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Biomedical subjects

I N Rosenberg

Publications and source records attributed to I N Rosenberg.

At least 19 recordsLinked to original sources

Oncogenic osteomalacia: clinical presentation, densitometric findings, and response to therapy.

A 72-yr-old white female who had previously enjoyed excellent health presented with global bone and muscle pain, and chronic fatigue. Her evaluation revealed an increased sedimentation rate and mild anemia, and a diagnosis of polymyalgia rheumatica was made. Prednisone therapy was of little benefit. A laboratory evaluation revealed mild hypocalcemia, marked hypophosphatemia, elevated alkaline phosphatase, normal 25- hydroxyvitamin D, and undectable 1,25-dihydroxyvitamin D. A diagnosis of oncogenic osteomalacia was made and the patient received calcitriol and neutraphos therapy. The patient's initial bone density by dual energy X-ray absorptiometry of the lumbar spine was 0.847 g/cm2 (T score -1.96) and of the femoral neck was 0.669 gm/cm2 (T score -2.89). After 40 mo of treatment with calcitriol and neutraphos, the bone mineral density of the lumbar spine and hip rose dramatically by 47.8 and 59.1%, respectively. Although oncogenic osteomalacia is a very rare metabolic bone disease, its recognition and appropriate treatment can have a dramatic effect not only on the bone mineral density of the patient, but also on the patient's general health and feeling of well-being.

Journal Article↗

Reconstitution of a purified and partially active low Km iodothyronine 5'-deiodinase with phospholipids.

We previously reported the detergent-extraction and purification to homogeneity from rat liver microsomes of an iodothyronine 5'-deiodinase which differed from the type I enzyme in its molecular size as well as its Km, thiol responsiveness, and its expected sensitivity to the antithyroid drug, propylthiouracil. The specific activity enhancement (22-fold) of the delipidated enzyme was, however, relatively modest. We report here that the purified enzyme can be activated a further 25-fold by reconstitution in microsomal phospholipids and, to a lesser extent, in soybean phospholipids. The data suggest that a native lipid environment is necessary for proper conformation of the enzyme for optimal activity.

Animals↗

Purification and partial characterization of iodothyronine 5'-deiodinase from rat liver microsomes.

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 characterization. The enzyme had an Mr of 56,000 with a single band in SDS-PAGE, suggesting absence of subunit structure. The high Km, and the GSH-responsive low Km, activities were co-purified, but the low Km enzyme lost GSH-responsiveness upon pretreatment with dithiothreitol (DTT) and urea. The enzyme was strongly inhibited by the iron chelator, alpha,alpha'-dipyridyl and showed a broad absorbance band at 410 nm. Spectral analysis with diethylpyrocarbonate (DEPC) revealed 5 histidine residues/mol enzyme, while enzyme activity was inhibited by DEPC in a pseudo-first order process with modification of 1 histidine residue/mol.

Animals↗

Regulation of iodothyronine 5'-deiodinases: effects of thiol blockers and altered substrate levels in vivo and in vitro.

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 (BAT) of hypothyroid rats (which contain predominantly the type II activity); the patterns of inhibition in these tissues were essentially similar, with a Ki of about 174 microM at 250 microM dithiothreitol. Hydroxyethyldisulfide was a strong inhibitor of the type II enzyme, with relatively little effect on the renal enzyme at both high concentrations (micromolar) of T4, i.e. predominantly type I activity, and low concentrations (nanomolar) to T4, i.e. both type I and low Km activity. Preincubation of cerebral microsomes with PTU, followed by removal of excess PTU, resulted in 70% inhibition of I-5'D activity in cerebral microsomes at 5 mM dithiothreitol; the corresponding inhibitions of the renal enzyme at high and low substrate concentrations were 66% and 48%, respectively. Specific binding of PTU to renal and cerebral microsomes was diminished by hydroxyethyldisulfide, but not by T4, suggesting that PTU binding was not dependent on substrate interaction. Administration of PTU in vivo (1 mg/100 g BW, ip; 1 h before killing) resulted in approximately 80% inhibition of I-5'D activity in renal microsomes at high T4, and 50-70% inhibition in pituitary, BAT, and renal microsomes at low T4, but no inhibition was observed in brain microsomes. HPLC analyses revealed a PTU content of 35-65 nmol/g wet wt in the pituitary, BAT, liver, and kidney, but no PTU was detected in the brain, suggesting that PTU may be excluded by the blood-brain barrier. Maintaining hypothyroid rats on 1 microgram T4/100 g BW.day for 5 days enhanced renal type I and low Km I-5'D with restoration of serum T3 to normal levels, although the type II I-5'Ds from all sources were severely depressed. A supraphysiological dose of T4 depressed all three I-5'Ds. The data indicate that the I-5'Ds are regulated in a qualitatively similar fashion.

