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T Flatmark

Publications and source records attributed to T Flatmark.

At least 127 records · Page 7Linked to original sources
PubMed1982

Inhibition of mitochondrial protein synthesis and energy coupling by fragment A of diphtheria toxin.

The effect of intact diphtheria toxin and its fragment A on energy-dependent functions in mouse liver mitochondria/mitoplasts has been studied. Fragment A was found to inhibit protein synthesis in mitoplasts to the same extent (approximately 80%) as the uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone, but had no similar effect in lysed mitoplasts. Intact diphtheria toxin had no effect in either case. Fragment A was found to function as a potent uncoupler in isolated mitochondria and mitoplasts, inhibiting oxidative phosphorylation by approximately 80% at a concentration (2 micrograms fragment A/mg of protein) that did not inhibit protein synthesis. In contrast, intact toxin slightly increased the tightness of energy coupling in isolated mitoplasts. 125I-labelled intact diphtheria toxin was bound to mitoplasts to about the same extent as labelled fragment A. At concentrations which efficiently inhibited mitochondrial protein synthesis, fragment A had no effect on the intramitochondrial concentration of nicotinamide adenine dinucleotides, nor was it capable of ADP-ribosylating mitochondrial proteins, indicating that the well known enzymatic activity of fragment A is not involved in the observed effect in mitochondria. The results indicate that fragment A of diphtheria toxin inhibits protein synthesis in mitochondria and mitoplasts by inhibiting mitochondrial energy transduction. The detailed mechanism of the uncoupling effect and its possible significance in intact cells remain to be elucidated.

Animals↗
PubMed1981

Acyl-CoA synthetase activity of rat liver microsomes. Substrate specificity with special reference to very-long-chain and isomeric fatty acids.

1. A fatty acid-depleted rat liver microsomal fraction has been used for the measurement of acyl-CoA synthetase (acid : CoA ligase (AMP-forming), EC 6.2.1.3) activity. The assay was based on measurement of the reaction product AMP by high-performance liquid chromatography (HPLC). The synthetase activity (V') revealed an optimum at 12 : 0 with saturated fatty acids as substrate, and at 14 : 1 with mono-unsaturated fatty acids. The apparent Michaelis constant, on the other hand, showed no systematic dependence on the fatty acid chain-length. 2. The mono-unsaturated fatty acids from 14 : 1 to 22 : 1 gave higher activities than the corresponding saturated fatty acids, and the relative differences were greatest with the very-long-chain fatty acids eicosaenoic (20 : 1 (11) (cis)) and docosaenoic acid (22 : 1 (11) (cis)). The synthetase activity with saturated and mono-unsaturated fatty acids was found to correlate to their capacity factor (k') on reversed phase chromatography (HPLC). This finding may indicate that the observed chain-length dependence of the activity largely reflects the partition of the fatty acids between a hydrophobic and a hydrophilic phase. In general, the position of the double bond and the cis/trans configuration had little effect on the V' values except for 22 : 1 (11)(cis) which revealed a 2-fold higher activity tha 22 : 1 (13) (cis). 3. The polyunsaturated fatty acid 22 : 6 (all cis) ;was notably found to be a much better substrate than other C22 fatty acids. 4. The present study does not support the idea of more than a single ATP-dependent acyl-CoA synthetase in the rat liver microsomal fraction.

Animals↗
PubMed1981

Effects of marine oil diet on peroxisomes and mitochondria of rat liver. A combined biochemical and morphometric study.

Feeding male rats a high cal% partially hydrogenated marine oil diet induced a response in the parenchymal liver cells at the cellular as well as the subcellular level. An adaptation in the lipid metabolism is indicated by an accumulation of triglycerides and a hypertrophy of the liver cells, and a rather selective increase (4.9-fold) in the specific activity of peroxisomal beta-oxidation. In addition, a slight proliferation of peroxisomes was observed; the volume fraction and the total number of peroxisomes increased 1.5-fold. The peroxisomes, however, preserved the biochemical and morphological homogeneity characteristic of peroxisomes in normal controls. At the mitochondrial level, a gradual development of megamitochondria was observed as the principal morphological change, with a 1.6-fold increase in the average size. The functional significance of this change remains to be determined. The effects of a partially hydrogenated marine oil diet on parenchymal liver cells appears to be manifestations with multiple causal factors. The high content of very long-chain trans fatty acids is of particular significance for the peroxisomal changes. An essential fatty acid deficiency, enforced by the presence of trans fatty acids in the diet, is most likely the determining factor for the development of megamitochondria.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1981

Polydispersity of rat liver peroxisomes induced by the hypolipidemic and carcinogenic agent clofibrate.

