Isadore N. Rosenberg's research while affiliated with University of Massachusetts Boston and other places

Publications (20)

Article
We have isolated and purified iodothyronine 5'-deiodinase from rat liver microsomes to homogeneity as judged by PAGE and analytical HPLC. The enzyme progressively lost activity after solubilization, and specific activity enhancement was a modest 22-fold, but the final preparation still had substantial activity and was used for molecular characteriz...
Article
To study the physiological regulation of the iodothyronine 5'-deiodinases (I-5'D), we have examined the effects of some thiol blockers and of thyroid status on I-5'D activities both in vitro and in vivo. At low (less than 5 mM) concentrations of dithiothreitol, propylthiouracil (PTU) inhibited I-5'D in the brain, pituitary, and brown adipose tissue...
Article
Although all iodothyronine 5'-deiodinases require thiol cofactors for activity, the type II variant has been suspected to contain no reactive thiol groups because of its resistance to inactivation by iodoacetate (IAC). We report here that, under suitable stoichiometric conditions for the alkylation reaction, the type II enzyme is substantially inac...
Article
When activated by dithiothreitol, iodothyronine 5'-deiodinase (I-5'D) activity in kidney microsomes is less sensitive to inhibition by propylthiouracil (PTU) and iopanoate (IOP) at nanomolar, compared to micromolar, substrate concentrations. The enzymatic activities at nanomolar substrate concentrations are, however, completely eliminated in the pr...
Article
At nanomolar substrate levels, physiological concentrations (less than or equal to 5 mM) of glutathione (GSH) activate a low Km iodothyronine 5'-deiodinase (I-5'D) activity in renal and hepatic microsomes, but not the low Km (type II) I-5'D in the pituitary, cerebral cortex, or brown adipose tissue. The latter enzyme as well as the type I enzyme ac...
Article
Thioredoxin (Thd) and NADPH-Thd reductase, purified to near homogeneity from rat liver cytosol, stimulated, in the presence of NADPH, the 5'-monodeiodination of rT3 by renal and hepatic microsomes at nanomolar, but not micromolar, substrate concentrations. T4 was not deiodinated at either concentration. Reduced Thd was effective at physiological co...
Article
Brown adipose tissue (BAT) of hypothyroid rats contains a low Km (type II) iodothyronine 5'-deiodinase (I-5'D) that has been characterized as being insensitive to inhibition by propylthiouracil (PTU), based mainly on observations with homogenates prepared in a medium containing 10 mM dithiothreitol (DTT) and enzymatic assays in the presence of 20 m...
Article
Full-text available
A protein has been purified from rat liver cytosol which promoted GSH-responsive iodothyronine 5'-deiodinase activities in rat kidney microsomes. The factor behaved as a basic protein with an Mr of 11,000. It was active as a GSH-disulfide transhydrogenase with beta-hydroxyethyl disulfide as an acceptor and was also active in stimulating calf thymus...
Article
Full-text available
Adipocyte membranes from hypothyroid rats showed increased low Km cAMP phosphodiesterase activity compared to normals, provided that the subcellular fractionations were done in isotonic, as opposed to hypotonic, buffers. The enhanced cAMP phosphodiesterase activity in hypothyroid membranes was nearly normalized by incubation with a 10-fold excess o...
Article
The iodotyrosine deiodinase represents a mechanism for glandular iodine conservation, the importance of which is evident from the occurrence of goiter and hypothyroidism if the enzyme is genetically deficient or impaired. The enzyme occurs principally in the particulate fraction of the thyroid. The naturally occurring cofactor is nicotinamide adeni...
Article
The naturally occurring dithiol, dihydrolipoamide (DHL), has been found to be 6-10 times as potent as the synthetic dithiol, dithiothreitol, in stimulating iodothyronine outer ring monodeiodinase activity in the rat kidney. In the presence of NADH, the oxidized form is also active in stimulating the enzymatic activity in whole homogenates and in th...
Article
Iodothyronine outer ring monodeiodinase activity in rat kidney microsomes is strongly inhibited by the coumarin anticoagulants dicoumarol and warfarin. The inhibition is competitive with respect to the substrates and uncompetitive with respect to the thiol activator, dithiothreitol. Kinetic studies reveal that the mechanism of this inhibition is di...