Adipose Tissue, Brown↗

Reactive thiols in type II iodothyronine 5'-deiodinases: inactivation by iodoacetate.

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 inactivated by IAC. The reaction follows pseudo-first-order kinetics with an inactivation rate constant of 0.08 min-1. Moreover, the enzyme is inhibited by hydroxyethyl disulfide and propylthiouracil. These reagents, but not thyroxine, also protect the enzyme from inactivation by IAC, The data suggest that IAC interacts with an essential thiol group in the active center domain.

Animals↗

Effects of glutathione on iodothyronine 5'-deiodinase activity.

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 activity at micromolar substrate concentrations required higher (greater than 10 mM) concentrations of GSH. However, GSH appeared to interact with the type I and type II enzymes even at subactivation levels, since it inhibited the activation of these enzymes by dithiothreitol (DTT). Activation of the renal and hepatic low Km I-5'D by GSH resembled that by DTT in 1) the similarity of Km values for both T4 (20 nM) and rT3 (2 nM), 2) the catalytic mechanism (ordered sequential with the iodothyronine as the second substrate), 3) the values for activation energies, and 4) sensitivity to inhibition by propylthiouracil. However, the low Km I-5'D activated by GSH was about 10-fold more sensitive to inhibition by iopanoate than when activated by DTT. The responsiveness of the low Km I-5'D's in renal and hepatic microsomes to physiological concentrations of GSH suggests their participation in the metabolism of iodothyronines in vivo.

Animals↗

Iodothyronine 5'-deiodination in rat kidney microsomes: sensitivity to propylthiouracil.

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 presence of a combination of 10 microM IOP and 100 microM PTU. In this report we present evidence that 1) the relative PTU insensitivity results from the residual activities of the high Km enzyme which, while being very sensitive to PTU inhibition at micromolar substrate concentrations, becomes progressively less PTU sensitive as substrate concentrations decline relative to its Km; and 2) the relative IOP insensitivity is due to the presence in kidney microsomes of a low Km enzyme which is relatively insensitive to IOP, but highly sensitive to inhibition by PTU. Classifying the deiodinases on the basis of PTU sensitivity, therefore, requires that not only the thiol concentrations, but, as in the case of the type I enzyme, also the substrate concentrations be specified. The PTU resistance of the type I enzyme at nanomolar substrate concentrations suggests a role of this enzyme in T3 neogenesis in PTU-treated animals.

Animals↗

Thioredoxin stimulates enzymatic outer ring monodeiodination of reverse triiodothronine.

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 concentrations in stimulating microsomal rT3 deiodination (EC50, approximately 15 microM); Thd-supported microsomal deiodination showed a maximum velocity approximately one third that in the presence of dithiothreitol (DTT), and Thd-supported deiodination, compared to that with DTT, was 10- and 2000-fold more sensitive to inhibition by propylthiouracil and iopanoate, respectively, than was the DTT-supported reaction. The Michaelis constants for rT3 (2.5 nM) were identical for the Thd- and DTT-activated reactions, suggesting that these thiols stimulated deiodination by the same enzyme. Arrhenius plots also revealed comparable activation energies for Thd- and DTT-mediated low Km rT3 monodeiodination; these activation energies were, moreover, distinct from those observed with the low Km T4 deiodination in the presence of DTT. The data suggest that renal and hepatic microsomes contain separate low Km rT3-specific and T4-specific 5'-monodeiodinases and that the rT3-specific monodeiodinase can use the reducing potential of NADPH, via the Thd system. Such an enzyme could mediate the disposal of rT3, the noncalorigenic metabolite of T4, independently of the conversion of T4 to T3.

