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T Flatmark

Publications and source records attributed to T Flatmark.

At least 91 records · Page 5Linked to original sources
PubMed1988

Phosphatidylinositol kinase of bovine adrenal chromaffin granules. Modulation by hydrophilic and amphiphilic cations.

The effects of hydrophilic and amphiphilic cations on the activity of phosphatidylinositol (PI) kinase (EC 2.7.1.67) of chromaffin granule ghosts were investigated. The cations studied can be divided into two groups, i.e. (i) compounds with a biphasic response (stimulation and inhibition), and (ii) those with a selective stimulatory effect on the enzyme activity. The cationic amphiphile trifluoperazine belongs to the first group, and stimulated the enzyme activity, maximal at 80 microM (2-fold), with a progressive inhibition at higher concentrations. This biphasic response was shared by a number of structurally related cationic amphiphiles, i.e. the tricyclic antidepressants, imipramine and desipramine, the phenothiazine, chlorpromazine, the miconazole derivative, calmidazolium, the beta-adrenergic agonist, propranolol, compound 48/80, as well as by the hydrophilic cations neomycin and poly-L-lysine. On the other hand, a pure stimulatory effect was observed with the amphiphilic polypeptide mastoparan and the polycationic compound spermidine, whereas ACTH1-39 and ACTH1-24 (peptides structurally related to mastoparan) revealed a slight inhibitory effect. We conclude that all the cations tested including Mg2+, stimulate PI kinase activity rather unspecifically by binding of the positively charged groups to a membrane component, probably the PI kinase itself. This site is different from that mediating the specific inhibition by calcium (Husebye ES and Flatmark T, Biochim Biophys Acta 968: 261-265, 1988). The inhibitory effect of cationic amphiphiles is correlated to their lipid solubility, and represents a perturbation of the membrane structure, but not a solubilization of enzyme or phosphoinositide from the membrane. The inhibitory effect of hydrophilic cations is due to complexation of ATP.

1-Phosphatidylinositol 4-Kinase↗
PubMed1988

On the mechanism of induction of the enzyme systems for peroxisomal beta-oxidation of fatty acids in rat liver by diets rich in partially hydrogenated fish oil.

In this paper, we describe the early biochemical changes in liver cells that occur in rats fed a semisynthetic diet containing 20% (w/w) partially hydrogenated fish oil. Within hours the level of ornithine decarboxylase (ODC) increased, peaked at about 24 h (11-fold increase) and returned to subnormal levels within 48 h. The diet evoked a similar rapid increase in the cellular level of mRNA for the bifunctional enzyme of peroxisomal beta-oxidation (enoyl-CoA hydratase: beta-hydroxyacyl-CoA dehydrogenase (HD)) (12-fold), followed by increases in the specific content of HD protein (3-fold) and the capacity for beta-oxidation in peroxisomes (5.3-fold). The cellular level of long-chain acyl-CoA increased 2.1-fold. By contrast, no significant changes were observed in the specific activities of ornithine decarboxylase, peroxisomal beta-oxidation activity and microsomal omega-hydroxylation as well as the level of long-chain acyl-CoA in livers of rats fed (1 week) diets containing 20% (w/w) soybean oil with added 3 or 6% (w/w) of either elaidic acid (18:1(11) (trans)), brassidic acid (22:1(13) (trans)) or erucic acid (22:1(13) (cis)). Expression of normal levels of mRNA for the bifunctional enzyme was also found. Morphometric analyses revealed no proliferation of peroxisomes in these fatty acid-supplemented diets, in contrast to that observed with the partially hydrogenated fish oil diet. These results are consistent with the proposal (Flatmark, T., Christiansen, E.N. and Kryvi, H. (1983) Biochim. Biohys. Acta 753, 460-466) that components in dietary oils, different from C22:1 cis and trans fatty acids, are responsible for the pleiotropic responses evoked in target cells. Thus, the pattern of response induced by partially hydrogenated fish oil mimics those induced by xenobiotic compounds collectively termed peroxisome proliferators.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1988

Soluble tyrosine hydroxylase (tyrosine 3-monooxygenase) from bovine adrenal medulla: large-scale purification and physicochemical properties.