Article
Enzymic activities catalyzing the reductive 5'-deiodination of thyroxine and 3,3',5'-triiodothyronine were solubilized from rat kidney microsomes by treatment with 0.2% deoxycholate. Deoxycholate reversibly inhibited the enzyme(s); removal of detergent restored activity and resulted in the formation of enzymatically active aggregates with a buoyant...
Article
Erythrocyte membranes obtained from chronically hypothyroid rats (propylthiouracil-treated), prepared after disruption of cells in iso-osmotic imidazole buffer, showed 1.5- to 2-fold higher (Ca2+ + Mg2+)-ATPase activity as compared to similar preparations obtained from normal rats. These differences disappeared when the membrane-bound activity was...
Article
Renal membranes (crude microsomal fraction) which catalyze the thiol-dependent outer ring deiodination of L-T4 with the formation of L-T3 are also capable of the 5'-deiodination of rT3 with the production of equivalent amount of I- and 3,3'-T2. rT3 deiodination was followed by measuring the amount of 125I-released from 125I-labeled (3'- or 5'-) L-r...
Article
T4 5'-deiodinase, found in cell membranes of kidney and liver, is a thiol-dependent enzyme inhibited by propylthiouracil (PTU). Pretreatment of enzymatically active kidney membrane preparations (Mx) with PTU (10 μM) and increasing concentrations of L-T4 under N2 resulted in increasing degrees of persistent inhibition, as assayed in the absence of p...
Article
Adipocytes isolated from normal, hypothyroid and hyperthyroid rats were characterized with respect to their lipolytic activity (assessed by glycerol release) and β-adrenergic receptors (assessed by binding of (-)[3H]alprenolol). Fat cells from hypo-and hyperthyroid rats showed the same affinity (K=1.4x1010M-1) and binding capacity (N=1.21 x 10-13 m...
Article
Enzymatic 5'-deiodination of T4 in various tissues is known to be stimulated by thiols and inhibited by propylthiouracil (PTU). Dithiothreitol (DTT) stimulation of rat kidney T4 5'-deiodinase followed saturation kinetics. Inhibition of the enzyme by PTU (10 (-5) M) was overcome in a concentration-dependent manner by DTT, methimazole (MMI), and thio...
Article
The enzymatic activity in rat kidney homogenates mediating the conversion of T4 to T2 has been previously shown to be particulate. The subcellular organelle responsible for T4 5'-deiodination, however, has not been identified. Enzymatic activity was assessed with outer ring 125I-labeled L-T4 as the substrate and the iodothyronines were separated by...
Article
Unlike the microsomal iodotyrosine deiodinase activity, which was reponsive to both NADPH and dithionite, the enzymatic activity solubilized and purified from bovine thyroid microsomes could no longer utilize NADPH for deiodination. The purified enzyme could, however, be activated by dithionite, as well as by methyl viologen (MV), reduced by NADPH...

Citations

... Similar effects of these inhibitors were also found in the renal rT3 5'-DI studies (Table 4), although the relative order of potency differed from that found with liver. Both the low-Km 5'-DI activated by CCS in the present study and the high-Km 5'-DI activated by DTT show similar sensitivities to several of these inhibitors[1,23,24]. DISCUSSION Our previously reported investigations of the 5'-DI cofactor system of rat liver cytosol utilized partially purified chromatographic Fractions A and B, together with NADPH[7][8][9]25], and indicated that this system resembled the thioredoxin system in the following respects: (a) NADPH-dependence, (b) requirement for a reductase of Mr > 60000 plus a dithiol of Mr approx. ...
... Next, DTN was tested as an alternative reductive cosubstrate (27). In comparison to NADPH, the rate of MIT deiodination was strongly increased by the use of DTN (Figure 2A), which is in accordance with the literature (28). ...
... These effects can be reversed by calcium channel antagonism (6,7). In addition, modulation of [Ca 2+ ] i plays a role in mediating both lipolytic and antilypolytic actions of hormones in adipocytes (25)(26)(27)(28). Therefore, increasing [Ca 2+ ] i appears to promote triglyceride storage in adipocytes by exerting coordinated control over lipogenesis and lipolysis, serving to simultaneously stimulate the former and suppress the latter, and thereby cause lipid filling. ...