Animals↗

Iodothyronine 5'-deiodinase in brown adipose tissue: thiol activation and propylthiouracil inhibition.

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 mM DTT in vitro. In the studies reported herein, BAT homogenates from hypothyroid rats prepared in a DTT-free medium were found to contain I-5'D activity at 20 mM DTT, comparable to that in homogenates prepared in a DTT-containing medium, and were activated by submillimolar concentrations of DTT with an EC50 of approximately 0.5 mM. Almost all of the homogenate activities could be accounted for in microsomal preparations. The activity was substantially inhibited by 1 mM PTU. The PTU inhibition was progressively alleviated with increasing concentrations of added DTT and was not seen at DTT concentrations higher than 10 mM. At 250 microM DTT, the Km and maximum velocity values for rT3 and T4 were 2.9 and 1 nM and 70 and 200 fmol/mg protein X h, respectively, with a Ki for PTU of approximately 200 microM. On administration of PTU in vivo (2 mg/100 g BW; 1 h before killing) and subsequent assay at 250 microM DTT, the I-5'D in the homogenates was about 50% inhibited, and the microsomes showed a state of persistent inhibition, with activity levels about 70% of the control value. The data show that BAT type II I-5'D can be substantially activated at submillimolar concentrations of DTT, and this activation is sensitive to inhibition by PTU administered both in vitro and in vivo.

Adipose Tissue, Brown↗

Purification and characterization of a cytosolic protein enhancing GSH-dependent microsomal iodothyronine 5'-monodeiodination.

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 ribonucleotide reductase with one-third the potency of native calf thymus glutaredoxin. Another basic protein, which degraded iodothyronines oxidatively, was also identified in the cytosolic preparations; this co-purified with soluble protein factor in the earlier purification stages and was partially separated from this factor by CM-cellulose chromatography. The glutaredoxin-like protein present in rat liver and kidney cytosol could provide a physiologic regulatory mechanism for GSH-dependent 5'-monodeiodination of iodothyronines.

Animals↗

Effects of thyroid status on membrane-bound low Km cyclic nucleotide phosphodiesterase activities in rat adipocytes.

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 of cGMP. Preincubation of hypothyroid adipocytes with cGMP also restored to normal the blunted lipolytic response to micromolar concentrations of epinephrine. DEAE-Sephacel chromatography of detergent-solubilized membrane-bound cAMP phosphodiesterase showed a 2.5-fold enhancement in hypothyroid membranes of a form of the enzyme that was completely inhibited by cGMP; the enzymatic elution profiles of the soluble fractions showed no difference between normal and hypothyroid fat pads. The results suggest a possible regulatory role of cGMP in adipocytes in the hypothyroid state.

3',5'-Cyclic-AMP Phosphodiesterases↗

Iodothyronine 5'-deiodinase in rat kidney microsomes. Kinetic behavior at low substrate concentrations.

The thiol-activated enzymatic outer-ring monodeiodination of iodothyronines by rat kidney microsomes at low (nanomolar) substrate concentrations shows an apparently sequential reaction mechanism and is further characterized by insensitivity to inhibition by dicoumarol, a moderate sensitivity to inhibition by propylthiouracil (Ki = 100 microM) and iopanoic acid (Ki = 0.9 mM), responsiveness to 5 mM glutathione (GSH), and a thermal activation profile that is concave downward with a Td of approximately 20 degrees C. In contrast, the activity at high (micromolar) substrate concentrations shows a ping-pong reaction mechanism, is inhibited by micromolar concentrations of propylthiouracil, iopanoic acid and dicoumarol, is unresponsive to 5 mM GSH, and shows a concave upward thermal activation profile. Analysis of the microsomal deiodinase reaction over a wide range of 3,3',5'-triiodothyronine (rT3) concentrations (0.1 nM to 10 microM) suggested the presence of two enzymatic activities, with apparent Michaelis constants (Km) of 0.5 microM and 2.5 nM. Lineweaver-Burk plots of reaction velocities at nanomolar substrate concentrations in presence of 100 microM propylthiouracil also revealed an operationally distinct enzymatic activity with Km's of 2.5 and 0.63 nM and maximum velocities (Vmax's) of 16 and 0.58 pmol/mg protein per h for rT3 and thyroxine (T4), respectively. These findings are consistent with the presence of a low Km iodothyronine 5'-deiodinase in rat kidney microsomes distinct from the well characterized high Km enzyme and suggest that at circulating levels of free T4 the postulated low Km enzyme could be physiologically important.