A new procedure that permits large-scale purification of tyrosine 3-monooxygenase (tyrosine hydroxylase) (L-tyrosine,tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2) from the cytosolic fraction of bovine adrenal medulla is described. The homogenous enzyme revealed a subunit Mr of 60,000 and a specific activity of 425 nmol.min-1.mg-1. The N-terminal amino-acid sequence (27 residues) revealed 89% homology with the human pheochromocytoma enzyme as deduced from its cDNA sequence. The pure enzyme contained 0.66 +/- 0.09 mol iron, 0.13 mol zinc and 0.62 +/- 0.04 mol phosphate per mol subunit of Mr = 60,000. A broad light absorption band with its maximum around 700 nm (epsilon 700 nm = 1.3 (mM monomer)-1.cm-1) explains its blue-green color. EPR spectra at 3.6 K revealed high-spin Fe(III) (S = 5/2) in an environment of nearly axial symmetry (g values at 7.2-6.7, 4.7-5.3 and 1.9-2.0). A close correlation was observed between the absorbance at 700 nm and the intensity of the axial type of EPR spectrum. The absorption peak at 700 nm is compatible with a ligand-to-iron charge-transfer transition as a result of catecholate coordination to the iron. Physicochemical studies suggest that the enzyme does not undergo such major substrate- or cofactor-induced conformational changes as have been reported for the related enzyme, phenylalanine hydroxylase.

Adrenal Medulla↗
PubMed1988

Inhibition of the H+-ATPase in bovine adrenal chromaffin granule ghosts by diethylstilbestrol. Evidence for a tight coupling between ATP hydrolysis and proton translocation.

Diethylstilbestrol (DES) was found to inhibit reversibly the hydrolysis of MgATP (80% at 100 microM) and proton pump activity (I50 approximately equal to 15 microM, complete at 100 microM) in chromaffin granule ghosts. The parallel inhibition suggests a tight kinetic coupling between the two activities. The Mg2+-ATPase activity, but not proton pumping, was partially restored by N,N'-dicyclohexylcarbodiimide, indicating that the two inhibitors in combination cause a partial uncoupling. The non-competitive type of inhibition shows that the action of DES is distal to the site of ATP binding and hydrolysis. Although unspecific, the interaction of DES with the chromaffin granule membrane seems primarily to affect the H+-ATPase.

Adenosine Triphosphate↗
PubMed1988

Phosphatidylinositol kinase of bovine adrenal chromaffin granules: kinetic properties and inhibition by low concentrations of Ca2+.

The kinetics and regulatory properties of phosphatidylinositol (PI) kinase were studied in chromaffin granule ghosts isolated from the bovine adrenal medulla. Phosphatidylinositol 4-phosphate (PIP) was the major 32P-labelled phospholipid formed when the isolated membranes were phosphorylated by [gamma-32P]ATP. The PI kinase activity was rather independent of pH, but highly dependent on Mg2+ with a maximal stimulation at 60 mM Mg2+. By contrast, KCl and NaCl had a slight inhibitory effect. The Km value for MgATP was 44 and 62 microM in the presence of 1 and 20 mM MgCl2, respectively. The PI kinase was almost fully and reversibly inhibited by free Ca2+ (calmodulin-independent) in the nanomolar and low micromolar range, depending on the concentration of Mg2+. The inhibition was not dependent on Ca2+-stimulated protein phosphorylation, and it could not be explained by a dephosphorylation of PIP.

1-Phosphatidylinositol 4-Kinase↗
PubMed1987

Isolation and characterization of tetrahydropterin oxidation products generated in the tyrosine 3-monooxygenase (tyrosine hydroxylase) reaction.

