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Biomedical subjects

G Hennemann

Publications and source records attributed to G Hennemann.

At least 91 records · Page 5Linked to original sources
PubMed1984

Inactivation and affinity-labeling of rat liver iodothyronine deiodinase with N-bromoacetyl-3,3',5-triiodothyronine.

The thyroid hormone derivative N-bromoacetyl-3,3',5-triiodothyronine (BrAcT3) acts as an active site-directed inhibitor of rat liver iodothyronine deiodinase. Lineweaver Burk analysis of enzyme kinetic measurements showed that BrAcT3 is a competitive inhibitor of the 5'-deiodination of 3,3',5'-triiodothyronine (rT3) with an apparent Ki value of 0.1 nM. Preincubations of enzyme with BrAcT3 indicated that inhibition by this compound is irreversible. The inactivation rate obeyed saturation kinetics with a limiting inactivation rate constant of 0.35 min-1. Substrates and substrate analogs protected against inactivation by BrAcT3. Covalent incorporation of 125I-labeled BrAcT3 into "substrate-protectable" sites was proportional to the loss of deiodinase activity. The results suggest that BrAcT3 is a very useful affinity label for rat liver iodothyronine deiodinase.

Affinity Labels↗
PubMed1984

Modification of rat liver iodothyronine 5'-deiodinase activity with diethylpyrocarbonate and rose bengal; evidence for an active site histidine residue.

Iodothyronine 5'-deiodinase activity of rat liver microsomes was rapidly and completely lost by treatment with diethylpyrocarbonate (DEP) and by photo-oxidation with Rose Bengal (RB). In both cases inactivation followed pseudo first order reaction kinetics. Inactivation by DEP was diminished in the presence of substrate or competitive inhibitors, and was reversed by hydroxylamine treatment. In addition to photo-oxidation, deiodinase activity was also inhibited by RB in the dark. This inhibition was reversible and competitive with substrate (Ki 60 nM). These results suggest the location of an essential histidine residue at or near the active site of rat liver iodothyronine deiodinase.

Animals↗
PubMed1984

Metabolism of 3,3'-diiodothyronine in rat hepatocytes: interaction of sulfation with deiodination.

Production of 3,3'-diiodothyronine (3,3'-T2) is an important step in the peripheral metabolism of thyroid hormone in man. The rapid clearance of 3,3'-T2 is accomplished to a large extent in the liver. We have studied in detail the mechanisms of this process using monolayers of freshly isolated rat hepatocytes. After incubation with 3,[3'-125I]T2, chromatographic analysis of the medium revealed two major metabolic routes: outer ring deiodination and sulfation. We recently demonstrated that sulfate conjugation precedes and in effect accelerates deiodination of 3,3'-T2. In media containing different serum concentrations the cellular clearance rate was determined by the nonprotein-bound fraction of 3,3'-T2. At substrate concentrations below 10(-8) M 125I- was the main product observed. At higher concentrations deiodination became saturated, and 3,3'-T2 sulfate (T2S) accumulated in the medium. Saturation of 3,3'-T2 clearance was found to occur only at very high (greater than 10(-6)M) substrate concentrations. The sulfating capacity of the cells exceeded that of deiodination by at least 20-fold. Deiodination was completely inhibited by 10(-4) M propylthiouracil or thiouracil, resulting in the accumulation of T2S while clearance of 3,3'-T2 was little affected. No effect was seen with methimazole. Hepatocytes from 72-h fasted rats showed a significant reduction of deiodination but unimpaired sulfation. Other iodothyronines interfered with 3,3'-T2 metabolism. Deiodination was strongly inhibited by 2 microM T4 and rT3 (80%) but little by T3 (15%), whereas the clearance of 3,3'-T2 was reduced by 27% (T4 and rT3) and 12% (T3). It is concluded that the rapid hepatic clearance of 3,3'-T2 is determined by the sulfate-transferring capacity of the liver cells. Subsequent outer ring deiodination of the intermediate T2S is inhibited by propylthiouracil and by fasting, essentially without an effect on overall 3,3'-T2 clearance.

Animals↗
PubMed1984

Sulfation facilitates hepatic deiodination of iodothyronines.

The metabolism of 3,3',5'-triiodothyronine (rT3), 3,3'-5-triiodothyronine (T3) and 3,3'-diiodothyronine (3,3'-T2) by isolated rat hepatocytes in primary culture was studied by radioimmunoassay and by Sephadex LH-20 chromatography. The first step in the metabolism of rT3 is outer ring deiodination which is inhibited by thiouracil. In incubations with outer ring-labeled 3,3'-T2 or T3 the main products observed are iodide in the absence of thiouracil and the sulfate conjugates in the presence of the inhibitor. Rates of sulfation and iodide formation were both determined by the phenol sulfotransferase activity of the cells. Inner ring deiodination of T3 sulfate and subsequent outer ring deiodination of 3,3'-T2 sulfate by rat liver microsomes are much faster than the corresponding reactions for the non-conjugated iodothyronines. The results strongly suggest that sulfation precedes and in effect accelerates hepatic deiodination of T3 and 3,3'-T2.

