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T Flatmark

Publications and source records attributed to T Flatmark.

At least 145 records · Page 8Linked to original sources
PubMed1979

Comparison of the ultrastructure of adrenaline and noradrenaline storage granules of bovine adrenal medulla.

The ultrastructure of the membranes of noradrenaline (NA) and adrenaline (A) granules of the bovine adrenal medulla (Terland, O., T. Flatmark, and H. Kryvi, Biochim, Biophys. Acta 553, 460--468 (1979)) was analyzed by transmission, negative staining and freeze-etch electron microscopy. The two types of storage granules can be distinguished mainly by two morphological criteria: (a) The NA-granules have a more electron dense matrix core than the A-granules, (b) the NA-granules revealed less asymmetry in the distribution of intramembrane particles (nPF:nEF = 4,5:1) than the A-granules (nPF:nEF = 9:1). Thus, the trilaminar structure, negative staining pattern and size distribution of the intramembrane particles of the two fracture faces on freeze-etch electron microscopy were very similar for the two types of granules. Freeze-etching revealed a wide range of the particle size distribution for both fracture faces in both types of granules, with an average diameter of 12.6 +/- 2.7 nm (A-granules) and 10.2 +/- 2.8 nm (NA-granules) for the E-fracture faces and 11.4 +/- 2.7 nm (A-granules) and 9.8 +/- 2.4 nm (NA-granules) for the P-fracture faces. Some of the particles on the P-fracture face (outer surface of the membrane) revealed a subunit structure, most clearly seen in the specimens of NA-granules. Morhpometric analyses of sectioned bovine adrenal medulla revealed that the chromaffin granules on an average account for approx. 13.5% of the cytoplasmic volume in the total population of chromaffin cells.

Adrenal Medulla↗
PubMed1978

Fluorimetric detection of octopamine in high-performance liquid chromatography and its application to the assay of dopamine beta-monooxygenase in human serum.

A high-performance liquid chromatographic procedure is described for the determination of octopamine. The method, which is based on the separation on a microparticulate bonded strong cation-exchange resin and measurement of the native fluorescence, has been applied to give a sensitive assay of dopamine beta-monooxygenase (EC 1.14.17.1) activity in human serum with tyramine as the substrate. The procedure, which has been designed for use with an automatic sampler, has a detection limit of about 50 pmoles of octopamine, and the analysis time is approximately 10 min per sample.

Autoanalysis↗
PubMed1978

Long-chain acyl-CoA synthetase and "outer" carnitine long-chain acyltransferase activities of intact brown adipose tissue mitochondria.

1. The activities of long-chain acyl-CoA synthetase (acid: CoA ligase (AMP-forming), EC 6.2.1.3) and the "outer" carnitine long-chain acyltransferase (palmitoyl-CoA: L-carnitine O-palmitoyltransferase, EC 2.3.1.21) have been estimated in intact brown adipose tissue mitochondria. The assay of both enzymes is based on a coupled reaction in which the intramitochondrial (matrix) CoASH is the final acyl acceptor and the oxidation-reduction state of the flavoproteins in the acyl-CoA dehydrogens pathway is used to determine the intramitochondrial level of acyl-CoA. 2. Using endogenous fatty acids as the substrate, the progress curve of acyl-CoA synthetase activity was in most mitochondrial preparations linear within the first 30 s. When initial rates were measured, the Km value for CoASH (2.4 micron) was lower than previously determined for the acyl-CoA synthetase in brown adipose tissue mitochondria as well as in mitochondria of other tissues. The pH activity curve indicates that the unprotonated form of the fatty acids represents the substrate of acyl-CoA synthetase, i.e. similar to the effect of pH on the binding of fatty acids to bovine serum albumin. 3. Experimental evidence is presented that at temperatures higher than the transition temperature of the acyl-CoA synthetase (i.e. Tt = 19 degrees C), this enzymic reaction is rate-limiting in the sequence of coupled reactions leading to beta-oxidation in the mitochondrial matrix. 4. The initial rate of the long-chain acyl-COA synthetase reaction was estimated to v = 119 +/- 16 nmol . min-1 . mg-1 protein (mean +/- S.D., n = 5) at an optimal concentration of palmitate which exceeds that of rat heart mitochondria by a factor of 10.