The catalytic mechanism of tyrosine 3-monooxygenase (tyrosine hydroxylase, EC 1.14.16.2), isolated from the cytosolic fraction of bovine adrenal medulla, was studied by new techniques of product isolation and characterization. Using either (6R)-tetrahydrobiopterin, (6RS)-tetrahydroneopterin, or 6-methyl-tetrahydropterin, as the cofactors, three enzymatic oxidation products could be isolated and identified from the reaction mixture by high-performance liquid chromatography and rapid-scanning spectroscopy: (a) the 4a-hydroxy derivatives, (b) the quinonoid dihydropterins, and (c) the stable 7,8-dihydropterins. Stopped-flow spectroscopy revealed that the formation of the 4a-hydroxy-tetrahydropterins preceded the formation of the quinonoid forms with both L-tyrosine and L-phenylalanine as the substrate. The formations of 4a-hydroxy-tetrahydropterins and hydroxylated amino acids were tightly coupled as recently shown in the phenylalanine 4-monooxygenase reaction [Haavik, J., Døskeland, A. P. & Flatmark, T. (1986) Eur. J. Biochem. 160, 1-8]. No detectable carbinolamine dehydratase activity was present in the enzyme preparation.

Adrenal Medulla↗
PubMed1987

Characterization of phospholipase activities in chromaffin granule ghosts isolated from the bovine adrenal medulla.

Highly purified chromaffin granule membranes contain high levels (100 nmol/mg protein) of long-chain free fatty acids (Husebye, E.S. and Flatmark, T. (1984) J. Biol. Chem. 259, 15272-15276), as well as lysophosphatidylcholine (268 nmol/mg protein) and lysophosphatidylethanolamine (92 nmol/mg protein). The release of saturated and unsaturated long-chain fatty acids from endogenous phospholipids was 38 and 28 nmol/mg protein per h, respectively, at 37 degrees C and pH 7.5 (alkaline pH optimum). p-Bromophenacyl bromide inhibited the release of palmitate and oleate by 88 and 65%, respectively. The deacylation of membrane phospholipids was not significantly affected by micromolar free Ca2+. Based on experiments with pancreatic phospholipase A2, stearate and arachidonate were found to be suitable markers for deacylation at the sn-1 and sn-2 positions, respectively. Experiments with exogenously added labeled phosphatidylcholines confirmed that chromaffin granule ghosts contain a phospholipase A2 activity (alkaline pH optimum). The preparations also revealed a phospholipase A1 activity (acid pH optimum). Finally, the ghosts contain a lysophospholipase activity (alkaline pH optimum), that accounts for the major part of the deacylation of membrane phospholipids, notably the release of saturated fatty acids (stearate and palmitate). It is unlikely that the high content of lysophospholipids is an artifact of the procedure by which the granule ghosts are isolated.

Acylation↗
PubMed1987

Studies on Mg2+-dependent ATPase in bovine adrenal chromaffin granules. With special reference to the effect of inhibitors and energy coupling.

The Mg2+-ATPase activities of bovine adrenal chromaffin granules were studied in highly purified preparations of granule ghosts and in intact organelles. The overall ATPase activity (150-250 nmol ADP min-1 mg-1) of the granule ghost preparations was inhibited less than 5% by the bathophenanthroline chelate of Fe(II), a potent inhibitor of mitochondrial F1-ATPase. This small inhibition can be accounted for by a very minor contamination with mitochondria or mitochondrial fragments. The overall ATPase activity of native granule ghosts was inhibited about 75% by N-ethylmaleimide, with half-maximal inhibition at about 20 microM. The titration curve was slightly shifted towards higher concentrations as compared to the inhibition curve for the proton pump activity, which was completely inhibited at 25 microM. N,N'-Dicyclohexylcarbodiimide inhibited the overall ATPase activity by 75-80% at 1.1 mumol/mg protein, a concentration that completely abolished the proton pump activity. Low concentrations (10 microM) of vanadate inhibited the overall ATPase activity by about 15% but had no effect on the proton pump activity, which was partly inhibited only at higher vanadate concentrations. Our attempts to assign a function to the vanadate-sensitive and N-ethylmaleimide-insensitive ATPase have so far been unsuccessful. In particular, our assay for ATP diphosphohydrolase activity was negative, although the chromaffin granule ghosts revealed a low Mg2+-ADPase activity (11.8 nmol AMP min-1 mg-1 protein). In intact chromaffin granules the specific Mg2+-ATPase activity (50-70 nmol ADP min-1 mg-1) was stimulated 2-fold by uncouplers, as compared to 1.6-1.7-fold in granule ghosts. The degree of energy coupling was rather independent of the external pH (6.5 less than pH less than 8.0) and temperature (20-45 degrees C). As expected, partial inhibition (about 15%) of the overall ATPase activity by 10 microM vanadate increased the ATPase control ratio. ADP was found to be a potent inhibitor of the proton pump activity with MgATP as the substrate, and the effect can partly be explained by a competitive type of inhibition of the hydrolytic reaction. This effect of ADP explains some of the kinetic data reported for MgATP-dependent (H+-ATPase-dependent) reactions in this organelle, notably the energy-dependent accumulation and storage of catecholamines.

