respective IC50 of 94.8 and 88.1 microM. The results of Lineweaver-Burk plots indicated that chlorogenic acid inhibited competitively the binding of NADPH to FAS I, while left those of acetyl-CoA and malonyl-CoA unaffected. Further kinetic studies showed that chlorogenic acid blocked the activity of FAS I mainly by inhibiting the ss-ketoacyl reductase domain, which catalyzed the same reaction as that done by FabG in the fatty acid synthesis. The ss-ketoacyl reduction reactions accomplished by both FAS I and FabG required nucleotide cofactor, NADPH. Furthermore, the Lineweaver-Burk and Yonetani-Theorell analyses implicated that chlorogenic acid filled competitively in the binding-pocket of NADPH in the ss-ketoacyl reductase domain of FAS I. The similar results were also obtained from the inhibition of FabG by chlorogenic acid. As observed in these results, the inhibitions of FAS I and FabG by chlorogenic acid were highly related to the interference of the inhibitor with NADPH, which was possibly due to the similarity between chlorogenic acid and some portion of NADPH, maybe the section consisting of the two ribose groups.     

The inhibition of enzymes related to pulmonary fatty acid and phospholipid synthesis by dietary deprivation in the rat.

Acute nutritional deprivation results in significant reduction in the activities of acetyl-CoA carboxylase, fatty acid synthetase, microsomal fatty acid elongation and choline phosphotransferase in rat lung. This data establishes the enzymatic basis for the known inhibition of pulmonary surfactant production by acute starvation.     

DNA strand breaks and DNA repair response in lymphocytes after chronic in vivo exposure to very low doses of ionizing radiation in mice

In contrast to the well-documented negative effects of high-dose oxidant exposure, accumulating evidence supports a positive, perhaps essential physiologic role for very low-level oxidant stress. For example, low-level oxidant exposure, within or below the physiologic range, has been reported to stimulate membrane signal transduction, proliferation, antioxidant defense and DNA repair. In the present study, we have examined whether whole-body exposure to low-dose radiation (LDR) results in an alteration in constitutive (steady state) levels of DNA-strand breaks and whether an adaptive increase in DNA-repair response is induced. C57B1/6J mice were exposed to 0.04 Gy (4 cGy) of gamma-radiation as a model of low level oxidant stress. End points measured after chronic in vivo LDR included: (1) constitutive expression of DNA-strand breaks in quiescent spleen cells; (2) sensitivity to DNA damage after high-dose radiation exposure in vitro; (3) repair of constitutive and radiation-induced DNA strand breaks after mitogen stimulation: (4) activity of the DNA-repair associated enzyme, poly(ADP-ribose)transferase (ADPRT) and its substrate, NAD. The results indicated that the constitutive expression of DNA-strand breaks is significantly decreased after chronic LDR; however, DNA-repair capacity after high-dose radiation exposure is not increased above that observed in sham-irradiated mice. Associated with the reduction in constitutive DNA-strand break accumulation was a decrease in resting levels of the DNA-repair-associated enzyme poly(ADP-ribose) transferase (ADPRT). These results are consistent with the interpretation that cumulative DNA damage and associated DNA-repair activity in unstimulated cells are both reduced after chronic LDR exposure.     

Studies on the synthesis of rat liver fatty acid synthetase.

The synthesis of the multienzyme complex rat liver fatty acid synthetase was investigated utilizing modifications of methods developed in the laboratory of Schimke (Schimke, R. T. (1964) J. Biol. Chem. 239, 3808-3817 and Arias, I. M., Doyle, D., and Schimke, R. T. (1969) J. Biol. Chem. 244, 3303-3315). The relative amounts of radioactivity from a pulse of labeled lysine appearing in polypeptides derived from purified synthetase complex can be measured compensating for the varying amounts of lysine per polypeptide chain. The results show that labeled amino acid is incorporated into polypeptides derived from the complex at heterogeneous rates. However, 10 to 15 hours after the administration of a pulse, the amount of label per lysine residue in these polypeptides is identical. The results support the previously proposed model of this multienzyme complex (Tweto, J., Dehlinger, P., and Larrabee, A. R. (1972) Biochem. Biophys. Res. Commun. 48, 1371-1377). The previous work and that reported here suggests the existence of a pool of synthetase subunits which is an obligatory intermediate in both synthesis and turnover of the complex. The results obtained in this work are consistent with this model if the exchange of subunits into the intact complex is a relatively slow process requiring several hours to reach equilibrium.     

In vivo and in vitro demonstration of reduced myelin synthesis following early postnatal exposure to ionizing radiation in the rat

Irradiation of the immature central nervous system has been demonstrated histopathologically to result in a reduction in the quantity of myelin seen at later developmental ages [S. A. Gilmore, J. Neuropathol. Exp. Neurol. 22, 294-301 (1963). J. A. Beal and J. L. Hall, J. Neuropathol. Exp. Neurol. 33, 128-143 (1974)]. The results from our investigation indicate that this reduction in myelin content can be attributed to a decrease in sulfatide synthesis. Rats received whole-brain irradiation with 0, 500, 1500, 2000, or 2500 rad at 4 days postnatal (dpn). All of the rats exposed to 2000 or 2500 rad and 70% of those exposed to 1500 rad died within 6 to 10 days. At 17 dpn, animals received single intraperitoneal injections of [35S]sodium sulfate. Myelin synthesis, as indexed by the incorporation of sulfate into total lipids and glycolipids, was reduced in a dose-related fashion. To demonstrate a direct effect of ionizing radiation on myelinogenesis, brain cell reaggregate cultures derived from fetal rats were exposed at 12 days in vitro (div) to 0, 250, 500, 1000, or 1500 rad. A dose-related reduction in [35S]sulfate incorporation through 21 div was demonstrated. Reaggregates exposed to 250 or 500 rad but not 1000 or 1500 rad resumed normal myelin synthesis by 28 div. These changes occurred in the absence of histopathological changes, changes in protein content, and changes in the rate of protein synthesis.     

Early effect of myo-inositol deficiency on fatty acid synthetic enzymes of rat liver

Young rats (100 g) were fed either a purified myo-inositol-deficient balanced diet or a control diet containing 0.5% by weight myo-inositol, ad libitum, for up to 2 weeks following a 48 h fast. Weight gain was the same for animals in both groups. Liver triacylglycerol levels in the deficient animals were 1.8-, 3.5- and 3.0-fold higher than the corresponding levels in the control animals after 4, 8 and 14 days of feeding, respectively. In the myo-inositol-deficient group the specific activities of liver fatty acid synthetase and acetyl-CoA carboxylase were elevated 1.5-2.0-fold over controls, reaching a maximum after 3-4 days of feeding. Subsequently, activities declined to control levels. Rates of fatty acid synthetase synthesis in the deficient group, as measured by [3H]leucine incorporation into immunoprecipitable fatty acid synthetase polypeptide, were significantly higher (1.5-2.0-fold) than controls after 12-18 h of feeding and then declined to control levels by 1 day. No difference was noted between groups in either the rate of total, soluble liver protein synthesis or the half-life of fatty acid synthetase over this time period. These results suggest that the liver lipodystrophy observed during myo-inositol deficiency in rats may be due in part to elevated levels of lipogenic enzymes in this tissue in the early stage of the deficiency.