Acyltransferases↗
PubMed1978

An adenosine 3':5'-monophosphate-adenosine binding protein from mouse liver: some physicochemical properties.

A number of physiochemical properties of the cyclic AMP-adenosine binding protein of mouse liver (Ueland, P.M. and Døskeland, S.O. (1977) J. Biol. Chem. 252, 677--686) have been studied. 1. The specific extinction coefficient, E1%280nm, was estimated to 13.0. 2. Amino acid and amide group analyses confirmed the acidic properties of the protein as determined by electrofocusing (pI = 5.7). Based on the estimated partial specific volume (v = 0.74 cm3/g) the minimum molecular weight of the native, tetrameric protein was recalculated to be 185 000 (s20,w = 8.8 . 10(-13) s and Stokes radius = 48 A). 3. No NH2-terminal amino acid was found by the dansyl method using [14C]-dansyl chloride, indicating that the NH2-terminal groups are blocked. 4. Amino acid analyses gave 6 half-cystine residues per subunit, and the same number of free sulfhydryl groups was found by titration of the denatured protein with 5,5'-dithiobis (2-nitrobenzoic acid). 5. The reactivity of the SH groups in the native protein with 5,5'-dithiobis (2-nitrobenzoic acid) revealed rapidly reacting (SHI), sluggishly reacting (SHII) and "masked" (SHIII) SH groups. ATP, adenosine, Mg2+ and KCl, factors known to affect the activation of cyclic AMP binding sites (Ueland, P.M. and Døskeland, S.O. (1978) Arch. Biochem. Biophys., in press) changed the reactivity of separate SH groups.

Adenosine↗
PubMed1978

On the rate-limiting step in the transfer of long-chain acyl groups across the inner membrane of brown adipose tissue mitochondria.

Brown adipose tissue mitochondria predominantly oxidize fatty acids in order to generate heat for non-shivering thermogenesis, and have an unusually high capacity for net transfer of long-chain fatty acyl groups from the outer to the inner (matrix) compartment. The activities of the "outer" and "inner" carnitine long-chain acyltransferases have been estimated in isolated mitochondria of cold-acclimated guinea pits by the continuous spectrophotometric recording of the redox level of flavoproteins in the acyl-CoA dehydrogenase pathway. This redox level is determined by the intramitochondrial content of acyl-CoA under the selected experimental conditions. The apparent initial rate of the "inner" acyltransferase (palmitoyl-L-carnitine added) is three order of magnitudes higher than the "outer" acyltransferase (palmitoyl-CoA added), and this difference is not influenced by the substrate concentration, pH and reaction temperature. Thus, the "outer" acyltransferase reaction is rate limiting in the transfer of long-chain acyl groups across the inner membrane of these mitochondria and catalyzes a non-equilibrium reaction in the intact organelle. Estimates of the absolute rate of the "outer" long-chain acyltransferase indicate that it exceeds that of rat liver mitochondria by a factor of 20.

Acclimatization↗
PubMed1976

Effect of the hydrophile-lipophile balance of non-ionic detergents (Triton X-series) on the solubilization of biological membranes and their integral b-type cytochromes.

The solubilization of four integral membrane proteins (i.e. cytochrome b-561 of the chromaffin granule membrane, cytochrome b5 of the endoplasmic reticulum and the mitochondrial b-type cytochrome(s) as well as cytochrome c oxidase) has been studied at 0 degrees C using the non-ionic detergents of the Triton X-series having the common hydrophobic 4(1,1,3,3-tetramethylbutyl)phenoxy (t-octyl-phenoxy) group and a variable average number (n) of polar ethylene oxide units added. Following a pre-extraction of peripheral membrane and matrix proteins with low and high salt concentration and a weak non-ionic detergent (Tween 20, average hydrophile-lipophile balance (HLB) = 16.7), the amount of heme proteins solubilized by subsequent Triton X-solutions was measured. With the detergents tested the degree of solubilization decreased in the sequence cytochrome b-561 greater than cytochrome b5 greater than mitochondrial cytochrome(s) b and parallelled the effect of the detergents on light scattering and the phospholipid to protein ratio of the three membranes. For all the b-cytochromes, the solubilizing power of the detergent increased with decreasing average length of the polar ethylene oxide chain and the hydrophile-lipophile balance as long as clouding did not occur (e.g. Triton X-114,n = 7.5 and HLB = 12.4). Thus, the greatest difference in the degree os solubilization of the three cytochromes was observed with Triton X-405 (n = 40 and HLB = 17.9). All the cytochromes were most efficiently solubilized (i.e. approx. 90%) by Triton X-100 (n = 9.5 and HLB = 13.5).