1. The present study has confirmed that the hypolipidemic and carcinogenic agent clofibrate induces a marked increase in the specific activity of some peroxisomal marker enzymes in rat liver homogenates, notably of the palmitoyl-CoA dependent dehydrogenase and catalase activities. 2. Clofibrate was found to induce a marked polydispersity of the peroxisomes as determined by analytical differential centrifugation of homogenates and morphometric analysis of hepatocytes. 3. Two major populations of peroxisomes were detected by analytical differential centrifugation under conditions which reduce the hydrostatic pressure effects on the organelle to a minimum. Using urate oxidase as the marker enzyme, the S4,B-values of the two populations were estimated to 1 1 860 S and 4240 S, both different from that of the homogenous population of peroxisomes in the control animals (S4,B approximately equal to 6680 S). The 4240 S-population induced by clofibrate revealed a high specific activity relative to that of of urate oxidase and particularly relative to that of catalase, which was very low. In addition, a less distinct population of particles (870 S lees than S lees than 4240 S) contained more than 50% of the total particle-bound palmitoyl-CoA dependent dehydrogenase activity sedimented at a centrifugal effect of t integral of 0 rmp(2)dt = 1.5 x 10(10) min(-1), but not urate oxidase and catalase activities. This fraction was not observed in the homogenates of normal rats. As in the normal controls, the palmitoyl-CoA dependent dehydrogenase activity was found to be particle-bound (S greater than 870 S). 4. Morphometric analyses of randomly selected hepatocytes revealed that after clofibrate treatment the relative volume fraction of the peroxisomes increased by a factor of 5.5 and thier average diameter and volume by a factor of 1.3 and 2.1, respectively. Furthermore, the frequency of electron-dense matrix cores decreased on clofibrate treatment. In contrast, no change was observed in the average size of the mitochondria, and their relative volume fraction increased only by a factor of 1.2. 5. The clofibrate induced changes in eh morphological and biochemical properties of rat liver peroxisomes appears to be a very useful model system in which to study the biogenesis as well as the biochemical and physiological role(s) of this organelle in mammalian cells.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1980

Acyl-CoA synthetase activity of brown adipose tissue mitochondria. Substrate specificity and its relation to the endogenous pool of long-chain fatty acids.

1. A new assay of acyl-CoA synthetase (acid:CoA ligase (AMP-forming), EC 6.2.1.3) activity was developed for application to measurements on isolated intact mitochondria. The assay was based on the formation of the product AMP as determined by high-performance liquid chromatography (HPLC), making corrections for the partial conversion of AMP to ADP by the adenylate kinase reaction. 2. The substrate specificity of the long-chain acyl-CoA synthetase was measured in brown adipose tissue mitochondria, isolated from cold-acclimated guinea-pigs. Using mitochondria largely deficient of endogenous fatty acids, the synthetase activity (V') revealed an optimum at 12 :0 and 13 : 0 with saturated fatty acids as the substrate. In contrast to other tissues, the highest V' values were observed with unsaturated fatty acids 18 : 1 (9) (cis) and 18 : 2 (9, 12) (all cis). The apparent Michaelis constant varied within a narrow range and revealed no systematic dependence on the chain-length as was the case for V'. Since the mitochondria have a high capacity of fatty acid binding, with unknown affinity, the estimated K'm values are, however, of questionable significance. 3. The pattern of chain-length specificity in the synthetase reaction of the mitochondria compares well with the pattern of endogenous long-chain fatty acids in which mainly four species contribute, i.e. 18 : 1 (42 mol%) and 18 : 2 (18 mol%), 16 : 0 (26 mol%) and 18 : 0 (14 mol%).

Acetate-CoA Ligase↗
PubMed1980

Oxidoreductase activities of chromaffin granule ghosts isolated from the bovine adrenal medulla.

1. Based on estimated s-values of subpopulations of bovine adrenal chromaffin granules (Bødtker-Naess, V., Slinde, E., Terland, O. and Flatmark, T. (1978) Biochim. Biophys. Acta 541, 124--134) a new large-scale procedure is described for the isolation of the total population of chromaffin granules by differential centrifugation in 0.25 M sucrose. 2. Using the total population of chromaffin granules obtained by differential centrifugation, final purification was achieved by density-gradient centrifugation in either sucrose or Percoll-sucrose. In either case, the isolated granule fractions were contaminated with mitochondria to about the same degree. 3. Chromaffin granule ghosts, obtained by hypoosmotic lysis of granules isolated by sucrose density-gradient, centrifugation, were subjected to centrifugation on a discontinuous density gradient (buffer/0.9 M sucrose). By this procedure a substantial purification of the ghosts was achieved as determined from measurements of protein and various marker enzymes. 4. In contrast to preparations of chromaffin granule ghosts prepared by previous standard procedures, those purified by gradient centrifugation (on 0.9 M sucrose) did not reveal any NADH-linked cytochrome b-561 reductase activity. However, experimental evidence is presented for the existence of an intrinsic NADH-oxidizing enzyme system in the granule membrane. 5. No significant difference was observed in the specific content of cytochrome b.561 and NADH:(acceptor) oxidoreductase activities between ghost preparations obtained from populations of heavy and light chromaffin granules. 6. The functional significance of cytochrome b-561 and the NADH:(acceptor) oxidoreductase activities of the granule membrane remains to be determined.