Animals↗
PubMed1983

Properties of detergent-dispersed iodothyronine 5- and 5'-deiodinase activities from rat liver.

In order to obtain more knowledge about the regulation and mechanism of thyroid hormone deiodination, some properties of detergent-solubilized iodothyronine deiodinase have been studied. Moreover, a starting point for its purification has been made. Several chromatography media were tested for their ability to purify the deiodinases. In some instances, a 4-fold purification was obtained. Treatment of cholate-solubilized microsomes with 35% ammonium sulphate resulted in quantitative precipitation of the deiodinase activities and concomitant removal of phospholipid. The pellet could be solubilized with 0.3% W-1 ether and the deiodinase in this ammonium sulphate extract exhibited approximately 10-fold higher apparent Km and Vmax values for its substrate compared with the cholate extract. Readdition of some phospholipids was shown to decrease enzyme activity. Isoelectric focusing of W-1 ether-solubilized microsomes resulted in a major activity peak around pH 6.4 and a minor peak at pH 5.2, while in the ammonium sulphate extract the deiodinase had an isoelectric point at pH 9.3. Refocusing of this activity peak yielded a preparation with a specific activity only 3-times higher than in the ammonium sulphate extract. However, after sodium dodecyl sulphate polyacrylamide gel electrophoresis only five bands could be detected. The elevated kinetic parameters as well as the higher isoelectric point of the deiodinase after ammonium sulphate treatment were caused by delipidation of the enzyme. Both the change in isoelectric point and the behaviour on several column materials were found to be similar for the 5- and 5'-deiodinase activities. These results suggest that a single enzyme is operative in the deiodination of iodothyronines in rat liver and that its activity may be regulated by phospholipids.

Animals↗
PubMed1982

Hyperthyroxinaemia due to decreased peripheral triiodothyronine production.

Two patients, a boy of 8 and a women of 60 years of age, had higher than normal levels of serum total thyroxine (T4), free T4, (FT4), FT4 index, and reverse triiodothyronine, but normal serum triiodothyronine (T3) levels. The pituitary-thyroid axis could be normally stimulated by thyrotropin-releasing hormone, suggesting euthyroidism at the pituitary level. High levels of serum T4-binding globulin decreased during T3 treatment in the boy. Studies show that in these patients a raised serum FT4 is necessary to produce in the peripheral tissues sufficient amounts of T3 for biological action. Two possible mechanisms for a basic defect underlying this newly recognised syndrome are proposed: inhibition of T4 transport into tissue cells and reduced intracellular 5'-deiodinase activity catalysing T4 to T3 conversion.

Child↗
PubMed1982

Evidence that the uptake of tri-iodo-L-thyronine by human erythrocytes is carrier-mediated but not energy-dependent.

We investigated 3,3',5-tri-iodo-l-thyronine transport by human erythrocytes and by ;ghosts' prepared from these cells. Uptake of tri-iodothyronine by erythrocytes at 37 degrees C was time-dependent with a maximum reached after 60min. Tracer analysis after incubation for 1min revealed only one saturable binding site, with K(m) 128+/-19nm (mean+/-s.e.m.; n=7) and V(max.) 4.6+/-0.7pmol of tri-iodothyronine/min per 6x10(7) cells. After 10min incubation K(m) 100+/-16nm (n=10) was found with V(max.) 7.7+/-1.2pmol of tri-iodothyronine/10min per 6x10(7) cells. At 0 degrees C the uptake system is still active, with K(m) 132+/-26nm and V(max.) 1.8+/-0.3pmol of tri-iodothyronine/10min per 6x10(7) cells. The V(max.) with intact cells is 5-fold greater than the V(max.) with membranes derived from the same amount of cells when uptake studies are performed in media with similar free tri-iodothyronine concentrations. This indicates that at least 80% of tri-iodothyronine taken up by the intact erythrocytes enters the cell. This saturable uptake system can be inhibited by X-ray-contrast agents in a dose-dependent fashion. (+/-)-Propranolol, but not atenolol, has the same effect, indicating that the membrane-stabilizing properties of (+/-)-propranolol are involved. Furthermore, there is no inhibition by ouabain or vanadate, which indicates that tri-iodothyronine uptake is not dependent on the activity of Na(+)+K(+)-dependent adenosine triphosphatase. We have prepared erythrocyte ;ghosts', resealed after 2.5min with 0mm-, 2mm- or 4mm-ATP inside. Inclusion of ATP and integrity of the membrane of the erythrocyte ;ghosts' were verified on the basis of an ATP-concentration-dependent functioning of the Ca(2+) pump. No difference was found in the uptake of tri-iodothyronine by erythrocyte ;ghosts' with or without ATP included, indicating that uptake of tri-iodothyronine is not ATP-dependent. The following conclusions are drawn. (1) Tri-iodothyronine enters human erythrocytes. (2) There is only one saturable uptake system present for tri-iodothyronine, which is neither energy (i.e. ATP)-dependent nor influenced by the absence of an Na(+) gradient across the plasma membrane. This mode of uptake of tri-iodothyronine by human erythrocytes is in sharp contrast with that of rat hepatocytes, which uptake system is energy-dependent and ouabain-sensitive [Krenning, Docter, Bernard, Visser & Hennemann (1978) FEBS Lett.91, 113-116; Krenning, Docter, Bernard, Visser & Hennemann (1980) FEBS Lett.119, 279-282]. (3) X-ray-contrast agents inhibit tri-iodothyronine uptake by erythrocytes in a similar fashion to that by which they inhibit the uptake of tri-iodothyronine by rat hepatocytes [Krenning, Docter, Bernard, Visser & Hennemann (1982) FEBS Lett.140, 229-233].