Adenosine Diphosphate↗
PubMed1987

Modulation by pterins of the phosphorylation and phenylalanine activation of phenylalanine 4-mono-oxygenase.

The interaction between phenylalanine 4-mono-oxygenase and analogues of the natural cofactor (6R)-tetrahydrobiopterin [(6R)-BH4] was studied. The rate of cyclic AMP-dependent phosphorylation of phenylalanine 4-mono-oxygenase was inhibited only by those pterins [(6R)-BH4, (6S)-BH4 and 7,8-dihydrobiopterin (BH2)] that were able to decrease the potency and efficiency of phenylalanine as an allosteric activator of the hydroxylase. Since BH2 lacks cofactor activity, this was not required to modulate either the phosphorylation or the phenylalanine-activation of the hydroxylase. Half-maximal inhibition of the phosphorylation was observed at 1.9 microM-(6R)-BH4, 9 microM-(6S)-BH4 and 17 microM-BH2. Competition experiments indicated that all three pterins acted through binding to the cofactor site of the hydroxylase. Since the phosphorylation site and the cofactor binding site are known to reside, respectively, in the N- and C-terminal domains of the hydroxylase, the pterins were able to induce an interdomain conformational change. BH2, whose dihydroxypropyl group is not subject to epimerization, and (6S)-BH4 both inhibited the phosphorylation less efficiently than did the (6R)-epimer of BH4. Pterins with different spatial arrangements of the dihydroxypropyl side chain thus appeared to elicit different conformations of the phosphorylation site. The hydroxylase reaction showed a higher apparent Km for (6S)-BH4 than for (6R)-BH4 both when the native and the phenylalanine-activated enzyme were tested. For the activated enzyme Vmax was 40% lower with the (6S)-epimer than the (6R)-epimer, also when the more rapid enzyme inactivation occurring with the former cofactor was taken into account.

Binding Sites↗
PubMed1987

Effect of a high-fat diet with partially hydrogenated fish oil on long-chain fatty acid metabolizing enzymes in subcellular fractions of rat liver.

Hepatic metabolism of long-chain fatty acids were studied in young male rats fed a semisynthetic diet containing 20% (w/w) partially hydrogenated fish oil (PHFO)2, with or without 2% (w/w) linoleic acid. The enzymic activities involved in the formation and breakdown of long-chain acyl-CoA were both increased in the animals fed the semisynthetic diet, compared to pellet-fed control animals. Thus, the specific palmitoyl-CoA synthetase activity increased slightly in both the mitochondrial (1.4-fold) and the microsomal (1.6-fold) fractions. In the peroxisome-enriched fraction the activity was increased (about 2.6-fold) only on addition of linoleic acid to the diet. The data are consistent with an increased catabolism of long-chain fatty acids by a peroxisomal and a mitochondrial pathway. Thus, the total carnitine palmitoyltransferase activity increased 2-fold in the mitochondrial fraction, and was partly prevented by added linoleic acid. Peroxisomal beta-oxidation activity was also increased (about 7-fold) in livers of PHFO-fed rats, but did not change when linoleic acid was added. The PHFO-fed rats also revealed elevated capacity for hydrolysis of palmitoyl-CoA in both the mitochondrial (2.4-fold) and the cytosolic (2.0-fold) fractions and the latter was almost completely and selectively prevented by added linoleic acid. The s values of mitochondria and peroxisomes varied with the dietary regime, and some of the observed changes in the specific activities of the fatty acid metabolizing enzymes with multiple subcellular localization can be explained as an effect of changes in the s values of the organelles. Thus, the s value of mitochondria increased 1.8-fold as a result of PHFO feeding, but was fully prevented by linoleic acid in the diet. On the other hand, the s values of peroxisomes decreased by about 50% on feeding a PHFO diet, and by about 25% with added linoleic acid.