... The location of Dio2 is critical for generating intracellular T3 for the THRcontaining nuclear compartment (Arrojo E Drigo and Bianco, 2011). Unlike Dio2, immunohistochemical and immunofluorescence studies have shown that Dio1 is localized in the plasma membrane with the catalytic site located in the cytosol (Leonard and Rosenberg, 1978;Baqui et al., 2000;Leonard et al., 2001) and has a hydrophobic N-terminal extension into the ER lumen. This N-terminus acts as a signal recognition sequence but does not contribute to the catalytic activity, although mutating the amino acids results in reduced efficiency of transient expression and optimal formation/folding of the protein (Toyoda et al., 1995;Bianco et al., 2002;Bianco and Larsen, 2005). ...
... Despite an initial hypothesis that sequential deiodination was performed by two distinct enzymes acting either in the phenolic or the tyrosyl ring, evidence soon demonstrated that a single enzyme, type 1 deiodinase (DIO1, DIO1), was responsible for both ORD and IRD (Fekkes et al. 1982, St Germain & Morganelli 1989, Mandel et al. 1992. This process was classically studied in the liver, kidney and the thyroid and was subjected to 6-propyl-2-thiouracil (PTU) inhibition (Visser et al. 1975, Leonard & Rosenberg 1978, Ishii et al. 1981. However, PTU did not inhibit the local deiodination of T 4 to T 3 in brain and pituitary tissues, suggesting the existence of two separate pathways of enzymatic ORD in these tissues. ...
... The endogenous intracellular thiols glutathione and thioredoxin have been shown to stimulate 5′-deiodination activities of tissue preparations and expressed Dio1 transcripts ( Yamada et al., 1981;Rosenberg, 1987, 1988;Bhat et al., 1989;Goswami and Rosenberg, 1990;Sharifi and St. Germain, 1992). Moreover, the activation of 5′-deiodination in a rat kidney microsomal preparation by glutathione, but not that by DTT, was lost upon preincubation by DTT ( Goswami and Rosenberg, 1990). ...
... The active site of ID1 contains selenocysteine [37,38]. In addition, the activity of this enzyme depends on the presence of sulfhydryl (eSH) group [39,40]. Chaurasia and Kar [36] postulated that the mechanism by which Pb induced inhibition of ID1 enzyme activity could be mediated through the high binding affinity of this heavy metal to essential sulfhydryl (eSH) group in ID1, substituting it, thereby hindering its enzymatic reaction [41,42]. ...
... Влияние ЙТГ на уровень циклических нуклеотидов Влияние гипертиреоза:-повышение содержания цАМФ в миоцитах сердца крыс [7];-увеличение концентрации цАМФ в крови, скелетных мышцах и жировой ткани человека [8];-введение L-тироксина (200 мкг на крысу через день в течение 30-ти дней)-повышение концентрации цАМФ в сердечной мышце крыс на 27% [9]. Влияние гипотиреоза:-введение пропилурацила (0,1% раствор в питьевой воде в течение 3-х недель)-увеличение активности цАМФ-фосфодиэстеразы (фермента, катализирующего расщепление цАМФ до АМФ) в адипоцитах крыс в 2,5 раза [10];-введение L-метил-2-меркаптоимидазола (1 мг на крысу ежедневно 30 дней)-снижение уровня цАМФ в сердце на 27% [9]. ...
... Interestingly, the low level of rT 3 ORD measured in sea bream kidney homogenates in the presence of DTT is almost insensitive to PTU, while the higher activity level in the absence of DTT is about 68% inhibited by 0 . 1 mM PTU. This finding shows some similarity with the fact that PTU is only efficient in blocking mammalian or chicken D1 activity when there is sufficient substrate turnover (Goswami & Rosenberg (1988) and own observations). It could mean that sea bream D1 can be classified as mildly PTU-sensitive, although less sensitive than mammalian D1s. ...
... Our data derived from in vivo experiments in rats, supports results of earlier in vitro studies (Lamirand et al., 2008). Other in vitro studies indicated that the exposure of neuronal cells to Met-Hg (Kim et al., 2005) or neuroblastoma cells to TM (James et al., 2005) results in a depletion of GSH which is both an antioxidant and a cofactor of deiodinases (Goswani and Rosenberg, 1988;Bhat et al., 1989;Croteau et al., 1998;Goemann et al., 2010). Thus, cerebellar D2 activity might be impaired due to a lack of the reducing cofactor. ...