Adenosine Triphosphate↗
PubMed1982

One enzyme for the 5'-deiodination of 3,3',5'-triiodothyronine and 3',5'-diiodothyronine in rat liver.

Many studies suggest that one enzyme is involved in the phenolic ring deiodination of iodothyronines in rat liver and kidney and another one in the tyrosyl ring deiodination. This study describes some characteristic of the phenolic ring (5'-) deiodination of rT3 and 3',5'-T2 by rat liver microsomes. At pH 7.2 the Km values of the 5'-deiodination of rT3 and 3',5'-T2 were 0.103 and 0.77 microM, respectively. 3',5'-T2 and rT3 inhibited the respective 5'-deiodination reactions competitively, the Ki values being 1.05 and 0.134 microM, respectively. Several radiographic contrast agents markedly inhibit the 5'-monodeiodination or rT3 and 3',5'-T2, the type of inhibition being competitive. Of these compounds iopanoic acid, ipodic acid and iophenoxic acid are the most potent inhibitors with Ki values of approximately 2 microM for both reactions. The non-iodine containing compound 8-anilino-1-naphthalene sulphonic acid (ANS) appeared to be a very strong competitive inhibitor of both 5'-deiodinations (Ki 4.3-4.7 microM), whereas salicylic acid, which as ANS inhibits the binding of iodothyronines to T4-binding globulin, inhibited these reactions to a much lesser extent (Ki 300-500 microM). On the other hand, diiodosalicylic acid was a very strong inhibitor. The beta-adrenergic blocker D,L-propranolol was a weak noncompetitive inhibitor of both 5'-deiodinations (Ki 0.4-0.7 mM). These reactions were also inhibited by various 2,6-diiodophenol derivatives, triiodophenol being the strongest and diiodotyrosine the weakest inhibitor tested. Comparing the Ki values of various inhibitors for the 5'-deiodination of rT3 and 3',5'-T2, a positive correlation between these values was found (r = 0.97). It was concluded that rT3 (to 3,3'-T2) and 3'-5'-T2 (to 3'-T1) monodeiodinating activities are very similar to each other and that there may just be one monodeiodinase catalyzing the 5'-deiodination of iodothyronines in rat liver.

Anilino Naphthalenesulfonates↗
PubMed1982

Evidence for a single enzyme in rat liver catalysing the deiodination of the tyrosyl and the phenolic ring of iodothyronines.

The enzymic 5'-deiodination of 3',5'-di-iodothyronine and 5-deiodination of 3,3',5-tri-iodothyronine by rat liver microsomal fractions were found to be characterized by apparent Km values of 0.77 and 17.4 microM respectively, 3',5'-Di-iodothyronine was a competitive inhibitor of 3,3',5-tri-iodothyronine 5-deiodination (Ki 0.65 microM) and 3,3',5-tri-iodothyronine was a competitive inhibitor of 3',5'-di-iodothyronine 5'-deiodination (Ki 19.6 microM). In addition, several radiographic contrast agents and iodothyronine analogues inhibited both reactions competitively and with equal potencies (r = 0.999). These results strongly suggest the existence of a single hepatic deiodinase acting on both the tyrosyl and phenolic ring of iodothyronines.

Animals↗
PubMed1981

Characteristics of active transport of thyroid hormone into rat hepatocytes.

Thyroid hormone uptake into primary cultured rat hepatocytes was studied using 1-min incubations with radio-iodine-labelled iodothyronines. (1) Uptake of thyroxine indicates two saturable sites with apparent Km values of 1.2 nM and 1.0 microM, and non-saturable uptake. Similar kinetics of triiodothyronine uptake have been observed. (2) The high-affinity systems of both hormones are energy-dependent (i.e., inhibited by KCN and oligomycin). It is postulated that these systems represent active transport of thyroid hormone into the cell. (3) Analysis of mutual inhibition by the substrates for the triiodothyronine and thyroxine transport systems indicates that triiodothyronine and thyroxine cross the cell membrane via separate transport systems. (4) Preincubation with ouabain resulted in a decrease in uptake of both triiodothyronine and thyroxine, suggesting that a sodium gradient is essential for this transport.

Animals↗