Animals↗
PubMed1987

Evidence that deletion of coding sequences in the 5' end of the thymidine kinase gene of herpes simplex virus type 1 affects the stability of the gene products.

The experiments described in the present work were designed to study the function of the N-terminal end of thymidine kinase (TK) encoded by herpes simplex virus type 1. Specifically we were interested to know whether this end was involved in binding of the enzyme to other molecules, had any influence on its subcellular localization or affected one or more of the activities associated with the enzyme. A parental enzyme and a deletion mutant, lacking the 45 N-terminal amino acids, derived from this strain, were used. Thymidine kinase from the parental virus bound to DNA-Sepharose, but the truncated enzyme did not. This was apparently not due to a specific ability to bind to DNA, since immunofluorescence studies indicated that both the normal and the deleted TK were mainly located in the cytoplasm, preferentially in the perinuclear region. Phosphorylation of thymidine as well as the amounts of TK polypeptides were markedly reduced at late times after infection with the mutant, but not to the same extent after infection with the wild-type. The deleted TK gene was efficiently transcribed as shown by hybridization of RNA to a probe specific for the gene, and this RNA directed the synthesis in vitro of TK polypeptides. Deletion of the 5' end of the gene seems to affect the stability of either the enzyme or TK-specific mRNA, or both. The TMP phosphorylating activity seems to be particularly destabilized relative to the thymidine phosphorylating activity.

Cell Compartmentation↗
PubMed1986

Studies on catalase compartmentation in digitonin-treated rat hepatocytes.

The cellular compartmentation of catalase was studied in digitonin-permeabilized rat hepatocytes. A biphasic dose-response curve was observed for the unmasking of catalase activity by digitonin in latency studies. About 40-60% of the total catalase activity was seen in the range of 5-200 microM digitonin compared to 13% free activity in control preparations without digitonin. The free catalase activity began to increase again above 300 micron digitonin, and all latency was lost around 500 microM and above. These results indicate that there exists in rat hepatocytes catalase with two levels of crypticity to digitonin, only one of which was seen in a mixed organelle preparation containing peroxisomes.

Animals↗
PubMed1986

Stereoselective effects in the interactions of pterin cofactors with rat-liver phenylalanine 4-monooxygenase.

The (6R) and (6S) epimers of l-erythro-tetrahydrobiopterin (BH4) and some of its structural analogs, were tested as cofactors and non-covalent effectors in the phenylalanine 4-monooxygenase (phenylalanine hydroxylase, EC 1.14.16.1) reaction. The oxidation-reduction potentials (Em,7) of the free (not enzyme-bound) form of the (6R) and (6S) epimers were rather similar (range 174-184 mV) for the oxidation of tetrahydropterins to quinonoid dihydropterins. Rapid-mixing kinetic experiments were performed at 20 degrees C under conditions which allow only a few turnover reactions of the enzyme. Three main oxidation products were identified spectroscopically at pH 6.8 for all three tetrahydropterins tested: the C(4a)-hydroxy derivatives, the quinonoid dihydropterins, and the stable 7,8-dihydropterins (in that sequence). The formation of the C(4a)-hydroxy forms closely paralleled that of tyrosine, and supports the proposal that this covalent adduct is formed as an immediate product on completion of the catalytic cycle. Assay of the initial rate of C(4a)-hydroxy derivative formation represents a new approach in kinetic studies of this enzyme, and the kinetic parameters obtained for the phenylalanine-activated enzyme are presented. The affinity of binding of (6R)-BH4 and (6S)-BH4 to phenylalanine hydroxylase was also estimated on the basis of their quenching of the intrinsic tryptophan fluorescence of the enzyme. The apparent affinities were found to correspond well to the Km values estimated in kinetic studies of the hydroxylation reaction with the phenylalanine activated enzyme, i.e. higher for (6R)-BH4 than for (6S)-BH4. The lower V value observed for the native enzyme with the (6R) epimer in steady-state kinetics is explained by its higher potency as a negative effector, since the oxidation-reduction potentials of the two diastereomers were similar. Dihydrobiopterin (BH2) was found to inhibit the hydroxylation reaction and quenched the intrinsic tryptophan fluorescence of the enzyme with the same concentration dependence as that observed with (6S)-BH4.