Adrenal Medulla↗
PubMed1980

Subcellular distribution of ascorbate in bovine adrenal medulla. Evidence for accumulation in chromaffin granules against a concentration gradient.

The subcellular distribution of ascorbate and catecholamines has been studied in homogenates of bovine adrenal medulla and cortex. 1. The recovery of the vitamin was found to be 4.10 +/- 0.22 and 9.57 +/- 1.37 mumol/g wet weight for the medulla and cortex, respectively. A major fraction (34.4%) of the vitamin was recovered in the particulate fraction of the medulla as compared to about 8% in the corresponding fraction of the cortex. In comparison, 78.9% of the catecholamines were found in the particulate fraction of the medulla. 2. Analytical differential centrifugation of medulla homogenates revealed a sedimentation profile of ascorbate which was identical to that obtained for noradrenalin and adrenalin. The co-sedimentation of these compounds indicates that ascorbate is an essential component of the heavy as well as the light population of chromaffin granules. The stoichiometry of catecholamines to ascorbate was approx. 25:1 in both subpopulations. 3. Based on an estimated volume fraction of approximately 13% for the chromaffin granules, as determined morphometrically (Kryvi, H., Flatmark, T. and Terland, O. (1979) Eur. J. Cell Biol. 20, 76-82), a concentration gradient (chromaffin granules:cytosol) of approx. 4 was estimated for ascorbate in the cells of adrenal medulla. 4. No ascorbate 2-sulfate was detected in any of the subcellular fractions isolated, and the content of dehydroascorbate in isolated chromaffin granules was less than 1% of the total ascorbate value.

Adrenal Cortex↗
PubMed1980

Mitochondrial iron not bound in heme and iron-sulfur centers. Estimation, compartmentation and redox state.

A method is described for the assay of total mitochondrial non-heme iron and a fraction which does not belong to the iron-sulfur proteins (FeS centers) of the outer and inner membrane. The assay of the latter fraction, which is termed 'non-heme non-FeS iron', is based on the formation of a chelate of Fe(II) with bathophenanthroline sulfonate in osmotically swollen mitochondria under conditions where the FeS centers are quite stable as determined by EPR spectroscopy at 20.4 K, 93 K and 123 K. The 'non-heme non-FeS iron', which in normal rat liver mitochondria amounts to approx. one third of the total mitochondrial iron (i.e. 1.7 +/- 0.3 nmol . mg-1 protein), does not represent a homogeneous pool of iron. Based on studies of its reaction with bathophenanthroline sulfonate and the dependency of this reaction on reducing agents in mitochondria and mitoplasts, evidence is presented that this non-heme iron is present in two major pools in which the inner membrane constitutes the barrier. A minor fraction (i.e. 0.4 +/- 0.2 nmol . mg-1 protein) is localized to the 'outer' compartment and a major fraction (i.e. 1.1 +/- 0.1 nmol . mg-1 protein) is localized to the 'inner' compartment and is equally distributed between the inner membrane and the matrix. The experiments described in this study also indicate that approximately half of the 'non-heme non-FeS iron' of the 'inner' pool is in the ferrous form in mitochondria as isolated, and this was not increased when oxidizable substrates were added to the mitochondria. Although the biological significance of this iron pool is not yet clear, it is likely that it represents a transit iron pool being the proximate iron donor for heme synthesis catalyzed by the enzyme ferrochelatase.

Animals↗
PubMed1980

Electron paramagnetic resonance of the copper in dopamine beta-monooxygenase. Rapid reduction by ascorbate, the steady-state redox level, chelation with EDTA, and reactivation of the apoenzyme by added copper.

The water-soluble form of dopamine beta-monooxygenase from bovine adrenal medulla was studied. Addition of excess CuSO4 to purified enzyme preparations followed by extensive ultrafiltration against copper-free buffer at pH 7.0, gave preparations with about four copper atoms per enzyme tetramer of Mr 290 000. The enzyme-bound copper was shown by the rapid-freeze technique and electron paramagnetic resonance (EPR) to be reduced by ascorbate at a rate which was faster than the overall catalytic rate; about 10% of the copper was oxidized to Cu(II) during steady-state catalysis in the presence of excess ascorbate. These results support an electron-transfer function of the enzyme-bound copper during catalysis and indicate that the reduction by ascorbate is not the rate-limiting step. The enzyme-bound copper was rapidly chelated by EDTA at pH 7.0., and the apoenzyme thus obtained after dialysis revealed no EPR-detectable copper. Addition of CuSO4 to the apoenzyme gave an EPR spectrum similar to that of the native enzyme, and the apoenzyme was fully reactivated by the optimal concentration of CuSO4 in less than 2 s.