Animals↗
PubMed1976

Studies on the ferrochelatase activity of isolated rat liver mitochondria with special reference to the effect of oxidizable substrates and oxygen concentration.

The mitochondrial ferrochelatase activity has been studied in coupled rat liver mitochondria using deuteroporphyrin IX (incorporated into liposomes of lecithin) and Fe(III) or Co(II) as the substrates. 1. It was found that respiring mitochondria catalyze the insertion of Fe(II) and Co(II) into deuteroporphyrin. When Fe(III) was used as the metal donor, the reaction revealed an absolute requirement for a supply of reducing equivalents supported by the respiratory chain. 2. A close correlation was found between the disappearance of porphyrin and the formation of heme which allows an accurate estimate of the extinction coefficient for the porphyrin to heme conversion. The value deltae (mM-1 - cm-1) = 3.5 for the wavelength pair 498 509 nm, is considerably lower than previously reported. 3. The maximal rate of deuteroheme synthesis was found to be approx. 1 nM - min-1 - mg-1 of protein at 37 degrees C, PH 7.4 and optimal substrate concentrations, i.e. 75 muM Fe(III) and 50 muM deuteroporphyrin. 4. Provided the mitochondria are supplemented with an oxidizable substrate, the presence of oxygen has no effect on the rate of deuteroheme synthesis.

Animals↗
PubMed1976

Purification and characterization of dopamine beta-hydroxylase from bovine adrenal medulla.

A new purification procedure that permits large-scale purification of dopamine beta-hydroxylase from bovine adrenal medulla was developed. Whole adrenal medullas were extracted with 0.1% Triton X-100, and the enzyme was purified by precipitation with polyethylene glycol, chromatography on DEAE-cellulose, and adsorption to concanavalin A linked to agarose. The yield of protein and the specific activity were high compared with previously published methods. The enzyme appeared essentially homogenous by the criteria of polyacrylamide gel electrophoresis in the presence or absence of dodecylsulfate, and sedimentation velocity analysis. The purified protein was subjected to amino acid and carbohydrate analyses, and the results were compared with previously published data. We found about 3 mol of copper per mol of protein (tetramer of 290000 daltons). No free sulfhydryl groups could be found. Analysis for NH2-terminal amino acids with [14C]dansyl chloride revealed 2 residues of alanine and 2 residues of serine per tetramer. We found the NH2-terminal amino acid of chromogranin A to be leucine. The results of our analysis for amino acid composition and NH2-terminal amino acids do not support the suggestion that dopamine beta-hydroxylase and chromogranin A contain identical peptide chains.

Adrenal Medulla↗
PubMed1975

Energy-dependent accumulation of iron by isolated rat liver mitochondria. Requirement of reducing equivalents and evidence for a unidirectional flux of Fe(II) across the inner membrane.

The relationship between the rate of endogenous respiration and the energy-dependent accumulation of iron and calcium was studied in rat liver mitochondria energized by external ATP and inhibited to a variable extent by rotenone. In contrast to the uptake of calcium, that of iron revealed an absolute requirement for reducing equivalents supported by the respiratory chain, which indicates that iron crosses the inner membrane only in the ferrous form. Experimental evidence is presented that a primary event of the energy-linked uptake of iron is that Fe(III) is bound to ligands on the C-side of the inner membrane, ligands which have a unique microenvironment giving the metal a half-reduction potential which is sufficiently high to establish a oxidation-reduction equilibrium with the respiratory chain at the level of cytochrome c. In addition, evidence is presented that this accumulation represents a unidirectional flux of Fe(II) from the C-side to the M-side of the inner membrane and the matrix where it is tightly bound to ligands (proteins?) not yet characterized. As expected, the energy-dependent accumulation of iron is accompanied by an internal alkalinization of the mitochondria analogous to that observed for calcium. A schematic model of the energized accumulation of iron by rat liver mitochondria is presented.

Adenosine Triphosphate↗