Animals↗

Stimulation of iodothyronine outer ring monodeiodinase by dihydrolipoamide.

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 the postnuclear and mitochondrial, but not in the microsomal fraction. The covalently bound lipoamide in the mitochondrial alpha-keto acid dehydrogenase complexes, when reduced with substrates or NADH, was, however, ineffective. The activation of the enzyme by DHL was very similar to that obtained with dithiothreitol in respect to temperature dependence and inhibition by propylthiouracil and by dicoumarol, suggesting that these thiols activate the same enzyme through similar mechanisms. The higher potency of DHL cannot be explained on the basis of its greater lipophilicity or relatively proximal dithiol structure. The occurrence of DHL in mitochondria raises the possibility of a role as a thiol cofactor in enzymatic outer ring deiodination of T4 or rT3, although attempts to stimulate deiodination in mitochondrial preparations by reducing endogenous bound lipoamide were unsuccessful.

Animals↗

Solubilization of a phospholipid-requiring enzyme, iodothyronine 5'-deiodinase, from rat kidney membranes.

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 density of 1.17 g/ml resembling that of membranes. Fractionation of the solubilized membrane components in the presence of 0.2% deoxycholate by either gel filtration or sucrose gradient centrifugation inactivated the enzyme(s) and activity could be restored by the addition of partially purified soybean phospholipids; this allowed some of the physical properties of the enzyme(s) to be determined. 5'-Deiodinating activity of both thyroxine and 3,3',5'-triiodothyronine was associated with protein(s) with S20,W of 3.5 S, Stokes' radius of 32 A, and a calculated molecular weight of 49 900. A partial specific volume of 0.74 cm3/g was calculated from sedimentation in 2H2O and H2O sucrose gradients. Phospholipid reactivation of lipid-depleted enzyme preparations was concentration-dependent, with near maximal restoration when sufficient phospholipid was added to restore the phospholipid:protein ratio to that of thyroxine and 3,3',5'-triiodothyronine could not be resolved by sedimentation or molecular sieving and showed similar behavior toward deoxycholate solubilization and phospholipid reconstitution.

Animals↗

Ferredoxin and ferredoxin reductase activities in bovine thyroid. Possible relationship to iodotyrosine deiodinase.

NADPH-ferredoxin reductase and ferredoxin activities have been identified in bovine thyroid particulate fractions (mainly mitochondria) after ultrasonication and DEAE-cellulose chromatography. The proteins were identified by their ability to reconstitute NADPH-cytochrome c reductase activity when used in combination. NADPH ferredoxin reductase and ferredoxin also catalyzed NADPH-dependent deiodination of L-diiodotyrosine; bovine adrenodoxin and adrenodoxin reductase could partially replace the thyroidal components in NADPH-dependent deiodination of L-diiodotyrosine. Both these reconstitutive activities were substantially inhibited by the iron chelators, alpha, alpha'-dipyridyl and o-phenanthroline. Deiodination by the NADPH-ferredoxin reductase-ferredoxin system was inhibited by the addition of a previously characterized dithionite-responsive flavoprotein iodotyrosine deiodinase (preparation F), isolated and purified from bovine thyroid particulate fractions after solubilization with steapsin. Ferredoxin reductase alone showed dithionite-responsive deiodinase activity and elution profiles of this activity on gel filtration before and after steapsin treatment suggest that preparation F may be a form of ferredoxin reductase modified by steapsin.

Animals↗