Adrenal Medulla↗
PubMed1980

Induction of megamitochondria by cuprizone(biscyclohexanone oxaldihydrazone). Evidence for an inhibition of the mitochondrial division process.

1. Feeding weanling mice a diet containing 0.5% (w/w) cuprizone (biscyclohexanone oxaldihydrazone) induced megamitochondria of a different type of that previously described (Suzuki, K., Science 163, 81-82 (1969). 2. As in the normal controls, a single population of mitochondria with a narrow size distribution, was detected by analytical differential centrifugation of liver homogenates from mice fed a cuprizone-diet. However, their average S-value increased 2.4fold, i.e. from 11 500 S to 27 500 S. The marked increase in the mitochondrial size (megamitochondria) were also detected by morphometric analysis of hepatocytes and isolated mitochondrial fractions, and the 1.6fold increase in the average mitochondrial diameter (as determined by scanning electron microscopy) was as expected from the increase in the S-value. On the other hand, the relative volume density or volume fraction of the mitochondria was unchanged. 3. The megamitochondria revealed a normal content of respiratory chain pigments, normal specific respiratory rates with succinate and NADH-linked substrates as well as normal energy coupling. 4. Removal of cuprizone from the diet normalized the size distribution of mitochondria in hepatocytes. The transition followed first-order kinetics with a half-life of about 30 h. During the period dividing mitochondria were frequently observed, and the division process appeared to follow both a transverse-partition and an elongation-constriction mechanism.

Animals↗
PubMed1979

In vitro binding of protoheme IX and protoporphyrin IX to components in the matrix of rat liver mitochondria.

The matrix fraction of rat liver mitochondria contains several components capable of binding protoheme IX and protoporphyrin IX in vitro. When binding is analyzed by sucrose density gradient centrifugation, two main groups of components can be separated; i.e. one of high and one of low molecular weight. Experimental evidence is presented indicating a protein nature of these components, and at least six species were detected by gel electrophoresis. The binding of protoheme IX and protoporphyrin IX to matrix components is also evident from a marked change in the heme and porphyrin absorbance spectra in the presence of added matrix. The data obtained indicate that the matrix fraction has a high total binding capacity for heme, but the affinity for heme of the various components seems to be different. Based on the separation of bound and free heme by gel electrophoresis it is concluded that the heme-binding capacity of the high affinity components does not exceed 14 nmol heme . mg-1 protein.

Animals↗
PubMed1979

Isolation and characterization of noradrenalin storage granules of bovine adrenal medulla.

A method is described for the preparation of (1) the heavy population of bovine adrenal chromaffin granules (SH (average sedimentation coefficient) = 12 400 S in 0.25 M sucrose) essentially free from contamination with mitochondria and other organelles, and (2) a subpopulation of this heavy population which is highly enriched in noradrenalin (greater than or approximately 95% of the total catecholamine is noradrenalin). The method is based on isopycnic gradient centrifugation using a self-generating gradient of polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.5 M sucrose medium. The isolated population of noradrenalin granules appeared highly electron dense in transmission electron microscopy and revealed a rather narrow size distribution. The specific content of amine and adenine nucleotides (with reference to total granule protein) was markedly higher than for the total population of heavy chromaffin granules. The molar ratio of amines to adenine nucleotides was, however, lower in the noradrenalin granules, i.e. 4.8 vs. 11.9.

Adenine Nucleotides↗
PubMed1979

Bovine dihydropteridine reductase: purification by affinity chromatography and comparison of enzymes from liver and adrenal medulla.

Dihydropteridine reductase has been purified to homogeneity from bovine liver and bovine adrenal medulla by precipitation with polyethylene glycol, ion exchange chromatography, gel filtration, and affinity chromatography on 5'-AMP-Sepharose 4B. The enzymes from the two tissues seem identical by the criteria of gel filtration chromatography, affinity chromatography, polyacrylamide gel electrophoresis in the presence and absence of dodecyl sulfate, isoelectric focusing, amino acid analysis, and binding of NADH. Fluorescence studies show two independent binding sites for NADH and a dissociation constant of 10 nM at pH 6.8. Isoelectric focusing of the enzyme as purified in the presence of NADH revealed three different bands, which by removal of this coenzyme were converted into a single band, corresponding to pI 5.7. The enzyme contains no carbohydrate or zinc.

Adrenal Medulla↗