Animals↗
PubMed1986

Effects of essential fatty acid deficiency on mitochondria and peroxisomes in rat hepatocytes with special reference to a partially hydrogenated fish oil diet.

Feeding male rats a high cal% partially hydrogenated fish oil diet induced morphological and biochemical changes in hepatocytes at the mitochondrial and peroxisomal level. At the mitochondrial level, formation of megamitochondria was related to the development of an essential fatty acid deficiency, as measured by a high 20:3/20:4 fatty acid ratio. These mitochondrial changes were fully prevented by adding linoleic acid to the partially hydrogenated fish oil diet. The megamitochondria revealed a normal specific content of respiratory chain pigments, normal specific respiratory rates and a normal energy coupling. At the peroxisomal level, feeding of the partially hydrogenated fish oil diet caused a considerable proliferation, which was unrelated to essential fatty acid deficiency. The total number of peroxisomes increased 1.9-fold, and 2.6-fold in the presence of added linoleic acid. Essential fatty acid deficiency seemed to result in an inhibition of peroxisomal biogenesis. It was concluded that the induction of megamitochondria by partially hydrogenated fish oil was fully attributable to essential fatty acid deficiency, whereas peroxisomal proliferation must be attributed to other factors in the diet.

Animals↗
PubMed1986

Relation between induction of ornithine decarboxylase and specific gene expression in rat liver in response to the tumor promoter agent clofibrate.

The time course of changes in a number of biochemical parameters in rat liver was studied during 10 days of clofibrate administration. Ornithine decarboxylase (ODC) and putrescine levels began to increase within hours of the first dose and reached maxima at about 36 h (40 and 10 times control levels, respectively) and then returned to normal levels by 48 h. This ODC induction by clofibrate is different from that seen in compensatory liver hyperplasia or diethylnitrosamine administration in that it was not accompanied by elevations in cAMP or increased activation of cytoplasmic cAMP-dependent protein kinases, type I or II. Messenger RNA levels, notably of the species coding for the enzymes of the peroxisomal beta-oxidation pathway, increased in parallel with ODC and putrescine to reach a maximum also at 36 h. The enzymes of the peroxisomal beta-oxidation pathway, on the other hand, increased more gradually over time to reach a plateau at approximately 7 - 10 days. The magnitude of increase in mRNA (about 7-fold) was comparable to that of peroxisomal beta-oxidation as measured by cyanide-insensitive palmitoyl-CoA-dependent NAD+ reductase activity; comparable increases in the specific content of enoyl-CoA hydratase: beta-hydroxyacyl-CoA dehydrogenase and of peroxisomal thiolase were observed, as determined by SDS electrophoresis. A gradual increase in long-chain acyl-CoA (1.5-fold) followed the increase in beta-oxidation, whereas a 2-fold increase in acid-soluble CoA (free CoA and short-chain acyl-CoA) was seen as early as 36 h. This sequence of changes is at variance with proposals that increased levels of long-chain acyl-CoA mediate induction of peroxisomal beta-oxidation.

Acyl Coenzyme A↗